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Sample records for rna aptamer sequences

  1. A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer.

    PubMed

    Álvarez-Martos, Isabel; Ferapontova, Elena E

    2017-08-05

    A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus, is not an aptamer and cannot be used neither for in vivo nor in situ analysis of dopamine in the presence of structurally related neurotransmitters. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence

    PubMed Central

    2017-01-01

    Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface. PMID:28194280

  3. Minimal RNA aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor.

    PubMed

    Sivaprakasam, Kannan; Pagán, Oné R; Hess, George P

    2010-02-01

    Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10(11) neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.

  4. RNA aptamer inhibitors of a restriction endonuclease.

    PubMed

    Mondragón, Estefanía; Maher, L James

    2015-09-03

    Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. RAPID-SELEX for RNA Aptamers

    PubMed Central

    Ozer, Abdullah; Pagano, John M.; White, Brian S.; Shalloway, David; Lis, John T.; Craighead, Harold G.

    2013-01-01

    Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5×1015 sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance. PMID:24376564

  6. Use of a Fluorescent Aptamer RNA as an Exonic Sequence to Analyze Self-Splicing Ability of a Group I Intron from Structured RNAs

    PubMed Central

    Furukawa, Airi; Tanaka, Takahiro; Furuta, Hiroyuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

    2016-01-01

    Group I self-splicing intron constitutes an important class of functional RNA molecules that can promote chemical transformation. Although the fundamental mechanism of the auto-excision from its precursor RNA has been established, convenient assay systems for its splicing activity are still useful for a further understanding of its detailed mechanism and of its application. Because some host RNA sequences, to which group I introns inserted form stable three-dimensional (3D) structures, the effects of the 3D structures of exonic elements on the splicing efficiency of group I introns are important but not a fully investigated issue. We developed an assay system for group I intron self-splicing by employing a fluorescent aptamer RNA (spinach RNA) as a model exonic sequence inserted by the Tetrahymena group I intron. We investigated self-splicing of the intron from spinach RNA, serving as a model exonic sequence with a 3D structure. PMID:27869660

  7. Use of a Fluorescent Aptamer RNA as an Exonic Sequence to Analyze Self-Splicing Ability of aGroup I Intron from Structured RNAs.

    PubMed

    Furukawa, Airi; Tanaka, Takahiro; Furuta, Hiroyuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

    2016-11-17

    Group I self-splicing intron constitutes an important class of functional RNA molecules that can promote chemical transformation. Although the fundamental mechanism of the auto-excision from its precursor RNA has been established, convenient assay systems for its splicing activity are still useful for a further understanding of its detailed mechanism and of its application. Because some host RNA sequences, to which group I introns inserted form stable three-dimensional (3D) structures, the effects of the 3D structures of exonic elements on the splicing efficiency of group I introns are important but not a fully investigated issue. We developed an assay system for group I intron self-splicing by employing a fluorescent aptamer RNA (spinach RNA) as a model exonic sequence inserted by the Tetrahymena group I intron. We investigated self-splicing of the intron from spinach RNA, serving as a model exonic sequence with a 3D structure.

  8. Evolution and Protein Packaging of Small Molecule RNA Aptamers

    PubMed Central

    Lau, Jolene L.; Baksh, Michael M.; Fiedler, Jason D.; Brown, Steven D.; Kussrow, Amanda; Bornhop, Darryl J.; Ordoukhanian, Phillip

    2011-01-01

    A high-affinity RNA aptamer (Kd = 50 nM) was efficiently identified by SELEX against a heteroaryl dihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (non-encapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique. PMID:21899290

  9. Investigating the malleability of RNA aptamers

    SciTech Connect

    Ilgu, Muslum; Wang, Tianjiao; Lamm, Monica H.; Nilsen-Hamilton, Marit

    2013-03-25

    Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics.The paper discusses the integration of biochemical analysis with NMR spectroscopy and computational modeling to explore the relation between ligand binding and structural malleability of some well-studied aptamers. Several methods for determining aptamer binding affinity and specificity are discussed, including isothermal titration calorimetry, steady state fluorescence of 2-aminopurine substituted aptamers, and dye displacement assays. Also considered are aspects of molecular dynamics simulations specific to aptamers including adding ions and simulating aptamer structure in the absence of ligand when NMR spectroscopy or X-ray crystallography structures of the unoccupied aptamer are not available. We focus specifically on RNA aptamers that bind small molecule ligands as would be applied in sensors or integrated into riboswitches such as to measure the products of metabolic activity.

  10. Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection

    PubMed Central

    Thiel, William H.; Bair, Thomas; Peek, Andrew S.; Liu, Xiuying; Dassie, Justin; Stockdale, Katie R.; Behlke, Mark A.; Miller, Francis J.; Giangrande, Paloma H.

    2012-01-01

    Background The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers. Methodology/Principal Findings We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs. Conclusions and Significance We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies. PMID:22962591

  11. RNA aptamer delivery through intact human skin.

    PubMed

    Lenn, Jon D; Neil, Jessica; Donahue, Christine; Demock, Kellie; Tibbetts, Caitlin Vestal; Cote-Sierra, Javier; Smith, Susan H; Rubenstein, David; Therrien, Jean-Philippe; Pendergrast, P Shannon; Killough, Jason; Brown, Marc B; Williams, Adrian C

    2017-09-20

    It is generally recognised that only relatively small molecular weight (typically < ∼500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (MW=20,395) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridisation assay quantified aptamer from the epidermis and dermis giving levels far exceeding the cellular IC50 values (> 100,000-fold) and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A Th17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and 22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very large molecular weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Structural Principles of Fluorescent RNA Aptamers.

    PubMed

    Trachman, Robert J; Truong, Lynda; Ferré-D'Amaré, Adrian R

    2017-10-01

    Several aptamer RNAs have been selected in vitro that bind to otherwise weakly fluorescent small molecules and enhance their fluorescence several thousand-fold. By genetically tagging cellular RNAs of interest with these aptamers and soaking cells in their cell-permeable cognate small-molecule fluorophores, it is possible to use them to study RNA localization and trafficking. These aptamers have also been fused to metabolite-binding RNAs to generate fluorescent biosensors. The 3D structures of three unrelated fluorogenic RNAs have been determined, and reveal a shared reliance on base quadruples (tetrads) to constrain the photo-excited chromophore. The structural diversity of fluorogenic RNAs and the chemical diversity of potential fluorophores to be activated are likely to yield a variety of future fluorogenic RNA tags that are optimized for different applications in RNA imaging and in the design of fluorescent RNA biosensors. Copyright © 2017. Published by Elsevier Ltd.

  13. RNA fluorescence with light-up aptamers

    NASA Astrophysics Data System (ADS)

    Ouellet, Jonathan

    2016-06-01

    Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way.

  14. Development of RNA aptamers for detection of Salmonella Enteritidis.

    PubMed

    Hyeon, Ji-Yeon; Chon, Jung-Whan; Choi, In-Soo; Park, Chankyu; Kim, Dong-Eun; Seo, Kun-Ho

    2012-04-01

    We developed and evaluated RNA aptamers to analyze their potential for use in detecting Salmonella Enteritidis. The selected aptamer was observed to specifically bind to Salmonella Enteritidis without any cross-reactivity to other Salmonella serovars. Thus, this study suggests that aptamers specific to Salmonella Enteritidis have a high potential for use in presumptive presumptive screening methods or alternative serotyping methods.

  15. Galaxy Workflows for Web-based Bioinformatics Analysis of Aptamer High-throughput Sequencing Data

    PubMed Central

    Thiel, William H

    2016-01-01

    Development of RNA and DNA aptamers for diagnostic and therapeutic applications is a rapidly growing field. Aptamers are identified through iterative rounds of selection in a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). High-throughput sequencing (HTS) revolutionized the modern SELEX process by identifying millions of aptamer sequences across multiple rounds of aptamer selection. However, these vast aptamer HTS datasets necessitated bioinformatics techniques. Herein, we describe a semiautomated approach to analyze aptamer HTS datasets using the Galaxy Project, a web-based open source collection of bioinformatics tools that were originally developed to analyze genome, exome, and transcriptome HTS data. Using a series of Workflows created in the Galaxy webserver, we demonstrate efficient processing of aptamer HTS data and compilation of a database of unique aptamer sequences. Additional Workflows were created to characterize the abundance and persistence of aptamer sequences within a selection and to filter sequences based on these parameters. A key advantage of this approach is that the online nature of the Galaxy webserver and its graphical interface allow for the analysis of HTS data without the need to compile code or install multiple programs. PMID:28131286

  16. IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    PubMed Central

    Boese, B. J.; Corbino, K.; Breaker, R. R.

    2017-01-01

    We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust affinity for cellulose in both the powdered and paper form, but did not show any significant affinity for closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using the glucosamine 6-phosphate to activate glmS ribozyme function. PMID:18696364

  17. Inhibition of Hirame rhabdovirus growth by RNA aptamers.

    PubMed

    Hwang, S D; Midorikawa, N; Punnarak, P; Kikuchi, Y; Kondo, H; Hirono, I; Aoki, T

    2012-12-01

    RNA aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. They are an effective tool for pharmaceutical research and development of antiviral agents. Here, we describe four Hirame rhabdovirus (HIRRV)-RNA aptamers (H1, H2, H3 and H4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (SELEX). The HIRRV-RNA aptamers specifically bind to HIRRV. Hirame natural embryo (HINAE) cells treated with virus and the RNA aptamer showed a decrease in appearance of cytopathic effect when compared with control (treated only with virus). Rhodovulum sulfidophilum was transformed with genes for the RNA aptamers, and the aptamers were detected in the culture medium, indicating that they were secreted from the cells. Thus, the recombinant R. sulfidophilum might be a powerful tool for the prevention of HIRRV in aquaculture.

  18. Structure and thermodynamics of Drug-RNA aptamer interactions.

    PubMed

    Da Costa, J B; Dieckmann, T

    2013-04-01

    This mini-review will provide an overview on the recent studies of structure and thermodynamics of RNA aptamers that target drug molecules. These aptamers are studied to provide insight into RNA drug interactions. This interaction is important due to the many roles RNA plays in cell biology.

  19. Aptamer mediated siRNA delivery

    PubMed Central

    Chu, Ted C.; Twu, Karen Y.; Ellington, Andrew D.; Levy, Matthew

    2006-01-01

    Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology. PMID:16740739

  20. Current Progress of RNA Aptamer-Based Therapeutics

    PubMed Central

    Zhou, Jiehua; Bobbin, Maggie L.; Burnett, John C.; Rossi, John J.

    2012-01-01

    Aptamers are single-stranded nucleic acids that specifically recognize and bind tightly to their cognate targets due to their stable three-dimensional structure. Nucleic acid aptamers have been developed for various applications, including diagnostics, molecular imaging, biomarker discovery, target validation, therapeutics, and drug delivery. Due to their high specificity and binding affinity, aptamers directly block or interrupt the functions of target proteins making them promising therapeutic agents for the treatment of human maladies. Additionally, aptamers that bind to cell surface proteins are well suited for the targeted delivery of other therapeutics, such as conjugated small interfering RNAs (siRNA) that induce RNA interference (RNAi). Thus, aptamer-siRNA chimeras may offer dual-functions, in which the aptamer inhibits a receptor function, while the siRNA internalizes into the cell to target a specific mRNA. This review focuses on the current progress and therapeutic potential of RNA aptamers, including the use of cell-internalizing aptamers as cell-type specific delivery vehicles for targeted RNAi. In particular, we discuss emerging aptamer-based therapeutics that provide unique clinical opportunities for the treatment various cancers and neurological diseases. PMID:23130020

  1. Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor

    PubMed Central

    Kahsai, Alem W.; Wisler, James W.; Lee, Jungmin; Ahn, Seungkirl; Cahill, Thomas J.; Dennison, S. Moses; Staus, Dean P.; Thomsen, Alex R. B.; Anasti, Kara M.; Pani, Biswaranjan; Wingler, Laura M.; Desai, Hemant; Bompiani, Kristin M.; Strachan, Ryan T.; Qin, Xiaoxia; Alam, S. Munir; Sullenger, Bruce A.; Lefkowitz, Robert J.

    2016-01-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs. PMID:27398998

  2. Aptamer-siRNA Chimeras: Discovery, Progress, and Future Prospects.

    PubMed

    Kruspe, Sven; Giangrande, Paloma H

    2017-08-09

    Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery tools for many therapeutic oligonucleotide-based drugs, including small interfering RNAs (siRNAs). In this review, we summarize recent progress in the aptamer selection technology that has made possible the identification of cell-specific, cell-internalizing aptamers for the cell-targeted delivery of therapeutic oligonucleotides. In addition, we review the original, proof-of-concept aptamer-siRNA delivery studies and discuss recent advances in aptamer-siRNA conjugate designs for applications ranging from cancer therapy to the development of targeted antivirals. Challenges and prospects of aptamer-targeted siRNA drugs for clinical development are further highlighted.

  3. Identification and Characterization of RNA Aptamers: A Long Aptamer Blocks the AMPA Receptor and a Short Aptamer Blocks Both AMPA and Kainate Receptors.

    PubMed

    Jaremko, William J; Huang, Zhen; Wen, Wei; Wu, Andrew; Karl, Nicholas; Niu, Li

    2017-03-21

    AMPA and kainate receptors, along with NMDA receptors, represent different subtypes of glutamate ion channels. AMPA and kainate receptors share a high degree of sequence and structural similarities, and excessive activity of these receptors has been implicated in neurological diseases such as epilepsy. Therefore, blocking detrimental activity of both receptor types could be therapeutically beneficial. Here, we report the use of an in vitro evolution approach involving systematic evolution of ligands by exponential enrichment with a single AMPA receptor target (i.e. GluA1/2R) to isolate RNA aptamers that can potentially inhibit both AMPA and kainate receptors. A full-length or 101-nucleotide (nt) aptamer selectively inhibited GluA1/2R with a KI of ~5 µM, along with GluA1 and GluA2 AMPA receptor subunits. Of note, its shorter version (55 nt) inhibited both AMPA and kainate receptors. In particular, this shorter aptamer blocked equally potently the activity of both the GluK1 and GluK2 kainate receptors. Using homologous binding and whole-cell recording assays, we found that an RNA aptamer most likely binds to the receptor's regulatory site and inhibits it noncompetitively. Our results suggest the potential of using a single receptor target to develop RNA aptamers with dual activity for effectively blocking both AMPA and kainate receptors.

  4. Aptamers and the RNA World, Past and Present

    PubMed Central

    Gold, Larry; Janjic, Nebojsa; Jarvis, Thale; Schneider, Dan; Walker, Jeffrey J.; Wilcox, Sheri K.; Zichi, Dom

    2012-01-01

    Summary Aptamers and the SELEX process were discovered over two decades ago. These discoveries have spawned a productive academic and commercial industry. The collective results provide insights into biology, past and present, through an in vitro evolutionary exploration of the nature of nucleic acids and their potential roles in ancient life. Aptamers have helped usher in an RNA renaissance. Here we explore some of the evolution of the aptamer field and the insights it has provided for conceptualizing an RNA world, from its nascence to our current endeavor employing aptamers in human proteomics to discover biomarkers of health and disease. PMID:21441582

  5. Structure and Sequence Search on Aptamer-Protein Docking

    NASA Astrophysics Data System (ADS)

    Xiao, Jiajie; Bonin, Keith; Guthold, Martin; Salsbury, Freddie

    2015-03-01

    Interactions between proteins and deoxyribonucleic acid (DNA) play a significant role in the living systems, especially through gene regulation. However, short nucleic acids sequences (aptamers) with specific binding affinity to specific proteins exhibit clinical potential as therapeutics. Our capillary and gel electrophoresis selection experiments show that specific sequences of aptamers can be selected that bind specific proteins. Computationally, given the experimentally-determined structure and sequence of a thrombin-binding aptamer, we can successfully dock the aptamer onto thrombin in agreement with experimental structures of the complex. In order to further study the conformational flexibility of this thrombin-binding aptamer and to potentially develop a predictive computational model of aptamer-binding, we use GPU-enabled molecular dynamics simulations to both examine the conformational flexibility of the aptamer in the absence of binding to thrombin, and to determine our ability to fold an aptamer. This study should help further de-novo predictions of aptamer sequences by enabling the study of structural and sequence-dependent effects on aptamer-protein docking specificity.

  6. Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

    PubMed

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T; Volk, David E

    2014-06-10

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects.

  7. Evolution and Characterization of a Benzylguanine-binding RNA Aptamer

    PubMed Central

    Xu, J.; Carrocci, T.J.; Hoskins, A. A.

    2016-01-01

    Repurposing the “protein-labeling toolkit” for RNA research could be a pragmatic approach for developing new RNA-labeling methods. We have evolved an RNA aptamer that tightly binds benzylguanine (bG), the key ligand for the protein SNAP-tag. The aptamer tightly binds bG fluorophores and can be purified from cellular RNA with bG agarose under native conditions. PMID:26538152

  8. Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast

    PubMed Central

    Weigand, Julia E.; Suess, Beatrix

    2007-01-01

    Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression. PMID:17567606

  9. On-chip synthesis of RNA aptamer microarrays for multiplexed protein biosensing with SPR imaging measurements.

    PubMed

    Chen, Yulin; Nakamoto, Kohei; Niwa, Osamu; Corn, Robert M

    2012-06-05

    Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.

  10. Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

    PubMed

    Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

    2011-02-01

    Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

  11. Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing.

    PubMed

    Valenzano, Stefania; De Girolamo, Annalisa; DeRosa, Maria C; McKeague, Maureen; Schena, Roberto; Catucci, Lucia; Pascale, Michelangelo

    2016-06-13

    Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. On the basis of the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by microequilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (Kd) values in the range of 0.2-152 μM with aptamer Tyr_10 as the most promising one followed by aptamer Tyr_14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust and innovative biosensor platforms for the detection of tyramine in food and beverages.

  12. RNA Aptamers as Effective Protein Antagonists in a Multicellular Organism

    NASA Astrophysics Data System (ADS)

    Shi, Hua; Hoffman, Bryan E.; Lis, John T.

    1999-08-01

    RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B53 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.

  13. Theophylline detection in serum using a self-assembling RNA aptamer-based gold nanoparticle sensor.

    PubMed

    Jiang, Hongyan; Ling, Kai; Tao, Xiaojun; Zhang, Qiqing

    2015-08-15

    Recently, DNA aptamer-gold nanoparticle (AuNP) conjugates have emerged as novel biosensing tools. Although RNA aptamers are more advantageous than DNA aptamers, their vulnerable nature during the construction of these conjugates restricts the development of RNA aptasensors. In this study, we developed an RNA aptamer-based AuNP sensor for the detection of theophylline in serum, combining the high binding affinity and selectivity of a theophylline RNA aptamer and the fluorescence quenching ability of AuNPs. In order to prevent nuclease degradation during the experimental process, the single strand of the theophylline RNA aptamer (33-mer) was split at the end loop region into two shorter halves, which were able to reassemble to form the theophylline-binding pocket. One fragment was linked to a DNA sequence that included a 15 thymine (T15) spacer and a polyadenine (polyA, A12) tail. The chimeric RNA/DNA oligonucleotide was attached to AuNPs within a few minutes via adsorption of the polyA tail. The other fragment was labeled with a fluorophore (Cy3). The two individual fragments self-assembled in the presence of theophylline. Upon ligand binding, the fragments came into close proximity, resulting in fluorescence quenching. This sensor exhibited a low detection limit of 0.05 µM, with a linear dynamic range from 0.1 to 10 µM in serum. Moreover, the sensor did not recognize theophylline-related compounds (e.g., caffeine and theobromine), demonstrating its high selectivity. This strategy offers new possibilities for the application of RNA aptasensors in clinical settings. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Anti-Transcription Factor RNA Aptamers as Potential Therapeutics

    PubMed Central

    Mondragón, Estefanía

    2016-01-01

    Transcription factors (TFs) are DNA-binding proteins that play critical roles in regulating gene expression. These proteins control all major cellular processes, including growth, development, and homeostasis. Because of their pivotal role, cells depend on proper TF function. It is, therefore, not surprising that TF deregulation is linked to disease. The therapeutic drug targeting of TFs has been proposed as a frontier in medicine. RNA aptamers make interesting candidates for TF modulation because of their unique characteristics. The products of in vitro selection, aptamers are short nucleic acids (DNA or RNA) that bind their targets with high affinity and specificity. Aptamers can be expressed on demand from transgenes and are intrinsically amenable to recognition by nucleic acid-binding proteins such as TFs. In this study, we review several natural prokaryotic and eukaryotic examples of RNAs that modulate the activity of TFs. These examples include 5S RNA, 6S RNA, 7SK, hepatitis delta virus-RNA (HDV-RNA), neuron restrictive silencer element (NRSE)-RNA, growth arrest-specific 5 (Gas5), steroid receptor RNA activator (SRA), trophoblast STAT utron (TSU), the 3′ untranslated region of caudal mRNA, and heat shock RNA-1 (HSR1). We then review examples of unnatural RNA aptamers selected to inhibit TFs nuclear factor-kappaB (NF-κB), TATA-binding protein (TBP), heat shock factor 1 (HSF1), and runt-related transcription factor 1 (RUNX1). The field of RNA aptamers for DNA-binding proteins continues to show promise. PMID:26509637

  15. Characterization of RNA aptamers that disrupt the RUNX1–CBFβ/DNA complex

    PubMed Central

    Barton, Jenny L.; Bunka, David H. J.; Knowling, Stuart E.; Lefevre, Pascal; Warren, Alan J.; Bonifer, Constanze; Stockley, Peter G.

    2009-01-01

    The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor β protein (CBFβ). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD–CBFβ complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity (∼100–160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFβ, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD–CBFβ complex and thus will further aid exploring the functions of this key transcription factor. PMID:19740763

  16. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay.

    PubMed

    Win, Maung Nyan; Klein, Joshua S; Smolke, Christina D

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with K(d) values of 2.50 and 4.00 microM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques.

  17. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

    PubMed Central

    Win, Maung Nyan; Klein, Joshua S.; Smolke, Christina D.

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with Kd values of 2.50 and 4.00 μM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques. PMID:17038331

  18. An RNA-aptamer-based two-color CRISPR labeling system

    PubMed Central

    Wang, Siyuan; Su, Jun-Han; Zhang, Feng; Zhuang, Xiaowei

    2016-01-01

    The spatial organization and dynamics of chromatin play important roles in essential biological functions. However, direct visualization of endogenous genomic loci in living cells has proven to be laborious until the recent development of CRISPR-Cas9-based chromatin labeling methods. These methods rely on the recognition of specific DNA sequences by CRISPR single-guide RNAs (sgRNAs) and fluorescent–protein-fused catalytically inactive Cas9 to label specific chromatin loci in cells. Previously, multicolor chromatin labeling has been achieved using orthogonal Cas9 proteins from different bacterial species fused to different fluorescent proteins. Here we report the development of an alternative two-color CRISPR labeling method using only the well-characterized Streptococcus pyogenes Cas9, by incorporating MS2 or PP7 RNA aptamers into the sgRNA. The MS2 or PP7 aptamers then recruit the corresponding MS2 or PP7 coat proteins fused with different fluorescent proteins to the target genomic loci. Here we demonstrate specific and orthogonal two-color labeling of repetitive sequences in living human cells using this method. By attaching the MS2 or PP7 aptamers to different locations on the sgRNA, we found that extending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers enhances the signal-to-background ratio of chromatin imaging. PMID:27229896

  19. Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells▿ †

    PubMed Central

    Bellecave, Pantxika; Cazenave, Christian; Rumi, Julie; Staedel, Cathy; Cosnefroy, Ophélie; Andreola, Marie-Line; Ventura, Michel; Tarrago-Litvak, Laura; Astier-Gin, Thérèse

    2008-01-01

    We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection. PMID:18347106

  20. Massively Parallel Interrogation of Aptamer Sequence, Structure and Function

    SciTech Connect

    Fischer, N O; Tok, J B; Tarasow, T M

    2008-02-08

    Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. Methodology/Principal Findings. High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and interchip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  1. Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

    PubMed Central

    Dieckmann, T; Suzuki, E; Nakamura, G K; Feigon, J

    1996-01-01

    In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures. PMID:8756406

  2. Spinach RNA aptamer detects lead (II) with high selectivity†

    PubMed Central

    DasGupta, Saurja; Shelke, Sandip A.; Li, Nan-sheng

    2015-01-01

    Spinach RNA aptamer contains a G-quadruplex motif that serves as a platform for binding and fluorescence activation of a GFP-like fluorophore. Here we show that Pb2+ induces formation of Spinach’s G-quadruplex and activates fluorescence with high selectivity and sensitivity. This device establishes the first example of an RNA-based sensor that provides a simple and inexpensive tool for Pb2+ detection. PMID:25940073

  3. Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

    PubMed Central

    2009-01-01

    Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

  4. Stabilization of bovine insulin against agitation-induced aggregation using RNA aptamers.

    PubMed

    Malik, Ravinder; Roy, Ipsita

    2013-08-16

    Stabilization of monomeric insulin is a primary requirement for preserving the efficacy of the final formulation. Degraded and/or aggregated protein as well as the presence of any of the conventional excipients can result in immunogenic or anaphylactic reactions, and reduced bioavailability of the protein drug. The aim of this work was to select novel RNA-based stabilizers of bovine insulin which would recognize and bind to the monomeric protein and help retain its bioactivity. RNA aptamers were selected by an in vitro selection method. They were screened for their ability to inhibit insulin fibrillation using agitation as a stress condition. The in vitro activity of insulin was determined by phosphorylation of downstream proteins in the cell. In vivo bioactivity was determined in a diabetic rat model. RNA aptamers, which bound to insulin with very low dissociation constants and high specificity, were selected. These sequences were aligned and consensus regions were found. The RNA sequences had no effect on the signalling cascade initiated by insulin. The bioactivity of insulin, as measured by its ability to lower plasma glucose level in a diabetic rat model, also remained unchanged. RNA aptamers are a novel class of protein stabilizers which have the ability to disrupt protein-protein interactions and hence inhibit protein aggregation. Their non-toxic and non-immunogenic nature makes such formulations safe for use.

  5. Regulation of photosensitisation processes by an RNA aptamer

    NASA Astrophysics Data System (ADS)

    Thoa, Tran Thi Thanh; Minagawa, Noriko; Aigaki, Toshiro; Ito, Yoshihiro; Uzawa, Takanori

    2017-02-01

    One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy)32+, an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy)32+ (Kd = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy)32+ chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions.

  6. Regulation of photosensitisation processes by an RNA aptamer.

    PubMed

    Thoa, Tran Thi Thanh; Minagawa, Noriko; Aigaki, Toshiro; Ito, Yoshihiro; Uzawa, Takanori

    2017-02-24

    One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy)3(2+), an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy)3(2+) (Kd = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy)3(2+) chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions.

  7. Regulation of photosensitisation processes by an RNA aptamer

    PubMed Central

    Thoa, Tran Thi Thanh; Minagawa, Noriko; Aigaki, Toshiro; Ito, Yoshihiro; Uzawa, Takanori

    2017-01-01

    One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy)32+, an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy)32+ (Kd = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy)32+ chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions. PMID:28233875

  8. Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

    PubMed Central

    Tian, Y; Adya, N; Wagner, S; Giam, C Z; Green, M R; Ellington, A D

    1995-01-01

    Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies. PMID:7489503

  9. RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

    PubMed

    Punnarak, Porntep; Santos, Mudjekeewis D; Hwang, Seong Don; Kondo, Hidehiro; Hirono, Ikuo; Kikuchi, Yo; Aoki, Takashi

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

  10. RNA aptamer evolution: two decades of SELEction.

    PubMed

    Aquino-Jarquin, Guillermo; Toscano-Garibay, Julia D

    2011-01-01

    Aptamers are small non-coding RNAs capable of recognizing, with high specificity and affinity, a wide variety of molecules in a manner that resembles antibodies. This class of nucleic acids is the resulting product of applying a well-established screening method known as SELEX. First developed in 1990, the SELEX process has become a powerful tool to select structured oligonucleotides for the recognition of targets, starting with small molecules, going through protein complexes until whole cells. SELEX has also evolved along with new technologies positioning itself as an alternative in the design of a new class of therapeutic agents in modern molecular medicine. This review is an historical follow-up of SELEX method over the two decades since its first appearance.

  11. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    NASA Astrophysics Data System (ADS)

    Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

    2012-03-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  12. A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.

    PubMed

    Alam, Khalid K; Tawiah, Kwaku D; Lichte, Matthew F; Porciani, David; Burke, Donald H

    2017-09-15

    RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

  13. Aptamer redesigned tRNA is nonfunctional and degraded in cells

    PubMed Central

    LEE, DENNIS; MCCLAIN, WILLIAM H.

    2004-01-01

    An RNA aptamer derived from tRNAGln isolated in vitro and a rationally redesigned tRNAGln were used to address the relationship between structure and function of tRNAGln aminoacylation in Escherichia coli. Two mutant tRNAGln sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNAGln in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNAGln knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro. PMID:14681579

  14. An l-RNA Aptamer with Expanded Chemical Functionality that Inhibits MicroRNA Biogenesis.

    PubMed

    Kabza, Adam M; Sczepanski, Jonathan T

    2017-09-19

    To facilitate isolation of l-aptamers with novel RNA-binding properties, we employed a cationic nucleotide, 5-aminoallyluridine, during the mirror image in vitro selection process. Through this effort, we identified a modified l-RNA aptamer (MlRA) capable of binding oncogenic precursor microRNA 19a (pre-miR-19a) with exceptional affinity, and we showed that cationic modification is absolutely critical for binding. Furthermore, formation of the MlRA-pre-miR-19a complex inhibited Dicer-mediated cleavage of the pre-miR, thus blocking formation of the mature functional microRNA. The MlRA reported here not only represents the first l-aptamer to be evolved by using modified nucleotides but also the first modified aptamer (of any type) to be selected against a structured RNA target. Our results demonstrate that functionalized l-aptamers, which are intrinsically nuclease-resistant, provide an attractive approach for developing robust RNA-binding reagents. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein

    PubMed Central

    Ellenbecker, Mary; St. Goddard, Jeremy; Sundet, Alec; Lanchy, Jean-Marc; Raiford, Douglas; Lodmell, J. Stephen

    2015-01-01

    Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensional scaling to compare the predicted secondary structures of known N-binding RNAs, or aptamers, that were isolated and characterized in previous in vitro evolution experiment. These aptamers did not exhibit overt sequence or predicted structure similarity, so we employed bioinformatic methods to propose novel aptamers based on analysis and clustering of secondary structures. We screened and scored the predicted secondary structures of novel randomly generated RNA sequences in silico and selected several of these putative N-binding RNAs whose secondary structures were similar to those of known N-binding RNAs. We found that overall the in silico generated RNA sequences bound well to N in vitro. Furthermore, introduction of these RNAs into cells prior to infection with RVFV inhibited viral replication in cell culture. This proof of concept study demonstrates how the predictive power of bioinformatics and the empirical power of biochemistry can be jointly harnessed to discover, synthesize, and test new RNA sequences that bind tightly to RVFV N protein. The approach would be easily generalizable to other applications. PMID:26141677

  16. Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein.

    PubMed

    Ellenbecker, Mary; St Goddard, Jeremy; Sundet, Alec; Lanchy, Jean-Marc; Raiford, Douglas; Lodmell, J Stephen

    2015-10-01

    Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensional scaling to compare the predicted secondary structures of known N-binding RNAs, or aptamers, that were isolated and characterized in previous in vitro evolution experiment. These aptamers did not exhibit overt sequence or predicted structure similarity, so we employed bioinformatic methods to propose novel aptamers based on analysis and clustering of secondary structures. We screened and scored the predicted secondary structures of novel randomly generated RNA sequences in silico and selected several of these putative N-binding RNAs whose secondary structures were similar to those of known N-binding RNAs. We found that overall the in silico generated RNA sequences bound well to N in vitro. Furthermore, introduction of these RNAs into cells prior to infection with RVFV inhibited viral replication in cell culture. This proof of concept study demonstrates how the predictive power of bioinformatics and the empirical power of biochemistry can be jointly harnessed to discover, synthesize, and test new RNA sequences that bind tightly to RVFV N protein. The approach would be easily generalizable to other applications.

  17. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers

    PubMed Central

    Espah Borujeni, Amin; Mishler, Dennis M.; Wang, Jingzhi; Huso, Walker; Salis, Howard M.

    2016-01-01

    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription–translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications. PMID:26621913

  18. Automated physics-based design of synthetic riboswitches from diverse RNA aptamers.

    PubMed

    Espah Borujeni, Amin; Mishler, Dennis M; Wang, Jingzhi; Huso, Walker; Salis, Howard M

    2016-01-08

    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications.

  19. In vivo expression and purification of aptamer-tagged small RNA regulators

    PubMed Central

    Said, Nelly; Rieder, Renate; Hurwitz, Robert; Deckert, Jochen; Urlaub, Henning; Vogel, Jörg

    2009-01-01

    Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria. PMID:19726584

  20. A self-assembling RNA aptamer-based graphene oxide sensor for the turn-on detection of theophylline in serum.

    PubMed

    Ling, Kai; Jiang, Hongyan; Li, Yang; Tao, Xiaojun; Qiu, Chen; Li, Fu-Rong

    2016-12-15

    To date, few effective fluorescent biosensors based on RNA aptamers have been developed because the intrinsic instability of RNA in the presence of nucleases precludes the application of RNA aptamers for the analysis of biological fluids. In this study, we developed a simple, sensitive, selective turn-on fluorescent aptasensor for theophylline detection in serum, utilizing ligand-induced self-assembling RNA aptamers and two different interaction stages of the aptamer fragments with graphene oxide (GO). A single strand of the theophylline RNA aptamer (33-mer) was split at the end loop region into two shorter fragments, one of which was labeled with a fluorophore (FAM). In the absence of theophylline, the adsorption of the two individual fragments on GO brought the fluorophore in close proximity to the GO surface, resulting in highly efficient quenching of fluorescence. The system showed very low background fluorescence. Conversely, the fragments self-assembled into an RNA aptamer/theophylline complex and were dissociated from GO. The quenched fluorescence was significantly recovered, and theophylline could be detected at a wide range of concentrations from 1 to 100μM, with a detection limit of 0.155μM and good selectivity in serum. Moreover, because of the shorter RNA fragments and the effective protection ability of GO from nuclease cleavage, the RNA sequences remained stable during the experiments. This design may serve as an example for the application of RNA aptasensors in the clinical setting. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. RNA-based networks: using RNA aptamers and ribozymes as synthetic genetic devices.

    PubMed

    Weigand, Julia E; Wittmann, Alexander; Suess, Beatrix

    2012-01-01

    Within the last few years, a set of synthetic riboswitches has been engineered, which expands the toolbox of genetic regulatory devices. Small molecule binding aptamers have been used for the design of such riboswitches by insertion into untranslated regions of mRNAs, exploiting the fact that upon ligand binding the RNA structure interferes either with translation initiation or pre-mRNA splicing in yeast. In combination with self-cleaving ribozymes, aptamers have been used to modulate RNA stability. In this chapter, we discuss the applicability of different aptamers, ways to identify novel genetic devices, the pros and cons of various insertion sites and the application of allosteric ribozymes. Our expertise help to apply synthetic riboswitches to engineer complex genetic circuits.

  2. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    PubMed Central

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-01-01

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. PMID:26389898

  3. Development of RNA Aptamer and its Ligand Binding Assay on Microchip Electrophoresis

    PubMed Central

    Ohno, Ken-ichi; Nakata, Chikara; Sano, Yoshihiro; Nishikawa, Fumiko; Nishikawa, Satoshi; Arakawa, Hidetoshi

    2012-01-01

    Microchip electrophoresis (ME) coupled with fluorescence detection was used to estimate the binding activity of aptamer in each systematic evolution of ligands by exponential enrichment (SELEX) round for a target molecule. This approach is a non-radioisotopic, rapid and simple platform, and electrophoretic separation appears to be an effective technique for aptamers of oligonucleotide molecules. We tried to obtain gonadotropin-specific RNA aptamer by the above approach. As a result, the peaks of aptamers based on the conformational differences between them were separated and detected on the electropherograms. Moreover, the intensity of peak of unbound aptamer was decreased with progression through the SELEX rounds, suggesting that RNA aptamer with high affinity was obtained by the proposed method. PMID:22291866

  4. Smooth Muscle Cell–targeted RNA Aptamer Inhibits Neointimal Formation

    PubMed Central

    Thiel, William H; Esposito, Carla L; Dickey, David D; Dassie, Justin P; Long, Matthew E; Adam, Joshua; Streeter, Jennifer; Schickling, Brandon; Takapoo, Maysam; Flenker, Katie S; Klesney-Tait, Julia; Franciscis, Vittorio de; Miller, Francis J; Giangrande, Paloma H

    2016-01-01

    Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease. PMID:26732878

  5. Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures

    PubMed Central

    Ellington, Andrew D.

    2009-01-01

    Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust. PMID:18594898

  6. Aptamers and aptamer targeted delivery

    PubMed Central

    Yan, Amy C.; Levy, Matthew

    2014-01-01

    When aptamers first emerged almost two decades ago, most were RNA species that bound and tagged or inhibited simple target ligands. Very soon after, the ‘selectionologists’ developing aptamer technology quickly realized more potential for the aptamer. In recent years, advances in aptamer techniques have enabled the use of aptamers as small molecule inhibitors, diagnostic tools and even therapeutics. Aptamers are now being employed in novel applications. We review, herein, some of the recent and exciting applications of aptamers in cell-specific recognition and delivery. PMID:19458497

  7. RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery.

    PubMed

    Pi, Fengmei; Zhang, Hui; Li, Hui; Thiviyanathan, Varatharasa; Gorenstein, David G; Sood, Anil K; Guo, Peixuan

    2017-04-01

    A novel modified nucleic acid nanoparticle harboring an annexin A2 aptamer for ovarian cancer cell targeting and a GC rich sequence for doxorubicin loading is designed and constructed. The system utilizes a highly stable three-way junction (3WJ) motif from phi29 packaging RNA as a core structure. A phosphorothioate-modified DNA aptamer targeting annexin A2, Endo28, was conjugated to one arm of the 3WJ. The pRNA-3WJ motif retains correct folding of attached aptamer, keeping its functions intact. It is of significant utility for aptamer-mediated targeted delivery. The DNA/RNA hybrid nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little accumulation in healthy organs 6 h post-injection. The Endo28-3WJ-Sph1/Dox intercalates selectively enhanced toxicity to annexin A2 positive ovarian cancer cells in vitro. The constructed RNA/DNA hybrid nanoparticles can potentially enhance the therapeutic efficiency of doxorubicin at low doses for ovarian cancer treatment through annexin A2 targeted drug delivery. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Biocompatible hydrogel membranes for the protection of RNA aptamer-based electrochemical sensors

    NASA Astrophysics Data System (ADS)

    Schoukroun-Barnes, Lauren R.; Wagan, Samiullah; Liu, Juan; Leach, Jennie B.; White, Ryan J.

    2013-05-01

    Electrochemical-aptamer based (E-AB) sensors represent a universal specific, selective, and sensitive sensing platform for the detection of small molecule targets. Their specific detection abilities are afforded by oligonucleotide (RNA or DNA) aptamers employed as electrode-bound biorecognition elements. Sensor signaling is predicated on bindinginduced changes in conformation and/or flexibility of the aptamer that is readily measurable electrochemically. While sensors fabricated using DNA aptamers can achieve specific and selective detection even in unadulterated sample matrices, such as blood serum, RNA-based sensors fail when challenged in the same sample matrix without significant sample pretreatment. This failure is at least partially a result of enzymatic degradation of the RNA sensing element. This degradation destroys the sensing aptamer inhibiting the quantitative measurement of the target analyte and thus limits the application of E-AB sensors constructed with RNA aptamer. To circumvent this, we demonstrate that a biocompatible hydrogel membrane protects the RNA aptamer sensor surface from enzymatic degradation for at least 3 hours - a remarkable improvement over the rapid (~minutes) degradation of unprotected sensors. To demonstrate this, we characterize the response of sensors fabricated with representative DNA and RNA aptamers directed against the aminoglycoside antibiotic, tobramycin in blood serum both protected and unprotected by a polyacrylamide membrane. Furthermore, we find encapsulation of the sensor surface with the hydrogel does not significantly impede the detection ability of aptamer-based sensors. This hydrogel-aptamer interface will thus likely prove useful for the long-term monitoring of therapeutics in complex biological media.

  9. Analytical applications of aptamers

    NASA Astrophysics Data System (ADS)

    Tombelli, S.; Minunni, M.; Mascini, M.

    2007-05-01

    Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. Aptamers are proposed as alternatives to antibodies as biorecognition elements in analytical devices with ever increasing frequency. This in order to satisfy the demand for quick, cheap, simple and highly reproducible analytical devices, especially for protein detection in the medical field or for the detection of smaller molecules in environmental and food analysis. In our recent experience, DNA and RNA aptamers, specific for three different proteins (Tat, IgE and thrombin), have been exploited as bio-recognition elements to develop specific biosensors (aptasensors). These recognition elements have been coupled to piezoelectric quartz crystals and surface plasmon resonance (SPR) devices as transducers where the aptamers have been immobilized on the gold surface of the crystals electrodes or on SPR chips, respectively.

  10. Rapid detection of food pathogens using RNA aptamers-immobilized slide.

    PubMed

    Maeng, Jin-Soo; Kim, Namsoo; Kim, Chong-Tai; Han, Seung Ryul; Lee, Young Ju; Lee, Seong-Wook; Lee, Myung-Hyun; Cho, Yong-Jin

    2012-07-01

    The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.

  11. Crystal structure of NF-κB (p50)2 complexed to a high-affinity RNA aptamer

    PubMed Central

    Huang, De-Bin; Vu, Don; Cassiday, Laura A.; Zimmerman, Jeff M.; Maher, L. James; Ghosh, Gourisankar

    2003-01-01

    We have recently identified an RNA aptamer for the transcription factor NF-κB p50 homodimer [(p50)2], which exhibits little sequence resemblance to the consensus DNA target for (p50)2, but binds (p50)2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem–loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3′ helix, together with the major groove of the irregular 3′ helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50)2. PMID:12886018

  12. Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

    NASA Astrophysics Data System (ADS)

    Sánchez-Luque, Francisco J.; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo

    2014-09-01

    The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

  13. RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX.

    PubMed

    Burke, D H; Gold, L

    1997-05-15

    We used in vitro selection (SELEX) to isolate RNA 'aptamers' to S-adenosyl methionine (SAM). Individual aptamer sequences conform to the structural element noted previously for adenosine binding in selections for aptamers to ATP and NAD+. When we compare the patterns of sequence conservation among 65 adenosine-binding sequences to the published structure of the adenosine aptamer, we find that the most highly conserved nucleotides contact the bound adenosine directly, and that one conserved nucleotide outside the binding pocket is in position to stabilize nucleotides within the binding pocket. The aptamer's ability to bind diverse adenosine-containing cofactors is easily understood in terms of its mode of binding, which leaves the 5'position exposed to solvent. We propose that aptamers that bind their targets away from the reactive moiety may be particularly well suited for catalysis. Finally, we estimate that one sequence in 10(11) may be able to form this structural motif, and that there may be many other adenosine-binding motifs that have escaped detection because of their lower representation in the starting random pools.

  14. Multivalent Aptamer-RNA Conjugates for Simple and Efficient Delivery of Doxorubicin/siRNA into Multidrug-Resistant Cells.

    PubMed

    Jeong, Hyosook; Lee, Soo Hyeon; Hwang, Yeonju; Yoo, Hyundong; Jung, Heesun; Kim, Sun Hwa; Mok, Hyejung

    2017-04-01

    Multivalent aptamer-siRNA conjugates containing multiple mucin-1 aptamers and BCL2-specific siRNA are synthesized, and doxorubicin, an anthracycline anticancer drug, is loaded into these conjugates through intercalation with nucleic acids. These doxorubicin-incorporated multivalent aptamer-siRNA conjugates are transfected to mucin-1 overexpressing MCF-7 breast cancer cells and their multidrug-resistant cell lines. Doxorubicin-incorporated multivalent aptamer-siRNA conjugates exert promising anticancer effects, such as activation of caspase-3/7 and decrease of cell viability, on multidrug-resistant cancer cells because of their high intracellular uptake efficiency. Thus, this delivery system is an efficient tool for combination oncotherapy with chemotherapeutics and nucleic acid drugs to overcome multidrug resistance.

  15. An aptamer-siRNA chimera suppresses HIV-1 viral loads and protects from helper CD4(+) T cell decline in humanized mice.

    PubMed

    Neff, Charles Preston; Zhou, Jiehua; Remling, Leila; Kuruvilla, Jes; Zhang, Jane; Li, Haitang; Smith, David D; Swiderski, Piotr; Rossi, John J; Akkina, Ramesh

    2011-01-19

    Therapeutic strategies designed to treat HIV infection with combinations of antiviral drugs have proven to be the best approach for slowing the progression to AIDS. Despite this progress, there are problems with viral drug resistance and toxicity, necessitating new approaches to combating HIV-1 infection. We have therefore developed a different combination approach for the treatment of HIV infection in which an RNA aptamer, with high binding affinity to the HIV-1 envelope (gp120) protein and virus neutralization properties, is attached to and delivers a small interfering RNA (siRNA) that triggers sequence-specific degradation of HIV RNAs. We have tested the antiviral activities of these chimeric RNAs in a humanized Rag2(-/-)γc(-/-) (RAG-hu) mouse model with multilineage human hematopoiesis. In this animal model, HIV-1 replication and CD4(+) T cell depletion mimic the situation seen in human HIV-infected patients. Our results show that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4(+) T cell decline. In comparison to the aptamer alone, the aptamer-siRNA combination provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose. The aptamer thus acts as a broad-spectrum HIV-neutralizing agent and an siRNA delivery vehicle. The combined aptamer-siRNA agent provides an attractive, nontoxic therapeutic approach for treatment of HIV infection.

  16. Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device

    PubMed Central

    Reinholt, Sarah J.; Ozer, Abdullah; Lis, John T.; Craighead, Harold G.

    2016-01-01

    We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA’s reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO’s exoribonuclease activity, and in studies monitoring DXO’s degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation. PMID:27432610

  17. First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins.

    PubMed

    Sapag, Amalia; Salinas-Luypaert, Catalina; Constenla-Muñoz, Carlos

    2014-03-26

    Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxosceles venom agents.

  18. Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers.

    PubMed

    Jeng, Sunny C Y; Chan, Hedy H Y; Booy, Evan P; McKenna, Sean A; Unrau, Peter J

    2016-12-01

    The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 10(3)-fold increase in fluorescence. We find that RNA Mango has a large increase in its thermal stability upon the addition of its TO1-Biotin ligand. Consistent with this thermal stabilization, RNA Mango can effectively discriminate TO1-Biotin from a broad range of small molecule fluorophores. In contrast, RNA Spinach, which is known to have a substantially more rigid G-quadruplex structure, was found to bind to this set of fluorophores, often with higher affinity than to its native ligand, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and did not exhibit thermal stabilization in the presence of the TO1-Biotin fluorophore. Our data suggest that RNA Mango is likely to use a concerted ligand-binding mechanism that allows it to simultaneously bind and recognize its TO1-Biotin ligand, whereas RNA Spinach appears to lack such a mechanism. The high binding affinity and fluorescent efficiency of RNA Mango provides a compelling alternative to RNA Spinach as an RNA reporter system and paves the way for the future development of small fluorophore RNA reporter systems. © 2016 Jeng et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Thermodynamics and kinetics of adaptive binding in the malachite green RNA aptamer.

    PubMed

    Da Costa, Jason B; Andreiev, Aurelia I; Dieckmann, Thorsten

    2013-09-24

    Adaptive binding, the ability of molecules to fold themselves around the structure of a ligand and thereby incorporating it into their three-dimensional fold, is a key feature of most RNA aptamers. The malachite green aptamer (MGA) has been shown to bind several closely related triphenyl dyes with planar and nonplanar structures in this manner. Competitive binding studies using isothermal titration calorimetry and stopped flow kinetics have been conducted with the aim of understanding the adaptive nature of RNA-ligand interaction. The results of these studies reveal that binding of one ligand can reduce the ability of the aptamer pocket to adapt to another ligand, even if this second ligand has a significantly higher affinity to the free aptamer. A similar effect is observed in the presence of Mg(2+) ions which stabilize the binding pocket in a more ligand bound-like conformation.

  20. An RNA aptamer that interferes with the DNA binding of the HSF transcription activator.

    PubMed

    Zhao, Xiaoching; Shi, Hua; Sevilimedu, Aarti; Liachko, Nicole; Nelson, Hillary C M; Lis, John T

    2006-01-01

    Heat shock factor (HSF) is a conserved and highly potent transcription activator. It is involved in a wide variety of important biological processes including the stress response and specific steps in normal development. Reagents that interfere with HSF function would be useful for both basic studies and practical applications. We selected an RNA aptamer that binds to HSF with high specificity. Deletion analysis defined the minimal binding motif of this aptamer to be two stems and one stem-loop joined by a three-way junction. This RNA aptamer interferes with normal interaction of HSF with its DNA element, which is a key regulatory step for HSF function. The DNA-binding domain plus a flanking linker region on the HSF (DL) is essential for the RNA binding. Additionally, this aptamer inhibits HSF-induced transcription in vitro in the complex milieu of a whole cell extract. In contrast to the previously characterized NF-kappaB aptamer, the HSF aptamer does not simply mimic DNA binding, but rather binds to HSF in a manner distinct from DNA binding to HSF.

  1. NMR resonance assignments for the tetramethylrhodamine binding RNA aptamer 3 in complex with the ligand 5-carboxy-tetramethylrhodamine.

    PubMed

    Duchardt-Ferner, Elke; Juen, Michael; Kreutz, Christoph; Wöhnert, Jens

    2017-04-01

    RNA aptamers are used in a wide range of biotechnological or biomedical applications. In many cases the high resolution structures of these aptamers in their ligand-complexes have revealed fundamental aspects of RNA folding and RNA small molecule interactions. Fluorescent RNA-ligand complexes in particular find applications as optical sensors or as endogenous fluorescent tags for RNA tracking in vivo. Structures of RNA aptamers and aptamer ligand complexes constitute the starting point for rational function directed optimization approaches. Here, we present the NMR resonance assignment of an RNA aptamer binding to the fluorescent ligand tetramethylrhodamine (TMR) in complex with the ligand 5-carboxy-tetramethylrhodamine (5-TAMRA) as a starting point for a high-resolution structure determination using NMR spectroscopy in solution.

  2. In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets

    SciTech Connect

    Wang, Ying; Li, Zhaohui; Weber, Thomas J.; Hu, Dehong; Lin, Chiann Tso; Li, Jinghong; Lin, Yuehe

    2013-07-23

    Adenosine-5’-triphosphate (ATP) and guanosine-5’-triphosphate (GTP) are primary energy resources and function coordinately for numerous reactions such as microtubule assembly, insulin secretion and ion channel regulation. We have developed a novel DNA/RNA aptamer- graphene oxide nanosheet (GO-nS) sensing platform that can selectively and simultaneously detect ATP and GTP in live cells. A fluorescent tag is covalently attached to aptamers and fluorescence is quenched upon binding of aptamer to the GO-nS. Fluorescently tagged aptamers that selectively bind ATP or GTP were isolated from an aptamer library and were adsorbed onto GO-nS. Upon incubation with targets (ATP and/or GTP), the aptamers readily dissociated from GO-nS and the fluorescent signal was recovered. By covalently attaching fluorophores, both ATP and GTP sensing aptamers could be exploited to simultaneously visualize aptamer dissociation in live cells. In addition, the GO-nS appear to be biocompatible and protect the adsorbed DNA/RNA aptamers from enzymatic cleavage. Our results support the application of aptamer/GO-nS as a sensing platform for nucleotides in living cells and have implications for the development of additional sensor platforms for other bio-molecules that show selective interactions with aptamers and other biomarkers.

  3. Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    PubMed Central

    Forrest, Sophie; Lear, Zoe; Herod, Morgan R.; Ryan, Martin; Rowlands, David J.

    2014-01-01

    We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose dependent. The 3Dpol aptamers appeared to be more effective than 2′-C-methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future. PMID:25096816

  4. Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

    PubMed Central

    Fan, Shaoan; Wu, Feng; Martiniuk, Frank; Hale, Martha L; Ellington, Andrew D; Tchou-Wong, Kam-Meng

    2008-01-01

    AIM: To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated. METHODS: To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. RESULTS: Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. CONCLUSION: We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication. PMID:19009652

  5. Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics.

    PubMed

    Moore, Michael D; Cookson, Jonathan; Coventry, Veronica K; Sproat, Brian; Rabe, Lorna; Cranston, Ross D; McGowan, Ian; James, William

    2011-01-28

    RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.

  6. Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

    PubMed Central

    Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

  7. Highly stable aptamers selected from a 2'-fully modified fGmH RNA library for targeting biomaterials.

    PubMed

    Friedman, Adam D; Kim, Dongwook; Liu, Rihe

    2015-01-01

    When developed as targeting ligands for the in vivo delivery of biomaterials to biological systems, RNA aptamers immediately face numerous obstacles, in particular nuclease degradation and post-selection 2' modification. This study aims to develop a novel class of highly stable, 2'-fully modified RNA aptamers that are ideal for the targeted delivery of biomaterials. We demonstrated the facile transcription of a fGmH (2'-F-dG, 2'-OMe-dA/dC/dU) RNA library with unexpected hydrophobicity, the direct selection of aptamers from a fGmH RNA library that bind Staphylococcus aureus Protein A (SpA) as a model target, and the superior nuclease and serum stability of these aptamers compared to 2'-partially modified RNA variants. Characterizations of fGmH RNA aptamers binding to purified SpA and to endogenous SpA present on the surface of S. aureus cells demonstrate fGmH RNA aptamer selectivity and stability. Significantly, fGmH RNA aptamers were able to functionalize, stabilize, and specifically deliver aggregation-prone silver nanoparticles (AgNPs) to S. aureus with SpA-dependent antimicrobial effects. This study describes a novel aptamer class with considerable potential to improve the in vivo applicability of nucleic acid-based affinity molecules to biomaterials.

  8. Inhibition of Japanese encephalitis virus (JEV) replication by specific RNA aptamer against JEV methyltransferase.

    PubMed

    Han, Seung Ryul; Lee, Seong-Wook

    2017-01-29

    Japanese encephalitis virus (JEV) is the most common etiological agent of epidemic viral encephalitis. JEV encodes a single methyltransferase (MTase) domain located at the N-terminal region of the viral nonstructural protein NS5. JEV MTase is essential for viral replication and specifically catalyzes methylation of the viral RNA cap, which occurs exclusively in the cytoplasm. Therefore, JEV MTase is a potential target for antiviral therapy. Here, we identified specific and avid RNA aptamer (Kd ∼ 12 nM) with modified 2'-O-methyl pyrimidines against JEV MTase. The RNA aptamer efficiently inhibited viral cap methylation activity of MTase and interfered with JEV production in cells. Moreover, we generated a 24-mer truncated aptamer that could specifically bind to JEV MTase with high affinity (Kd ∼16 nM). The 24-mer aptamer efficiently inhibited JEV production and replication in cells. Therefore, MTase-specific RNA aptamer might be useful as an anti-JEV agent.

  9. An inhibitory RNA aptamer against the lambda cI repressor shows transcriptional activator activity in vivo.

    PubMed

    Ohuchi, Shoji; Suess, Beatrix

    2017-04-13

    An RNA aptamer is one of the promising components for constructing artificial genetic circuits. In this study, we developed a transcriptional activator based on an RNA aptamer against one of the most frequently applied repressor proteins, lambda phage cI. In vitro selection (SELEX), followed by in vivo screening identified an RNA aptamer with the intended transcriptional activator activity from an RNA pool containing a 40-nucleotide long random region. Quantitative analysis showed 35-fold elevation of reporter expression upon aptamer expression. These results suggest that the diversity of artificial transcriptional activators can be extended by employing RNA aptamers against repressor proteins to broaden the tools available for constructing genetic circuits. This article is protected by copyright. All rights reserved.

  10. An Aptamer Bio-barCode (ABC) assay using SPR, RNase H, and probes with RNA and gold-nanorods for anti-cancer drug screening.

    PubMed

    Loo, Jacky Fong-Chuen; Yang, Chengbin; Tsang, Hing Lun; Lau, Pui Man; Yong, Ken-Tye; Ho, Ho Pui; Kong, Siu Kai

    2017-10-07

    With modifications to an ultra-sensitive bio-barcode (BBC) assay, we have developed a next generation aptamer-based bio-barcode (ABC) assay to detect cytochrome-c (Cyto-c), a cell death marker released from cancer cells, for anti-cancer drug screening. An aptamer is a short single-stranded DNA selected from a synthetic DNA library that is capable of binding to its target with high affinity and specificity based on its unique DNA sequence and 3D structure after folding. Similar to the BBC assay, Cyto-c is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Ab) and an aptamer specifically against Cyto-c to form sandwich structures ([MMP-Ab]-[Cyto-c]-[Aptamer]). After washing and melting, our aptamers, acting as a DNA bio-barcode, are released from the sandwiches and hybridized with the probes specially designed for RNase H for surface plasmon resonance (SPR) sensing. In an aptamer-probe duplex, RNase H digests the RNA in the probe and releases the intact aptamer for another round of hybridization and digestion. With signal enhancement effects from gold-nanorods (Au-NRs) on probes for SPR sensing, the detection limit was found to be 1 nM for the aptamer and 80 pM for Cyto-c. Without the time-consuming DNA amplification steps by PCR, the detection process of this new ABC assay can be completed within three hours. As a proof-of-concept, phenylarsine oxide was found to be a potent agent to kill liver cancer cells with multi-drug resistance at the nano-molar level. This approach thus provides a fast, sensitive and robust tool for anti-cancer drug screening.

  11. Aptamer delivery of siRNA, radiopharmaceutics and chemotherapy agents in cancer.

    PubMed

    de Almeida, Carlos E B; Alves, Lais Nascimento; Rocha, Henrique F; Cabral-Neto, Januário Bispo; Missailidis, Sotiris

    2017-06-20

    Aptamers are oligonucleotide reagents with high affinity and specificity, which among other therapeutic and diagnostic applications have the capability of acting as delivery agents. Thus, aptamers are capable of carrying small molecules, nanoparticles, radiopharmaceuticals or fluorescent agents as well as nucleic acid therapeutics specifically to their target cells. In most cases, the molecules may possess interesting therapeutic properties, but their lack of specificity for a particular cell type, or ability to internalise in such a cell, hinders their clinical development, or cause unwanted side effects. Thus, chemotherapy or radiotherapy agents, famous for their side effects, can be coupled to aptamers for specific delivery. Equally, siRNA have great therapeutic potential and specificity, but one of their shortcomings remain the delivery and internalisation into cells. Various methodologies have been proposed to date, including aptamers, to resolve this problem. Therapeutic or imaging reagents benefit from the adaptability and ease of chemical manipulation of aptamers, their high affinity for the specific marker of a cell type, and their internalisation ability via cell mediated endocytosis. In this review paper, we explore the potential of the aptamers as delivery agents and offer an update on current status and latest advancements. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Entropy and Mg2+ control ligand affinity and specificity in the malachite green binding RNA aptamer.

    PubMed

    Bernard Da Costa, Jason; Dieckmann, Thorsten

    2011-07-01

    The binding of small molecule targets by RNA aptamers provides an excellent model to study the versatility of RNA function. The malachite green aptamer binds and recognizes its ligand via stacking and electrostatic interactions. The binding of the aptamer to its original selection target and three related molecules was determined by isothermal titration calorimetry, equilibrium dialysis, and fluorescence titration. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. These data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands whereas the complex with the original selection target is stable at low salt and in the absence of divalent metal ions.

  13. NMR characterization of a kissing complex formed between the TAR RNA element of HIV-1 and a DNA aptamer

    PubMed Central

    Collin, D.; van Heijenoort, C.; Boiziau, C.; Toulmé, J.-J.; Guittet, E.

    2000-01-01

    This work presents the first structural analysis of an RNA–DNA complex consisting of an 18 nt RNA hairpin and a 20 nt DNA aptamer. The DNA molecule was previously selected, from a randomly synthesized library, against the transactivation response element (TAR) involved in transcriptional regulation of the HIV genome. The DNA aptamer used in the present study is an imperfect stem–loop with the sequence 5′-ACTCCCAT-3′, characteristic of the selected candidates, in the apical loop. This octameric motif contains five bases complementary to the TAR loop sequence 5′-CUGGGA-3′. The use of homo- and heteronuclear NMR spectroscopy allowed assignment of the complex resonances and resolution of its secondary structure. Evidence is given for a kissing complex fold, which consists of a quasi-continuous helix formed by one stem of DNA, one stem of RNA and a central hybrid helix comprising 5 bp. Two out of helices residues of DNA and one of RNA connect the DNA–RNA loop–loop helix to the stem of either partner in the complex. In addition, two thymines of the DNA stem are engaged in a non-canonical T·T base pair. PMID:10954609

  14. Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

    PubMed Central

    Nguyen Quang, Nam; Perret, Gérald; Ducongé, Frédéric

    2016-01-01

    Aptamers are identified through an iterative process of evolutionary selection starting from a random pool containing billions of sequences. Simultaneously to the amplification of high-affinity candidates, the diversity in the pool is exponentially reduced after several rounds of in vitro selection. Until now, cloning and Sanger sequencing of about 100 sequences was usually used to identify the enriched candidates. However, High-Throughput Sequencing (HTS) is now extensively used to replace such low throughput sequencing approaches. Providing a deeper analysis of the library, HTS is expected to accelerate the identification of aptamers as well as to identify aptamers with higher affinity. It is also expected that it can provide important information on the binding site of the aptamers. Nevertheless, HTS requires handling a large amount of data that is only possible through the development of new in silico methods. Here, this review presents these different strategies that have been recently developed to improve the identification and characterization of aptamers using HTS. PMID:27973417

  15. Inhibiting the intrinsic pathway of coagulation with a FXII-targeting RNA Aptamer

    PubMed Central

    Woodruff, R. S.; Xu, Y.; Layzer, J.; Wu, W.; Ogletreee, M.L.; Sullenger, B.A.

    2013-01-01

    Background Exposure of the plasma protein factor XII to an anionic surface generates activated factor XII that not only triggers the intrinsic pathway of blood coagulation through the activatio of factor XI, but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of factor XII are not associated with excessive bleeding, thrombosis models in factor deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, factor XII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage. Objectives Utilizing an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease resistant RNA aptamer that binds specifically to factor XII and directly inhibits factor XII coagulant function. Methods and Results Herein, we describe the isolation and characterization of a high affinity RNA aptamer targeting factor XII/XIIa that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of factor XII, as well as inhibiting intrinsic pathway activation (factor XI activation). However, the aptamer does not affect the factor XIIa-mediated activation of the proinflammatory kallikrein-kinin system (plasma kallikrein activation). Conclusions We have generated a specific and potent factor XII/XIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation. PMID:23692437

  16. Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer.

    PubMed

    Woodruff, R S; Xu, Y; Layzer, J; Wu, W; Ogletree, M L; Sullenger, B A

    2013-07-01

    Exposure of the plasma protein factor XII (FXII) to an anionic surface generates activated FXII that not only triggers the intrinsic pathway of blood coagulation through the activation of FXI but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of FXII are not associated with excessive bleeding, thrombosis models in factor-deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, FXII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage. Using an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease-resistant RNA aptamer that binds specifically to FXII and directly inhibits FXII coagulant function. We describe the isolation and characterization of a high-affinity RNA aptamer targeting FXII/activated FXII (FXIIa) that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of FXII, as well as inhibiting intrinsic pathway activation (FXI activation). However, the aptamer does not affect the FXIIa-mediated activation of the proinflammatory kallikrein-kinin system (plasma kallikrein activation). We have generated a specific and potent FXII/FXIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation. © 2013 International Society on Thrombosis and Haemostasis.

  17. In vitro selection of DNA-based aptamers that exhibit RNA-like conformations using a chimeric oligonucleotide library that contains two different xeno-nucleic acids.

    PubMed

    Hagiwara, Kenta; Fujita, Hiroto; Kasahara, Yuuya; Irisawa, Yuuta; Obika, Satoshi; Kuwahara, Masayasu

    2015-01-01

    We successfully generated chimeric DNA aptamers that contained six nucleoside analogs of 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) in the primer region and multiple guanosine analogs of 2'-deoxy-2'-fluoro-ribonucleic acid (FNA) in the non-primer region using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). Active species enrichment became saturated only after five selection rounds, and we obtained DNA-based xeno-nucleic acid (XNA) aptamers that had high binding affinities for the target human thrombin, with dissociation constant (Kd) values of ≥10 nanomolar. Based on sequence and circular dichroism (CD) analyses, these XNA aptamers exhibited RNA-like conformations, which could cause DNA-based strands to adopt structurally diverse conformations.

  18. Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer

    PubMed Central

    HUANG, ZHEN; SZOSTAK, JACK W.

    2003-01-01

    Small changes in target specificity can sometimes be achieved, without changing aptamer structure, through mutation of a few bases. Larger changes in target geometry or chemistry may require more radical changes in an aptamer. In the latter case, it is unknown whether structural and functional solutions can still be found in the region of sequence space close to the original aptamer. To investigate these questions, we designed an in vitro selection experiment aimed at evolving specificity of an ATP aptamer. The ATP aptamer makes contacts with both the nucleobase and the sugar. We used an affinity matrix in which GTP was immobilized through the sugar, thus requiring extensive changes in or loss of sugar contact, as well as changes in recognition of the nucleobase. After just five rounds of selection, the pool was dominated by new aptamers falling into three major classes, each with secondary structures distinct from that of the ATP aptamer. The average sequence identity between the original aptamer and new aptamers is 76%. Most of the mutations appear to play roles either in disrupting the original secondary structure or in forming the new secondary structure or the new recognition loops. Our results show that there are novel structures that recognize a significantly different ligand in the region of sequence space close to the ATP aptamer. These examples of the emergence of novel functions and structures from an RNA molecule with a defined specificity and fold provide a new perspective on the evolutionary flexibility and adaptability of RNA. PMID:14624002

  19. RNA aptamer against autoantibodies associated with multiple sclerosis and bioluminescent detection probe on its basis.

    PubMed

    Vorobjeva, Maria A; Krasitskaya, Vasilisa V; Fokina, Alesya A; Timoshenko, Valentina V; Nevinsky, Georgy A; Venyaminova, Alya G; Frank, Ludmila A

    2014-03-04

    Nowadays, there are no specific laboratory tests for establishing the diagnosis of multiple sclerosis (MS). The presence of proteolytic autoantibodies against myelin basic protein is now considered as a characteristic feature of MS. New 2'-F-containing RNA aptamer of high affinity and specificity to these antibodies was selected. Covalent conjugate of this aptamer and Ca(2+)-regulated photoprotein obelin was obtained for the first time and applied as a label in bioluminescent microplate assay to detect target antibodies. The developed model solid-phase microassay is simple, fast, and highly sensitive.

  20. Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

    PubMed Central

    White, Rebekah R.; Shan, Siqing; Rusconi, Christopher P.; Shetty, Geetha; Dewhirst, Mark W.; Kontos, Christopher D.; Sullenger, Bruce A.

    2003-01-01

    Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent. PMID:12692304

  1. Organic additives stabilize RNA aptamer binding of malachite green.

    PubMed

    Zhou, Yubin; Chi, Hong; Wu, Yuanyuan; Marks, Robert S; Steele, Terry W J

    2016-11-01

    Aptamer-ligand binding has been utilized for biological applications due to its specific binding and synthetic nature. However, the applications will be limited if the binding or the ligand is unstable. Malachite green aptamer (MGA) and its labile ligand malachite green (MG) were found to have increasing apparent dissociation constants (Kd) as determined through the first order rate loss of emission intensity of the MGA-MG fluorescent complex. The fluorescent intensity loss was hypothesized to be from the hydrolysis of MG into malachite green carbinol base (MGOH). Random screening organic additives were found to reduce or retain the fluorescence emission and the calculated apparent Kd of MGA-MG binding. The protective effect became more apparent as the percentage of organic additives increased up to 10% v/v. The mechanism behind the organic additive protective effects was primarily from a ~5X increase in first order rate kinetics of MGOH→MG (kMGOH→MG), which significantly changed the equilibrium constant (Keq), favoring the generation of MG, versus MGOH without organic additives. A simple way has been developed to stabilize the apparent Kd of MGA-MG binding over 24h, which may be beneficial in stabilizing other triphenylmethane or carbocation ligand-aptamer interactions that are susceptible to SN1 hydrolysis.

  2. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

    PubMed Central

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M.; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-01-01

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice. PMID:26216949

  3. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation.

    PubMed

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-08-11

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.

  4. A Novel PEGylation Method for Improving the Pharmacokinetic Properties of Anti-Interleukin-17A RNA Aptamers.

    PubMed

    Haruta, Kazuhiko; Otaki, Natsuki; Nagamine, Masakazu; Kayo, Tomoyoshi; Sasaki, Asako; Hiramoto, Shinsuke; Takahashi, Masayuki; Hota, Kuniyoshi; Sato, Hideaki; Yamazaki, Hiroaki

    2017-02-01

    The obstacles to the development of therapeutic aptamers for systemic inflammatory diseases, such as nuclease degradation and renal clearance, have not been fully overcome. Here, we report a novel PEGylation method, sbC-PEGylation, which improves the pharmacokinetic properties of RNA aptamers that act against interleukin-17A (IL-17A) in mice and monkeys. sbC-PEGylated aptamers were synthesized by coupling the symmetrical branching molecule 2-cyanoethyl-N,N-diisopropyl phosphoroamidite to the 5' end of the aptamer, before conjugating two polyethylene glycol (PEG) molecules to the aptamer. Pharmacokinetic studies showed that compared with conventionally PEGylated aptamers, the sbC-PEGylated aptamer exhibited excellent stability in the blood circulation of mice and monkeys. In addition, one of the sbC-PEGylated aptamers, 17M-382, inhibited the interleukin-6 (IL-6) production induced by IL-17A in NIH3T3 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration of sbC-PEGylated 17M-382 was two times lower than that of non-PEGylated 17M-382. Furthermore, the intraperitoneal administration of sbC-PEGylated 17M-382 significantly inhibited the IL-6 production induced by IL-17A in a mouse air pouch model. Our findings suggest that the novel PEGylation method described in this study, sbC-PEGylation, could be used to develop anti-IL-17A aptamers as a therapeutic option for systemic inflammatory disease.

  5. A Novel PEGylation Method for Improving the Pharmacokinetic Properties of Anti-Interleukin-17A RNA Aptamers

    PubMed Central

    Otaki, Natsuki; Nagamine, Masakazu; Kayo, Tomoyoshi; Sasaki, Asako; Hiramoto, Shinsuke; Takahashi, Masayuki; Hota, Kuniyoshi; Sato, Hideaki; Yamazaki, Hiroaki

    2017-01-01

    The obstacles to the development of therapeutic aptamers for systemic inflammatory diseases, such as nuclease degradation and renal clearance, have not been fully overcome. Here, we report a novel PEGylation method, sbC-PEGylation, which improves the pharmacokinetic properties of RNA aptamers that act against interleukin-17A (IL-17A) in mice and monkeys. sbC-PEGylated aptamers were synthesized by coupling the symmetrical branching molecule 2-cyanoethyl-N,N-diisopropyl phosphoroamidite to the 5′ end of the aptamer, before conjugating two polyethylene glycol (PEG) molecules to the aptamer. Pharmacokinetic studies showed that compared with conventionally PEGylated aptamers, the sbC-PEGylated aptamer exhibited excellent stability in the blood circulation of mice and monkeys. In addition, one of the sbC-PEGylated aptamers, 17M-382, inhibited the interleukin-6 (IL-6) production induced by IL-17A in NIH3T3 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration of sbC-PEGylated 17M-382 was two times lower than that of non-PEGylated 17M-382. Furthermore, the intraperitoneal administration of sbC-PEGylated 17M-382 significantly inhibited the IL-6 production induced by IL-17A in a mouse air pouch model. Our findings suggest that the novel PEGylation method described in this study, sbC-PEGylation, could be used to develop anti-IL-17A aptamers as a therapeutic option for systemic inflammatory disease. PMID:27827561

  6. Chemically Modified, α-Amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) Receptor RNA Aptamers Designed for in Vivo Use.

    PubMed

    Huang, Zhen; Wen, Wei; Wu, Andrew; Niu, Li

    2017-09-05

    Glutamate ion channels have three subtypes, that is, α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors. Excessive activity of these receptor subtypes either individually or collectively is involved in various neurological disorders. RNA aptamers as antagonists of these receptors are potential therapeutics. For developing aptamer therapeutics, the RNA aptamers must be chemically modified to become ribonuclease-resistant or stable in biological fluids. Using systematic evolution of ligands by exponential enrichment (SELEX) and a chemically modified library, prepared enzymatically (i.e., the library contains RNAs with 2'-fluoro modified nucleoside triphosphates or ATPs, CTPs and UTPs, but regular GTPs), we have isolated an aptamer. The short aptamer (69 nucleotides) FN1040s selectively inhibits the GluA1 and GluA2Qflip AMPA receptor subunits, whereas the full-length aptamer (101 nucleotides) FN1040 additionally inhibits GluK1, but not GluK2, kainate receptor, and GluN1a/2A and GluN1a/2B, the two major native NMDA receptors. The two aptamers show similar potency (2-4 μM) and are stable with a half-life of at least 2 days in serum-containing medium or cerebrospinal fluid. Therefore, these two aptamers are amenable for in vivo use.

  7. mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach.

    PubMed

    Guzmán-Zapata, Daniel; Domínguez-Anaya, Yael; Macedo-Osorio, Karla S; Tovar-Aguilar, Andrea; Castrejón-Flores, José L; Durán-Figueroa, Noé V; Badillo-Corona, Jesús A

    2017-03-27

    Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of C. reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae.

  8. In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9

    PubMed Central

    Mi, Jing; Ray, Partha; Liu, Jenny; Kuan, Chien-Tsun; Xu, Jennifer; Hsu, David; Sullenger, Bruce A; White, Rebekah R; Clary, Bryan M

    2016-01-01

    The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs) that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery. PMID:27115840

  9. Predicting candidate genomic sequences that correspond to synthetic functional RNA motifs

    PubMed Central

    Laserson, Uri; Gan, Hin Hark; Schlick, Tamar

    2005-01-01

    Riboswitches and RNA interference are important emerging mechanisms found in many organisms to control gene expression. To enhance our understanding of such RNA roles, finding small regulatory motifs in genomes presents a challenge on a wide scale. Many simple functional RNA motifs have been found by in vitro selection experiments, which produce synthetic target-binding aptamers as well as catalytic RNAs, including the hammerhead ribozyme. Motivated by the prediction of Piganeau and Schroeder [(2003) Chem. Biol., 10, 103–104] that synthetic RNAs may have natural counterparts, we develop and apply an efficient computational protocol for identifying aptamer-like motifs in genomes. We define motifs from the sequence and structural information of synthetic aptamers, search for sequences in genomes that will produce motif matches, and then evaluate the structural stability and statistical significance of the potential hits. Our application to aptamers for streptomycin, chloramphenicol, neomycin B and ATP identifies 37 candidate sequences (in coding and non-coding regions) that fold to the target aptamer structures in bacterial and archaeal genomes. Further energetic screening reveals that several candidates exhibit energetic properties and sequence conservation patterns that are characteristic of functional motifs. Besides providing candidates for experimental testing, our computational protocol offers an avenue for expanding natural RNA's functional repertoire. PMID:16254081

  10. A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

    NASA Astrophysics Data System (ADS)

    Liu, Hong Yan; Gao, Xiaohu

    2013-11-01

    siRNA-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of siRNA due to the modularity of their diblock RNA structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. However, additional challenges must be addressed for efficient RNA interference (RNAi), in particular, endosomal escape. Currently, vast majority of siRNA delivery vehicles are based on cationic materials, which form complexes with negatively charged siRNA. Unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. The protein selectively tags along the siRNA block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. More interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to 18 amino acids completely abolishes the RNA binding function of dsRBD.

  11. A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

    PubMed Central

    Liu, Hong Yan; Gao, Xiaohu

    2013-01-01

    siRNA-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of siRNA due to the modularity of their diblock RNA structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. However, additional challenges must be addressed for efficient RNA interference (RNAi), in particular, endosomal escape. Currently, vast majority of siRNA delivery vehicles are based on cationic materials, which form complexes with negatively charged siRNA. Unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. The protein selectively tags along the siRNA block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. More interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to 18 amino acids completely abolishes the RNA binding function of dsRBD. PMID:24196104

  12. Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein.

    PubMed

    Amano, Ryo; Takada, Kenta; Tanaka, Yoichiro; Nakamura, Yoshikazu; Kawai, Gota; Kozu, Tomoko; Sakamoto, Taiichi

    2016-11-15

    AML1 (RUNX1) protein is an essential transcription factor involved in the development of hematopoietic cells. Several genetic aberrations that disrupt the function of AML1 have been frequently observed in human leukemia. AML1 contains a DNA-binding domain known as the Runt domain (RD), which recognizes the RD-binding double-stranded DNA element of target genes. In this study, we identified high-affinity RNA aptamers that bind to RD by systematic evolution of ligands by exponential enrichment. The binding assay using surface plasmon resonance indicated that a shortened aptamer retained the ability to bind to RD when 1 M potassium acetate was used. A thermodynamic study using isothermal titration calorimetry (ITC) showed that the aptamer-RD interaction is driven by a large enthalpy change, and its unfavorable entropy change is compensated by a favorable enthalpy change. Furthermore, the binding heat capacity change was identified from the ITC data at various temperatures. The aptamer binding showed a large negative heat capacity change, which suggests that a large apolar surface is buried upon such binding. Thus, we proposed that the aptamer binds to RD with long-range electrostatic force in the early stage of the association and then changes its conformation and recognizes a large surface area of RD. These findings about the biophysics of aptamer binding should be useful for understanding the mechanism of RNA-protein interaction and optimizing and modifying RNA aptamers.

  13. Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing.

    PubMed

    Kupakuwana, Gillian V; Crill, James E; McPike, Mark P; Borer, Philip N

    2011-01-01

    Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2-6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (K(d) = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (K(d) = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets.

  14. Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

    PubMed Central

    Kupakuwana, Gillian V.; Crill, James E.; McPike, Mark P.; Borer, Philip N.

    2011-01-01

    Background Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. Methodology/Principal Findings We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2–6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (Kd = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (Kd = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. Conclusions/Significance Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets. PMID:21625587

  15. A Conjugate of Two tPA-Binding RNA Aptamers Efficiently Inhibits Fibrinolysis.

    PubMed

    Bjerregaard, Nils; Dupont, Daniel M; Andreasen, Peter A

    2017-04-01

    Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.

  16. A RNA-DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug Delivery

    PubMed Central

    Leach, John C.; Wang, Andrew; Ye, Kaiming; Jin, Sha

    2016-01-01

    The side effects of radio- and chemo-therapy pose long-term challenges on a cancer patient’s health. It is, therefore, highly desirable to develop more effective therapies that can specifically target carcinoma cells without damaging normal and healthy cells. Tremendous efforts have been made in the past to develop targeted drug delivery systems for solid cancer treatment. In this study, a new aptamer, A10-3-J1, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), was designed. A super paramagnetic iron oxide nanoparticle-aptamer-doxorubicin (SPIO-Apt-Dox) was fabricated and employed as a targeted drug delivery platform for cancer therapy. This DNA RNA hybridized aptamer antitumor agent was able to enhance the cytotoxicity of targeted cells while minimizing collateral damage to non-targeted cells. This SPIO-Apt-Dox nanoparticle has specificity to PSMA+ prostate cancer cells. Aptamer inhibited nonspecific uptake of membrane-permeable doxorubic to the non-target cells, leading to reduced untargeted cytotoxicity and endocytic uptake while enhancing targeted cytotoxicity and endocytic uptake. The experimental results indicate that the drug delivery platform can yield statistically significant effectiveness being more cytotoxic to the targeted cells as opposed to the non-targeted cells. PMID:26985893

  17. A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication

    PubMed Central

    Feng, Hui; Beck, Jürgen; Nassal, Michael; Hu, Kang-hong

    2011-01-01

    Background The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ε RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. Methodology/Principal Findings Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. Conclusions/Significance This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B. PMID:22125633

  18. Aptamer Database

    PubMed Central

    Lee, Jennifer F.; Hesselberth, Jay R.; Meyers, Lauren Ancel; Ellington, Andrew D.

    2004-01-01

    The aptamer database is designed to contain comprehensive sequence information on aptamers and unnatural ribozymes that have been generated by in vitro selection methods. Such data are not normally collected in ‘natural’ sequence databases, such as GenBank. Besides serving as a storehouse of sequences that may have diagnostic or therapeutic utility, the database serves as a valuable resource for theoretical biologists who describe and explore fitness landscapes. The database is updated monthly and is publicly available at http://aptamer.icmb.utexas.edu/. PMID:14681367

  19. Distinct transcriptional responses of RNA polymerases I, II and III to aptamers that bind TBP

    PubMed Central

    Fan, Xiaochun; Shi, Hua; Lis, John T.

    2005-01-01

    The TATA-binding protein (TBP) is a general factor that is involved in transcription by all three types of nuclear RNA polymerase. To delineate the roles played by the DNA-binding surface of TBP in these transcription reactions, we used a set of RNA aptamers directed against TBP and examined their ability to perturb transcription in vitro by the different RNA polymerases. Distinct responses to the TBP aptamers were observed for transcription by different types of polymerase at either the initiation, reinitiation or both stages of the transcription cycle. We further probed the TBP interactions in the TFIIIB•DNA complex to elucidate the mechanism for the different sensitivity of Pol III dependent transcription before and after preinitiation complex (PIC) formation. Lastly, the aptamers were employed to measure the time required for Pol III PIC formation in vitro. This approach can be generalized to define the involvement of a particular region on the surface of a protein at particular stages in a biological process. PMID:15701755

  20. Cellular delivery of shRNA using aptamer-conjugated PLL-alkyl-PEI nanoparticles.

    PubMed

    Askarian, Saeedeh; Abnous, Khalil; Taghavi, Sahar; Oskuee, Reza Kazemi; Ramezani, Mohammad

    2015-12-01

    Introduction of an efficient gene delivery vector is still the main challenge of gene therapy. Both polyethylenimine (PEI) and poly(l-lysine) (PLL) comprise disadvantages which limited their application. To explore whether their deficiencies could be compensated by preparing copolymers consisting of both PLL and PEI, we generated several combinations of PLL-alkyl-PEI copolymers conjugated to aptamer and evaluated their both gene delivery efficiency and down-regulation of Bcl-XL, an anti-apoptotic gene, in lung cancer cell line. PLL was conjugated to either 10% or 50% of PEI by grafting different percentages of PEI to alkylated-PLL as core. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. According to transfection results, aptamer conjugated PLL-alkyl-10%-PEI (PLPE8%) was selected for further gene silencing study by plasmid shRNA. Decrease in Bcl-XL gene expression was estimated by both RT-PCR and western-blot experiments. The obtained results revealed that the new copolymers had appropriate nano-scale size (117-128 nm) even after aptamer conjugation (168-183 nm). Moreover, they exhibited increased transfection efficiencies by up to 1.8-5 folds and acceptable cytotoxicity. The apoptosis was induced in transfected cells by shRNA-aptamer-copolymer due to the down-regulation of mRNA and protein levels. This study suggested a new vector for targeted non-viral gene delivery with high transfection efficiency in lung cancer or pulmonary systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Selection of DNA aptamers with two modified bases

    PubMed Central

    Gawande, Bharat N.; Rohloff, John C.; Carter, Jeffrey D.; von Carlowitz, Ira; Zhang, Chi; Schneider, Daniel J.; Janjic, Nebojsa

    2017-01-01

    The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications. PMID:28265062

  2. Selection of DNA aptamers with two modified bases.

    PubMed

    Gawande, Bharat N; Rohloff, John C; Carter, Jeffrey D; von Carlowitz, Ira; Zhang, Chi; Schneider, Daniel J; Janjic, Nebojsa

    2017-03-14

    The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid-like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

  3. iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications

    PubMed Central

    Autour, Alexis; Westhof, Eric; Ryckelynck, Michael

    2016-01-01

    Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme. PMID:26932363

  4. Nuclear RNA Isolation and Sequencing.

    PubMed

    Dhaliwal, Navroop K; Mitchell, Jennifer A

    2016-01-01

    Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression. Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long noncoding RNAs. Here, we describe nuclear-RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long noncoding RNAs.

  5. Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

    PubMed Central

    Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

    2012-01-01

    Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

  6. Effect of serum on an RNA aptamer-based electrochemical sensor for theophylline.

    PubMed

    Ferapontova, Elena E; Gothelf, Kurt V

    2009-04-21

    Electrochemical performance of the ferrocene (Fc) redox-labeled RNA aptamer based sensor for theophylline (Th) is essentially inhibited in serum, but is restored in serum-free buffer solutions. This phenomenon is inconsistent with the data on methylene-blue-labeled aptamer beacon systems, which operational potential window is more negative compared to the Fc redox label. Electrochemical studies with a ferricyanide redox probe, having redox potential close to the Fc redox couple, and interfacial capacitance measurements unambiguously demonstrate that it is adsorption of serum proteins at positively charged electrode surface that slows down the kinetics of the electrode reactions in serum and interferes with the biosensor performance. In filtered serum solutions, in the absence of serum proteins, the Fc-labeled aptamer-based biosensor performed similarly to the pure buffer solutions, ad the signal for Th could be linearly calibrated versus Th concentration. These results on interfacial effects of serum are of particular importance for future research and development of the beacon-type biosensors for in vivo applications.

  7. A Nuclease-Resistant RNA Aptamer Specifically Inhibits Angiopoietin-1-Mediated Tie2 Activation and Function

    PubMed Central

    White, Rebekah R.; Roy, Julie A.; Viles, Kristi D.; Sullenger, Bruce A.; Kontos, Christopher D.

    2009-01-01

    Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and that plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2′-fluoro-modified ribonucleotides for Ang1-binding aptamers. After 9 rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (Kd 2.8 nM) but not Ang2 (Kd >1 μM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases. PMID:19037734

  8. Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

    PubMed

    Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

    2014-10-01

    The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

  9. Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

    PubMed Central

    Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores

    2014-01-01

    The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3′end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection. PMID:25111024

  10. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers

    PubMed Central

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques

    2016-01-01

    Loop–loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop–loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 106 RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5′CCNY and 5′RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch–ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop–loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch–aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. PMID:27067541

  11. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers.

    PubMed

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques

    2016-05-19

    Loop-loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop-loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 10(6) RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5'CCNY and 5'RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch-ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop-loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch-aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    PubMed

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology.

  13. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  14. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  15. Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing

    PubMed Central

    Stuart, Christopher H; Riley, Kathryn R; Boyacioglu, Olcay; Herpai, Denise M; Debinski, Waldemar; Qasem, Shadi; Marini, Frank C; Colyer, Christa L.; Gmeiner, William H

    2016-01-01

    Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10−6mol/l) relative to uncoated plates (2.4 × 10−6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10−6 mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC. PMID:27845768

  16. Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing.

    PubMed

    Stuart, Christopher H; Riley, Kathryn R; Boyacioglu, Olcay; Herpai, Denise M; Debinski, Waldemar; Qasem, Shadi; Marini, Frank C; Colyer, Christa L; Gmeiner, William H

    2016-11-15

    Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5(°)C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10(-6)mol/l) relative to uncoated plates (2.4 × 10(-6) mol/l), or plates coated with the related protein fibronectin (2.1 × 10(-6) mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.

  17. CD4 aptamer-RORγt shRNA chimera inhibits IL-17 synthesis by human CD4(+) T cells.

    PubMed

    Song, Pingfang; Chou, Yuan K; Zhang, Xiaowei; Meza-Romero, Roberto; Yomogida, Kentaro; Benedek, Gil; Chu, Cong-Qiu

    2014-10-03

    Cell type specific delivery of RNAi to T cells has remained to be a challenge. Here we describe an aptamer mediated delivery of shRNA to CD4(+) T cells targeting RORγt to suppress Th17 cells. A cDNA encoding CD4 aptamer and RORγt shRNA was constructed and the chimeric CD4 aptamer-RORγt shRNA (CD4-AshR-RORγt) was generated using in vitro T7 RNA transcription. 2'-F-dCTP and 2'-F-dUTP were incorporated into CD4-AshR-RORγt for RNase resistance. CD4-AshR-RORγt was specifically uptaken by CD4(+) Karpas 299 cells and primary human CD4(+) T cells. The RORγt shRNA moiety of CD4-AshR-RORγt chimera was cleaved and released by Dicer. Furthermore, CD4-AshR-RORγt suppressed RORγt gene expression in Karpas 299 cells and CD4(+) T cells and consequently inhibited Th17 cell differentiation and IL-17 production. These results demonstrate that aptamer-facilitated cell specific delivery of shRNA represents a novel approach for efficient RNAi delivery and is potentially to be developed for therapeutics targeting specific T cells subtypes.

  18. Recent Progress in Aptamer-Based Functional Probes for Bioanalysis and Biomedicine.

    PubMed

    Zhang, Huimin; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong

    2016-07-11

    Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer-based functional probes in the fields of bioanalysis and biomedicine.

  19. Thermodynamics of Ligand Binding to a Heterogeneous RNA Population in the Malachite Green Aptamer

    PubMed Central

    Sokoloski, Joshua E.; Dombrowski, Sarah E.; Bevilacqua, Philip C.

    2011-01-01

    The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with near equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structure, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics for binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15 to 60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4, and another from 50 to 60 °C where MG bound with positive heat capacity and n~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and n~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that binding stoichiometry of MG was improved not only by raising the temperature, but also by lowering the concentration of Mg2+ or increasing the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics. PMID:22192051

  20. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    SciTech Connect

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  1. Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

    PubMed

    Santosh, Baby; Yadava, Pramod K

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug "Macugen" is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions.

  2. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics

    PubMed Central

    Yadava, Pramod K.

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Macugen” is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions. PMID:25050359

  3. A kallikrein-targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release.

    PubMed

    Steen Burrell, K-A; Layzer, J; Sullenger, B A

    2017-09-01

    Essentials Kallikrein amplifies contact activation and is a potential target for preventing thrombosis. We developed and characterized a kallikrein aptamer using convergent evolution and kinetic assays. Kall1-T4 prolongs intrinsic clotting time by inhibiting factor XIIa-mediated prekallikrein activation. Kall1-T4 decreases high-molecular-weight kininogen cleavage and bradykinin release. Background Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non-enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose-dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein

  4. P41IDENTIFICATION OF GLIOMA SPECIFIC APTAMER TARGETS

    PubMed Central

    Arora, Mohit; Alder, Jane; Lawrence, Clare; Davis, Charles; Dawson, Tim; Hall, Greg; Shaw, Lisa

    2014-01-01

    INTRODUCTION: Aptamers are in vitro generated DNA and RNA sequences which are randomly created as a library, with multiple permutations and combinations. These are then exposed to the target structure against which we want an aptamer ‘selected’ using Sequential Enumeration of Ligands by Exponential enrichment (SELEX). METHOD: Commercially available glioma and glial cell lines and in-house generated primary glioma cultures were used. Modified aptamers based on published sequences against glioma cell lines and newly generated sequences were used in the project to identify their binding targets. Cy3 or biotin- conjugated aptamers were incubated with live glioma cell cultures and imaged using confocal or light microscopy.To determine the target ligand, aptamers were then reacted with glial cell lysate and subjected to precipitation using streptavidin agarose beads and SDS polyacrylamide electrophoresis. Proteins were analysed by mass spectroscopy. RESULTS: Known and unknown aptamer protein ligands were co-precipitated. Ku70, Ku80 were precipitated along with nucleolin and related proteins. CONCLUSION: The aptamer has shown preferential binding to glioma cells and could act as a delivery system for therapeutic payloads. The aptamer targets Ku70 and Ku80, which are known to be over expressed in other forms of cancer but their role in gliomagenesis has not been fully elucidated. Other novel proteins have also been identified. Thus the aptamer co-precipitation technique has identified potential glioma biomarkers that may be of clinical significance.

  5. A theophylline quartz crystal microbalance biosensor based on recognition of RNA aptamer and amplification of signal.

    PubMed

    Dong, Zong-Mu; Zhao, Guang-Chao

    2013-04-21

    A quartz crystal microbalance (QCM) biosensor for theophylline was developed by recognition of RNA aptamer and gold nanoparticle amplification technique. Firstly, a designed small single-stranded RNA, RNA1, was immobilized onto the QCM electrode through a thiol linker. Then, the complementary stranded RNA2, which can combine with RNA1 to form a double-stranded RNA with a recognition unit of theophylline, could be self-assembled on the QCM electrode surface through a hybrid reaction in the presence of theophylline. The recognition process could cause a frequency change of QCM to give the signal related to theophylline. When RNA2 was tethered to gold nanoparticles, the signal could be amplified to further enhance the sensitivity of the designed sensor. Under the optimal conditions, the QCM-based biosensor showed excellent sensitivity (limit of detection, 8.2 nM) and specificity with a dissociation constant of Kd = 5.26 × 10(-7) M. The sensor can be used to quantitatively detect theophylline in serum, suggesting that it can be applied in complex biological samples.

  6. RNA-aptamers that modulate the RhoGEF activity of Tiam1.

    PubMed

    Niebel, Björn; Wosnitza, Christine I; Famulok, Michael

    2013-10-15

    Rho GTPases regulate the actin cytoskeleton and thereby control cell migration, cell morphology, cell motility, and other cellular functions. The gene product of the oncogene Tiam1 acts as a guanine nucleotide exchange factor (GEF) for the Rho GTPase Rac. Like other RhoGEFs, Tiam1 is involved in cancer progression, but it also counteracts invasion in different cancer cell types. Hence, further investigations are required to unravel the functions of Tiam1 in the context of cancer initiation and progression, which appear to be cell specific. Although RhoGEFs in general seem to be attractive therapeutic targets, not many inhibitors have been described, yet. Here we report the identification and characterization of inhibitory RNA aptamers that specifically target Tiam1. After 16 selection rounds three aptamers sharing a 15 nucleotides consensus motif were identified. The clones K91 and K11 inhibited the Tiam1-mediated activation of the GTPase Rac2 in vitro. The tightest binder K91 neither bound the Rho GEF Vav1 nor the Arf GEF Cytohesin-2. In the presence of Rac1, the binding of K91 to Tiam1 was impaired indicating that the binding motif on Tiam1 overlaps with the GTPase binding site. K91 and K11 are the first reported inhibitory molecules targeting the GEF function of Tiam1. Due to their specificity over related GEF proteins they may represent promising tools for further elucidation of the biological functions of Tiam1. We anticipated that these aptamers will prove useful in validating the ambiguous roles of Tiam1 in cancer biology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells.

    PubMed

    Varshney, Akhil; Bala, Jyoti; Santosh, Baby; Bhaskar, Ashima; Kumar, Suresh; Yadava, Pramod K

    2017-03-01

    Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.

  8. A Universal Aptamer Chimera for the Delivery of Functional microRNA-126.

    PubMed

    Rohde, Jan-H; Weigand, Julia E; Suess, Beatrix; Dimmeler, Stefanie

    2015-06-01

    microRNAs (miRs) regulate vascular diseases such as atherosclerosis and cancer. miR-126 is important for endothelial cell signaling and promotes angiogenesis, protects against atherosclerosis, and reduces breast cancer cell growth and metastasis. The overexpression of miR-126, therefore, may be an attractive therapeutic strategy for the treatment of cardiovascular disease or cancer. Here we report a novel strategy to deliver miR-126 to endothelial and breast cancer cells. We tested three different strategies to deliver miR-126 by linking the miR to an aptamer for the ubiquitously expressed transferrin receptor (transferrin receptor aptamer, TRA). Linking the precursor of miR-126 (pre-miR-126) to the TRA by annealing of a complementary stick led to efficient uptake and processing of miR-126, resulting in the delivery of 1.6×10(6)±0.3×10(6) copies miR-126-3p per ng RNA in human endothelial cells and 7.4×10(5)±2×10(5) copies miR-126-3p per ng in MCF7 breast cancer cells. The functionality of the active TRA-miR-126 chimera was further demonstrated by showing that the chimera represses the known miR-126 target VCAM-1 and improved endothelial cell sprouting in a spheroid assay. Moreover, the TRA-miR-126 chimera reduced proliferation and paracrine endothelial cell recruitment of breast cancer cells to a similar extent as miR-126-3p mimics introduced by conventional liposome-based transfection. Together, this data demonstrates that pre-miR-126 can be delivered by a non-specific aptamer to exert biological functions in two different cell models. The use of the TRA-miR-126 chimera or the combination of the delivery strategy with other endothelial or tumor specific aptamers may provide an interesting therapeutic option to treat vascular disease or cancers.

  9. High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency.

    PubMed

    Takahashi, Mayumi; Wu, Xiwei; Ho, Michelle; Chomchan, Pritsana; Rossi, John J; Burnett, John C; Zhou, Jiehua

    2016-09-22

    The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct "biased sequences" and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the "biased sequences" was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.

  10. Compilation of small RNA sequences.

    PubMed

    Shumyatsky, G; Reddy, R

    1992-05-11

    This is an update containing small RNA sequences published during 1991. Approximately two hundred small RNA sequences are available in this and earlier compilations. The hard copy print out of this set will be available directly from us (inquiries should be addressed to R. Reddy). These files are also available on GenBank computer. Sequences from various sources covered in earlier compilations (see Reddy, R. Nucl. Acids Res. 16:r71; Reddy, R. and Gupta, S. Nucl Acids Res. 1990 Supplement, 18:2231 and 1991 Supplement, 19:2073) are not included in this update but are listed below.

  11. Aptamers: Biomedical Interest and Applications.

    PubMed

    Romero-López, Cristina; Berzal-Herranz, Alfredo

    2017-03-16

    Aptamers are short DNA or RNA oligonucleotides specialized in the specific and efficient binding to a target molecule. They are obtained by in vitro selection or evolution processes. It was in 1990 that two independent research groups described the bases of a new in vitro technology for the identification of RNA molecules able to specifically bind to a target [1,2]. Tuerk and Gold established the principals of the in vitro selection process that was named SELEX (Systematic Evolution of Ligands by Exponential enrichment), which is based on iterative cycles of binding, partitioning, and amplification of oligonucleotides from a pool of variant sequences [2]. Ellington and Szostak coined the term aptamer to define the selected molecules by the application of this method [1]. To date, numerous reports have described the isolation of aptamers directed against a great variety of targets covering a wide diversity of molecules varying in nature, size, and complexity ranging from ions to whole cells, including small molecules (e.g., aminoacids, nucleotides, antibiotics), peptides, proteins, nucleic acids, and viruses, among others (for example, see [3-6]). Modifications and optimization of the SELEX procedure aimed to get newly modified aptamers has also attracted much interest (examples can be found in [7,8]). These advances along with the parallel progresses in the nucleic acids chemistry and cellular delivery fields have allowed for the rise of a new hope in developing aptamers as efficient molecular tools for diagnostics and therapeutics (for recent comprehensive reviews, see [9-11]).

  12. Co-targeting EGFR and survivin with a bivalent aptamer-dual siRNA chimera effectively suppresses prostate cancer

    PubMed Central

    Liu, Hong Yan; Yu, Xiaolin; Liu, Haitao; Wu, Daqing; She, Jin-Xiong

    2016-01-01

    Current targeted therapies using small kinase inhibitors and antibodies have limited efficacy in treating prostate cancer (PCa), a leading cause of cancer death in American men. We have developed a novel strategy by engineering an RNA-based aptamer-siRNA chimera, in which a bivalent aptamer specifically binds prostate-specific membrane antigen (PSMA) via an antibody-like structure to promote siRNA internalization in PCa cells, and two siRNAs specific to EGFR and survivin are fused between two aptamers. The chimera is able to inhibit EGFR and survivin simultaneously and induce apoptosis effectively in vitro and in vivo. In the C4-2 PCa xenograft model, the treatment with the chimera significantly suppresses tumor growth and angiogenesis. The inhibition of angiogenesis is mediated by an EGFR-HIF1α-VEGF-dependent mechanism. Our results support that the bivalent aptamer-driven delivery of two siRNAs could be a new combination therapeutic strategy to effectively inhibit multiple and conventionally “undruggable” targets. PMID:27456457

  13. An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters.

    PubMed

    McCluskey, K; Carlos Penedo, J

    2017-03-08

    Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps or even contradictions between common definitions of the two terms, and we will conclude by proposing a more robust framework that considers the range of RNA behaviors unveiled in recent years as a reality to be described, rather than an increasingly unwieldy set of exceptions to the traditional models.

  14. RNA Aptamer That Specifically Binds to Mycolactone and Serves as a Diagnostic Tool for Diagnosis of Buruli Ulcer

    PubMed Central

    Aboagye, Samuel Yaw; Otchere, Isaac Darko; Liao, Albert M.; Caltagirone, Thomas G.; Yeboah-Manu, Dorothy

    2016-01-01

    most promising aptamer, Apt-3683 showed a discernible cleavage difference relative to the non-specific autocatalysis over a 3-minute time course. Conclusion This preliminary proof-of-concept indicates that diagnosis of BUD with RNA aptamers is feasible and can be used as point of care upon incorporation into a diagnostic platform. PMID:27776120

  15. Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment

    PubMed Central

    Sugiyama, Shigeru; Nomura, Yusuke; Sakamoto, Taiichi; Kitatani, Tomoya; Kobayashi, Asako; Miyakawa, Shin; Takahashi, Yoshinori; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Nakamura, Yoshikazu; Matsumura, Hiroyoshi

    2008-01-01

    Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 Å. A data set has been collected to 2.2 Å resolution. PMID:18931441

  16. Selection of DNA Aptamers for Ovarian Cancer Biomarker CA125 Using One-Pot SELEX and High-Throughput Sequencing

    PubMed Central

    Scoville, Delia J.; Uhm, Tae Kyu Brian; Shallcross, Jamie A.

    2017-01-01

    CA125 is a mucin glycoprotein whose concentration in serum correlates with a woman's risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient samples is of great clinical relevance. We suggest that powerful new diagnostic tools may be enabled by the development of nucleic acid aptamers with affinity for CA125. Here, we report on our use of One-Pot SELEX to isolate single-stranded DNA aptamers with affinity for CA125, followed by high-throughput sequencing of the selected oligonucleotides. This data-rich approach, combined with bioinformatics tools, enabled the entire selection process to be characterized. Using fluorescence anisotropy and affinity probe capillary electrophoresis, the binding affinities of four aptamer candidates were evaluated. Two aptamers, CA125_1 and CA125_12, both without primers, were found to bind to clinically relevant concentrations of the protein target. Binding was differently influenced by the presence of Mg2+ ions, being required for binding of CA125_1 and abrogating binding of CA125_12. In conclusion, One-Pot SELEX was found to be a promising selection method that yielded DNA aptamers to a clinically important protein target. PMID:28280637

  17. Quantitative assessment of RNA-protein interactions with high-throughput sequencing-RNA affinity profiling.

    PubMed

    Ozer, Abdullah; Tome, Jacob M; Friedman, Robin C; Gheba, Dan; Schroth, Gary P; Lis, John T

    2015-08-01

    Because RNA-protein interactions have a central role in a wide array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay that couples sequencing on an Illumina GAIIx genome analyzer with the quantitative assessment of protein-RNA interactions. This assay is able to analyze interactions between one or possibly several proteins with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of the EGFP and negative elongation factor subunit E (NELF-E) proteins with their corresponding canonical and mutant RNA aptamers. Here we provide a detailed protocol for HiTS-RAP that can be completed in about a month (8 d hands-on time). This includes the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, HiTS and protein binding with a GAIIx instrument, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, quantitative analysis of RNA on a massively parallel array (RNA-MaP) and RNA Bind-n-Seq (RBNS), for quantitative analysis of RNA-protein interactions.

  18. High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency

    PubMed Central

    Takahashi, Mayumi; Wu, Xiwei; Ho, Michelle; Chomchan, Pritsana; Rossi, John J.; Burnett, John C.; Zhou, Jiehua

    2016-01-01

    The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct “biased sequences” and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the “biased sequences” was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy. PMID:27652575

  19. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

    PubMed Central

    Jolly, Pawan; Estrela, Pedro

    2016-01-01

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  20. Chimeric aptamers in cancer cell-targeted drug delivery

    PubMed Central

    Kanwar, Jagat R; Roy, Kislay; Kanwar, Rupinder K

    2011-01-01

    Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern Pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities. PMID:21955150

  1. Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats

    PubMed Central

    Dua, Pooja; Ren, Shuo; Lee, Sang Wook; Kim, Joon-Ki; Shin, Hye-su; Jeong, OK-Chan; Kim, Soyoun; Lee, Dong-Ki

    2016-01-01

    Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2′F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application. PMID:27871171

  2. Deciphering the RNA landscape by RNAome sequencing

    PubMed Central

    Derks, Kasper WJ; Misovic, Branislav; van den Hout, Mirjam CGN; Kockx, Christel EM; Payan Gomez, Cesar; Brouwer, Rutger WW; Vrieling, Harry; Hoeijmakers, Jan HJ; van IJcken, Wilfred FJ; Pothof, Joris

    2015-01-01

    Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. PMID:25826412

  3. Aptamers-based assays for diagnostics, environmental and food analysis.

    PubMed

    Tombelli, Sara; Minunni, Maria; Mascini, Marco

    2007-06-01

    Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a huge library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. In addition to the very important aspect of having an unlimited source of identical affinity recognition molecules available due to the selection process, aptamers can offer advantages over antibodies that make them very promising for analytical applications. The use of aptamers as therapeutic tools is nowadays well established. On the contrary, the analytical application of aptamers in diagnostic devices or in systems for environmental and food analysis, is still under investigation and the scientific community still need further research to demonstrate the advancements brought by this new kind of ligands. This review will focus on these latter applications with particular attention to the detection of food pathogens, terrorism threat agents, thrombin and cytokines.

  4. Aptamer-miRNA-212 Conjugate Sensitizes NSCLC Cells to TRAIL

    PubMed Central

    Iaboni, Margherita; Russo, Valentina; Fontanella, Raffaela; Roscigno, Giuseppina; Fiore, Danilo; Donnarumma, Elvira; Esposito, Carla Lucia; Quintavalle, Cristina; Giangrande, Paloma H; de Franciscis, Vittorio; Condorelli, Gerolama

    2016-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent for its remarkable ability to selectively induce apoptosis in cancer cells, without affecting the viability of healthy bystander cells. The TRAIL tumor suppressor pathway is deregulated in many human malignancies including lung cancer. In human non-small cell lung cancer (NSCLC) cells, sensitization to TRAIL therapy can be restored by increasing the expression levels of the tumor suppressor microRNA-212 (miR-212) leading to inhibition of the anti-apoptotic protein PED/PEA-15 implicated in treatment resistance. In this study, we exploited a previously described RNA aptamer inhibitor of the tyrosine kinase receptor Axl (GL21.T) expressed on lung cancer cells, as a means to deliver miR-212 into human NSCLC cells expressing Axl. We demonstrate efficient delivery of miR-212 following conjugation of the miR to GL21.T (GL21.T-miR212 chimera). We show that the chimera downregulates PED and restores TRAIL-mediate cytotoxicity in cancer cells. Importantly, treatment of Axl+ lung cancer cells with the chimera resulted in (i) an increase in caspase activation and (ii) a reduction of cell viability in combination with TRAIL therapy. In conclusion, we demonstrate that the GL21.T-miR212 chimera can be employed as an adjuvant to TRAIL therapy for the treatment of lung cancer. PMID:27111415

  5. In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules

    PubMed Central

    Ahirwar, Rajesh; Nahar, Smita; Aggarwal, Shikha; Ramachandran, Srinivasan; Maiti, Souvik; Nahar, Pradip

    2016-01-01

    Aptamers, the chemical-antibody substitute to conventional antibodies, are primarily discovered through SELEX technology involving multi-round selections and enrichment. Circumventing conventional methodology, here we report an in silico selection of aptamers to estrogen receptor alpha (ERα) using RNA analogs of human estrogen response elements (EREs). The inverted repeat nature of ERE and the ability to form stable hairpins were used as criteria to obtain aptamer-alike sequences. Near-native RNA analogs of selected single stranded EREs were modelled and their likelihood to emerge as ERα aptamer was examined using AutoDock Vina, HADDOCK and PatchDock docking. These in silico predictions were validated by measuring the thermodynamic parameters of ERα -RNA interactions using isothermal titration calorimetry. Based on the in silico and in vitro results, we selected a candidate RNA (ERaptR4; 5′-GGGGUCAAGGUGACCCC-3′) having a binding constant (Ka) of 1.02 ± 0.1 × 108 M−1 as an ERα-aptamer. Target-specificity of the selected ERaptR4 aptamer was confirmed through cytochemistry and solid-phase immunoassays. Furthermore, stability analyses identified ERaptR4 resistant to serum and RNase A degradation in presence of ERα. Taken together, an efficient ERα-RNA aptamer is identified using a non-SELEX procedure of aptamer selection. The high-affinity and specificity can be utilized in detection of ERα in breast cancer and related diseases. PMID:26899418

  6. Aptamers in Therapeutics

    PubMed Central

    2016-01-01

    Aptamers are single strand DNA or RNA molecules, selected by an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Due to various advantages of aptamers such as high temperature stability, animal free, cost effective production and its high affinity and selectivity for its target make them attractive alternatives to monoclonal antibody for use in diagnostic and therapeutic purposes. Aptamer has been generated against vesicular endothelial growth factor 165 involved in age related macular degeneracy. Macugen was the first FDA approved aptamer based drug that was commercialized. Later other aptamers were also developed against blood clotting proteins, cancer proteins, antibody E, agents involved in diabetes nephropathy, autoantibodies involved in autoimmune disorders, etc. Aptamers have also been developed against viruses and could work with other antiviral agents in treating infections. PMID:27504277

  7. The RNA-binding domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner

    PubMed Central

    Marc, Daniel; Barbachou, Sosthène; Soubieux, Denis

    2013-01-01

    Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5′ terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5′-GUAAC / 3′-CUUAG double-stranded motif, which closely resembles the canonical 5′-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar KD values) that leads to oligomerization of the RBD on its RNA ligands. PMID:23093596

  8. RNA aptamer-based electrochemical aptasensor for C-reactive protein detection using functionalized silica microspheres as immunoprobes.

    PubMed

    Wang, Junchun; Guo, Jinjin; Zhang, Junjun; Zhang, Wenjuan; Zhang, Yuzhong

    2017-09-15

    C-reactive protein (CRP) is a widely accepted biomarker of cardiovascular disease and inflammation. In this study, a RNA aptamer-based electrochemical sandwich type aptasensor for CRP detection was described using the functionalized silica microspheres as immunoprobes. Silica microspheres (Si MSs), which have good monodispersity and uniform shape, were firstly synthesized. The silica microspheres functionlized with gold nanoparticles (Au NPs) provided large surface area for immobilizing signal molecules (Zinc ions, Zn(2+)) and antibodies (Ab). RNA aptamers, which were specific recognized to CRP, were assembled on the surface of Au NPs modified electrode via gold-sulfur affinity. In the presence of CRP, a sandwich structure of aptamer-CRP-immunoprobe was formed. Square wave voltammetry (SWV) was employed to record the sensing signal, and a clearly reductive peak corresponding to Zn(2+) at about -1.16V (vs. SCE) was obtained. Under optimal conditions, the aptasensor showed wide linear range (0.005ngmL(-1) to 125ngmL(-1)) and low detection limit (0.0017ngmL(-1) at a signal-to-noise ratio of 3). Some possible interfering substance was also investigated, and the results obtained showed that the aptasensor possessed good selectivity. When the aptasensor was applied to real serum samples analysis, the satisfied results were obtained, indicating that the aptasensor possessed potential real application ability. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Blocking the maturation of OncomiRNAs using pri-miRNA-17∼92 aptamer in retinoblastoma.

    PubMed

    Subramanian, Nithya; Kanwar, Jagat R; Kanwar, Rupinder K; Krishnakumar, Subramanian

    2015-02-01

    The miR-17∼92. or oncomiR-1, cluster encodes oncogenic microRNAs (miRNAs), and it also promotes retinoblastoma (RB) tumor formation. Antagomir and miRNA mimics based approaches are widely tried against oncogenic and tumor suppressive miRNAs. Other methods for targeting cancer related miRNAs are still under development. In the current study, we focused on the pri-miRNA-17∼92 aptamer (pri-apt), which can potentially replace the mix of five antagomirs by one aptamer that function to abrogate the maturation of miR-17, miR-18a, and miR-19b (P<0.05) for targeting RB. We used RB cell lines WERI-Rb1 and Y79 as an in vitro model. Cellular changes upon transfecting the pri-apt led to S-phase arrest in WERI-Rb1 cells and onset of apoptosis in both Y79 and WERI-Rb1 cell lines. There was increased cytotoxicity as measured by lactate dehydrogenase activity in pri-apt treated Y79 cells (P<0.05), and significant inhibition of cell proliferation was observed in both of the cell lines. Thus we showed the antiproliferative property of pri-apt in RB cell lines, which can be readily modified by developing appropriate vectors for the delivery of the aptamer specifically to cancer cells.

  10. Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.

    PubMed

    Chang, Tzuu-Wang; Janardhanan, Pavithra; Mello, Charlene M; Singh, Bal Ram; Cai, Shuowei

    2016-09-01

    Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.

  11. Development and characterization of Sindbis virus with encoded fluorescent RNA aptamer Spinach2 for imaging of replication and immune-mediated changes in intracellular viral RNA.

    PubMed

    Nilaratanakul, Voraphoj; Hauer, Debra A; Griffin, Diane E

    2017-05-01

    Viral RNA studies often rely on in situ hybridization and reverse transcriptase-PCR to provide snapshots of RNA dynamics in infected cells. To facilitate analysis of cellular RNAs, aptamers Spinach and Spinach2 that bind and activate the conditional fluorophore 3, 5-difluoro-4-hydroxybenzylidene imidazolinon have been developed. To determine the feasibility of applying this technology to viral RNA, we have used cDNA clones of the TE strain of Sindbis virus (SINV) to construct multiple viruses containing one or two copies of tRNA-scaffolded Spinach2 after a second subgenomic promoter, TEds-1Sp and TEds-2Sp within the 3'UTR, TE-1UTRSp, or after a second subgenomic promoter and in the 3'UTR, TEds-1Sp+1 UTRSp. TEds-1Sp+1 UTRSp gave the brightest signal and replicated well in cell culture, while TEds-2Sp was the dimmest and replicated poorly. Selection of baby hamster kidney cells infected with TEds-1Sp+1 UTRSp for improved signal intensity identified a virus with a stronger signal and point mutations in the tRNA scaffold. Imaging of SINV in BHK cells showed RNA to be concentrated in filopodia that contacted and transferred RNA to adjacent cells. The effect of treatment with anti-E2 antibody, which effects non-cytolytic clearance of SINV from neurons, on viral RNA was cell-type-dependent. In antibody-treated BHK cells, intracellular viral RNA increased and spread of infection continued. In undifferentiated and differentiated AP7 neuronal cells antibody treatment induced viral RNA clearance. Both viruses with two inserted aptamers were prone to deletion. These studies form the basis for further development of aptamer-labelled viral RNAs that will facilitate functional studies on the dynamics of infection and clearance.

  12. RNA sequence analysis using covariance models.

    PubMed Central

    Eddy, S R; Durbin, R

    1994-01-01

    We describe a general approach to several RNA sequence analysis problems using probabilistic models that flexibly describe the secondary structure and primary sequence consensus of an RNA sequence family. We call these models 'covariance models'. A covariance model of tRNA sequences is an extremely sensitive and discriminative tool for searching for additional tRNAs and tRNA-related sequences in sequence databases. A model can be built automatically from an existing sequence alignment. We also describe an algorithm for learning a model and hence a consensus secondary structure from initially unaligned example sequences and no prior structural information. Models trained on unaligned tRNA examples correctly predict tRNA secondary structure and produce high-quality multiple alignments. The approach may be applied to any family of small RNA sequences. Images PMID:8029015

  13. Improvement in the drug delivery and anti-tumor efficacy of PEGylated liposomal doxorubicin by targeting RNA aptamers in mice bearing breast tumor model.

    PubMed

    Moosavian, Seyedeh Alia; Abnous, Khalil; Badiee, Ali; Jaafari, Mahmoud Reza

    2016-03-01

    Targeted delivery by ligands such as aptamers, is a promising method to increase the efficiency of PEGylated-liposomal doxorubicin (PL-Dox). In this study, we have successfully conjugated our recently developed anti-breast cancer RNA aptamer (TSA14) to the surface of PL-Dox and characterized for their size, zeta potential, Dox percent encapsulation and release properties in the presence of fetal bovine serum. In vitro experiments showed that aptamer could improve cellular uptake and cytotoxicity of PL-Dox in TUBO breast cell line. In mice bearing TUBO breast tumor, although, the doxorubicin plasma level of liposomal doxorubicin did not significantly change after modification of nanoparticles with aptamer, however, much higher tumor accumulation of Dox as compared with non-targeted liposomes proved the tumor-targeting capability of aptamers. In the same way, aptamer-PL-Dox improved anti-tumor efficiency of liposomes in TUBO breast tumor in mice compared to non-targeted liposomes. Overall, the results showed that aptamer decoration of PL-Dox could significantly improve selectivity and the therapeutic efficacy of liposomal DOX and merits further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Advances in aptamers.

    PubMed

    Syed, Muhammad Ali; Pervaiz, Saima

    2010-10-01

    Aptamers are nucleic acid sequences synthesized through in vitro selection and amplification technique, possessing a broader range of applications in therapeutics, biosensing, diagnostics, and research. Aptamers offer a number of advantages over their antibodies counterpart, one of them is their ability to undergo chemical derivatization to increase their life in the body fluids and bioavailability in animals. Although aptamers were discovered in 1990s, they have become one of the most widely investigated molecules, with a huge number of publications in the last decade. This article presents an overview of the advancements that have been made in aptamers. We mainly focused on articles published since 2005.

  15. Escort Aptamers: New Tools for the Targeted Delivery of Therapeutics into Cells

    PubMed Central

    Davydova, A.S.; Vorobjeva, M.A.; Venyaminova, A.G.

    2011-01-01

    Escort aptamers are DNA or RNA sequences with high affinity to certain cell-surface proteins, which can be used for targeted delivery of various agents into cells of a definite type. The peculiarities of the selection of escort aptamers are discussed in this review. The methods used in selection of escort aptamers via the SELEX technique are considered, including selection against isolated cell-surface proteins, cell fragments, living eukaryotic cells, and bacteria. Particular attention is given to the design and chemical modification of escort aptamers. The different fields of application of escort aptamers are described, including the targeted delivery of siRNAs, nanoparticles, toxins, and photoagents, as well as the identification of specific cell markers and the detection or isolation of cells of a definite type. The potential for the application of escort aptamers in the development of new therapeutic agents and diagnostic systems is also discussed. PMID:22649701

  16. NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

    PubMed Central

    Latham, Michael P.; Zimmermann, Grant R.; Pardi, Arthur

    2009-01-01

    The apparent on- and off-rate constants for theophylline binding to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H-1H NMR ZZ-exchange spectroscopy. Analysis of the build-up rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M-1 s-1. This small apparent kon results from the free RNA existing as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data here also explain a very unusual property of this RNA-theophylline system, slow exchange on the NMR chemical shift timescale for a weak-binding complex. To our knowledge, it is unprecedented to have such a weak binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift timescale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand-receptor interactions are presented. PMID:19317486

  17. Multiplex Aptamer Discovery through Apta-Seq and Its Application to ATP Aptamers Derived from Human-Genomic SELEX.

    PubMed

    Abdelsayed, Michael M; Ho, Bao T; Vu, Michael M K; Polanco, Julio; Spitale, Robert C; Lupták, Andrej

    2017-08-18

    Laboratory-evolved RNAs bind a wide variety of targets and serve highly diverse functions, including as diagnostic and therapeutic aptamers. The majority of aptamers have been identified using in vitro selection (SELEX), a molecular evolution technique based on selecting target-binding RNAs from highly diverse pools through serial rounds of enrichment and amplification. In vitro selection typically yields multiple distinct motifs of highly variable abundance and target-binding affinities. The discovery of new aptamers is often limited by the difficulty of characterizing the selected motifs, because testing of individual sequences tends to be a tedious process. To facilitate the discovery of new aptamers within in vitro selected pools, we developed Apta-Seq, a multiplex analysis based on quantitative, ligand-dependent 2' acylation of solvent-accessible regions of the selected RNA pools, followed by reverse transcription (SHAPE) and deep sequencing. The method reveals, in a single sequencing experiment, the identity, structural features, and target dissociation constants for aptamers present in the selected pool. Application of Apta-Seq to a human genomic pool enriched for ATP-binding RNAs yielded three new aptamers, which together with previously identified human aptamers suggest that ligand-binding RNAs may be common in mammals.

  18. Protein synthesis editing by a DNA aptamer.

    PubMed Central

    Hale, S P; Schimmel, P

    1996-01-01

    Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response. Images Fig. 3 Fig. 4 PMID:8610114

  19. An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition.

    PubMed Central

    Kiga, D; Futamura, Y; Sakamoto, K; Yokoyama, S

    1998-01-01

    RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 10(12) random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA) of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 microM by equilibrium filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil. The K d values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding. PMID:9512549

  20. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.

    PubMed

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E

    2014-04-01

    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  1. Computational generation and screening of RNA motifs in large nucleotide sequence pools

    PubMed Central

    Kim, Namhee; Izzo, Joseph A.; Elmetwaly, Shereef; Gan, Hin Hark; Schlick, Tamar

    2010-01-01

    Although identification of active motifs in large random sequence pools is central to RNA in vitro selection, no systematic computational equivalent of this process has yet been developed. We develop a computational approach that combines target pool generation, motif scanning and motif screening using secondary structure analysis for applications to 1012–1014-sequence pools; large pool sizes are made possible using program redesign and supercomputing resources. We use the new protocol to search for aptamer and ribozyme motifs in pools up to experimental pool size (1014 sequences). We show that motif scanning, structure matching and flanking sequence analysis, respectively, reduce the initial sequence pool by 6–8, 1–2 and 1 orders of magnitude, consistent with the rare occurrence of active motifs in random pools. The final yields match the theoretical yields from probability theory for simple motifs and overestimate experimental yields, which constitute lower bounds, for aptamers because screening analyses beyond secondary structure information are not considered systematically. We also show that designed pools using our nucleotide transition probability matrices can produce higher yields for RNA ligase motifs than random pools. Our methods for generating, analyzing and designing large pools can help improve RNA design via simulation of aspects of in vitro selection. PMID:20448026

  2. Exploring Connectivity in Sequence Space of Functional RNA

    NASA Technical Reports Server (NTRS)

    Wei, Chenyu; Pohorille, Andrzej; Popovic, Milena; Ditzler, Mark

    2017-01-01

    Emergence of replicable genetic molecules was one of the marking points in the origin of life, evolution of which can be conceptualized as a walk through the space of all possible sequences. A theoretical concept of fitness landscape helps to understand evolutionary processes through assigning a value of fitness to each genotype. Then, evolution of a phenotype is viewed as a series of consecutive, single-point mutations. Natural selection biases evolution toward peaks of high fitness and away from valleys of low fitness. whereas neutral drift occurs in the sequence space without direction as mutations are introduced at random. Large networks of neutral or near-neutral mutations on a fitness landscape, especially for sufficiently long genomes, are possible or even inevitable. Their detection in experiments, however, has been elusive. Although a few near-neutral evolutionary pathways have been found, recent experimental evidence indicates landscapes consist of largely isolated islands. The generality of these results, however, is not clear, as the genome length or the fraction of functional molecules in the genotypic space might have been insufficient for the emergence of large, neutral networks. Thorough investigation on the structure of the fitness landscape is essential to understand the mechanisms of evolution of early genomes. RNA molecules are commonly assumed to play the pivotal role in the origin of genetic systems. They are widely believed to be early, if not the earliest, genetic and catalytic molecules, with abundant biochemical activities as aptamers and ribozymes, i.e. RNA molecules capable, respectively, to bind small molecules or catalyze chemical reactions. Here, we present results of our recent studies on the structure of the sequence space of RNA ligase ribozymes selected through in vitro evolution. Several hundred thousands of sequences active to a different degree were obtained by way of deep sequencing. Analysis of these sequences revealed

  3. Potent and Selective Inhibition of a Single α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Subunit by an RNA Aptamer*

    PubMed Central

    Park, Jae-Seon; Wang, Congzhou; Han, Yan; Huang, Zhen; Niu, Li

    2011-01-01

    Inhibitors of AMPA-type glutamate ion channels are useful as biochemical probes for structure-function studies and as drug candidates for a number of neurological disorders and diseases. Here, we describe the identification of an RNA inhibitor or aptamer by an in vitro evolution approach and a characterization of its mechanism of inhibition on the sites of interaction by equilibrium binding and on the receptor channel opening rate by a laser-pulse photolysis technique. Our results show that the aptamer is a noncompetitive inhibitor that selectively inhibits the GluA2Qflip AMPA receptor subunit without any effect on other AMPA receptor subunits or kainate or NMDA receptors. On the GluA2 subunit, this aptamer preferentially inhibits the flip variant. Furthermore, the aptamer preferentially inhibits the closed-channel state of GluA2Qflip with a KI = 1.5 μm or by ∼15-fold over the open-channel state. The potency and selectivity of this aptamer rival those of small molecule inhibitors. Together, these properties make this aptamer a promising candidate for the development of water-soluble, highly potent, and GluA2 subunit-selective drugs. PMID:21402710

  4. SSCP screening of individual aptamers.

    PubMed

    Gening, L V; Klincheva, S A; Gusev, A S; Surovoy, A Y; Potapov, V K

    2001-10-01

    Aptamers are specific binding nucleic acids that emerge from in vitro selection. During the systematic evolution of ligands by exponential enrichment (SELEX) procedure, analysis of the sequences of the numerous selected individual molecules becomes an important step in the final stage of aptamer selection. The sequencing of cloned aptamers from the selected pool generally reveals groups of identical sequences and rarely occurring individual aptamers. This study demonstrates an approach similar to the single strand conformation polymorphism (SSCP) method used for mutation testing in genes. Human angiotensin I-specific aptamers have been used to show the efficiency of the SSCP method to classify selected individual sequences into identity groups, which minimizes sequencing efforts. Additionally this approach allows the rapid isolation and identification of aptamers from a mixture.

  5. HPLC purification of RNA aptamers up to 59 nucleotides with single-nucleotide resolution.

    PubMed

    Huang, Zhen; Lin, Chi-Yen; Jaremko, William; Niu, Li

    2015-01-01

    An RNA sample is usually heterogeneous. RNA heterogeneity refers to difference in length or size (i.e., number of nucleotides [nt]), sequence, or alternative but coexisting conformations. Separation and purification of RNA is generally required for investigating the structure and function of RNA, such as RNA catalysis and RNA structure determination by nuclear magnetic resonance or crystallography. Separation and purification of RNA is also required for using RNAs as functional probes and therapeutics as well as building blocks for RNA nanoparticles. Previously established protocols are limited in separating RNAs longer than 25 nt by single-nucleotide resolution. When the length of RNAs becomes longer, single-nucleotide separation of RNAs becomes more challenging. Here we describe protocols, by the use of ion-pair, reverse-phase high-performance liquid chromatography (HPLC), to extend our ability to separate regular RNAs up to 59 nt with single-nucleotide resolution. For chemically modified RNAs at 2' positions on the ribose, we can resolve RNAs of similar sizes even with a 26 Da difference. This is much less than 320 Da, an average single-nucleotide molecular weight difference.

  6. Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state.

    PubMed

    Warfield, Becka M; Anderson, Peter C

    2017-01-01

    RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population

  7. Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state

    PubMed Central

    Warfield, Becka M.

    2017-01-01

    RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population

  8. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

    PubMed

    Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

    2016-12-01

    Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

  9. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

    PubMed Central

    Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N.

    2016-01-01

    ABSTRACT Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries. PMID:27715478

  10. A specific aptamer-cell penetrating peptides complex delivered siRNA efficiently and suppressed prostate tumor growth in vivo

    PubMed Central

    Diao, Yanjun; Liu, Jiayun; Ma, Yueyun; Su, Mingquan; Zhang, Hongyi; Hao, Xiaoke

    2016-01-01

    ABSTRACT Specific and efficient delivery of siRNA into intended tumor cells remains as a challenge, even though RNAi has been exploited as a new strategy for prostate cancer therapy. This work aims to address both specificity and efficiency of SURVIVIN-siRNA delivery by constructing a therapeutic complex using combinatorial strategies. A fusion protein STD was first expressed to serve as a backbone, consisting of streptavidin, a cell-penetrating peptide called Trans-Activator of Transcription (TAT) and a double-stranded RNA binding domain. A biotinylated Prostate Specific Membrane Antigen (PSMA) specific aptamer A10 and SURVIVIN-siRNA were then linked to STD protein to form the therapeutic complex. This complex could specifically targeted PSMA+ tumor cells. Compared to lipofectamine and A10-siRNA chimera, it demonstrated higher efficiency in delivering siRNA into target cells by 19.2% and 59.9%, and increased apoptosis by 16.8% and 26.1% respectively. Upon systemic administration, this complex also showed significant efficacy in suppressing tumor growth in athymic mice (p <0.001). We conclude that this therapeutic complex could specifically and efficiently deliver SURVIVIN-siRNA to target cells and suppressed tumor growth in vivo, which indicates its potential to be used as a new strategy in prostate cancer therapy PMID:26954374

  11. antaRNA: ant colony-based RNA sequence design

    PubMed Central

    Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

    2015-01-01

    Motivation: RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found, inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology, reliable RNA sequence design becomes a crucial step to generate novel biochemical components. Results: In this article, the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution, specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. Availability and implementation: http://www.bioinf.uni-freiburg.de/Software/antaRNA Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26023105

  12. Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins

    PubMed Central

    Perepelyuk, Maryna; Maher, Christina; Lakshmikuttyamma, Ashakumary; Shoyele, Sunday A

    2016-01-01

    MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer. PMID:27555773

  13. Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

    PubMed Central

    Lebars, Isabelle; Legrand, Pierre; Aimé, Ahissan; Pinaud, Noël; Fribourg, Sébastien; Di Primo, Carmelo

    2008-01-01

    In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability. PMID:18996893

  14. Experimental investigation of an RNA sequence space

    NASA Astrophysics Data System (ADS)

    Lee, Youn-Hyung; Dsouza, Lisa; Fox, George E.

    1993-12-01

    Modern rRNAs are the historic consequence of an ongoing evolutionary exploration of a sequence space. These extant sequences belong to a special subset of the sequence space that is comprised only of those primary sequences that can validly perform the biological function(s) required of the particular RNA. If it were possible to readily identify all such valid sequences, stochastic predictions could be made about the relative likelihood of various evolutionary pathways available to an RNA. Herein an experimental system which can assess whether a particular sequence is likely to have validity as a eubacterial 5S rRNA is described. A total of ten naturally occurring, and hence known to be valid, sequences and two point mutants of unknown validity were used to test the usefulness of the approach. Nine of the ten valid sequences tested positive whereas both mutants tested as clearly defective. The tenth valid sequence gave results that would be interpreted as reflecting a borderline status were the answer not known. These results demonstrate that it is possible to experimentally determine which sequences in local regions of the sequence space are potentially valid 5S rRNAs. This approach will allow direct study of the constraints governing RNA evolution and allow inquiry into how the last common ancestor of extant life apparently came to have very complex ribosomal RNAs that subsequently were very conserved.

  15. Experimental investigation of an RNA sequence space.

    PubMed

    Lee, Y H; Dsouza, L; Fox, G E

    1993-12-01

    Modern rRNAs are the historic consequence of an ongoing evolutionary exploration of a sequence space. These extant sequences belong to a special subset of the sequence space that is comprised only of those primary sequences that can validly perform the biological function(s) required of the particular RNA. If it were possible to readily identify all such valid sequences, stochastic predictions could be made about the relative likelihood of various evolutionary pathways available to an RNA. Herein an experimental system which can assess whether a particular sequence is likely to have validity as a eubacterial 5S rRNA is described. A total of ten naturally occurring and hence known to be valid, sequences and two point mutants of unknown validity were used to test the usefulness of the approach. Nine of the ten valid sequences tested positive whereas both mutants tested as clearly defective. The tenth valid sequence gave results that would be interpreted as reflecting a borderline status were the answer not known. These results demonstrate that it is possible to experimentally determine which sequences in local regions of the sequence space are potentially valid 5S rRNAs. This approach will allow direct study of the constraints governing RNA evolution and allow inquiry into how the last common ancestor of extant life apparently came to have very complex ribosomal RNAs that subsequently were very conserved.

  16. The Toolbox for Modified Aptamers.

    PubMed

    Lapa, Sergey A; Chudinov, Alexander V; Timofeev, Edward N

    2016-02-01

    Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well.

  17. Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

    PubMed Central

    Ozer, Abdullah; Tome, Jacob M.; Friedman, Robin C.; Gheba, Dan; Schroth, Gary P.; Lis, John T.

    2016-01-01

    Because RNA-protein interactions play a central role in a wide-array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the High Throughput Sequencing-RNA Affinity Profiling (HiTS-RAP) assay, which couples sequencing on an Illumina GAIIx with the quantitative assessment of one or several proteins’ interactions with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of EGFP and NELF-E proteins with their corresponding canonical and mutant RNA aptamers. Here, we provide a detailed protocol for HiTS-RAP, which can be completed in about a month (8 days hands-on time) including the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, high-throughput sequencing and protein binding with GAIIx, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, RNA-MaP and RBNS. A successful HiTS-RAP experiment provides the sequence and binding curves for approximately 200 million RNAs in a single experiment. PMID:26182240

  18. Experimental investigation of an RNA sequence space

    NASA Technical Reports Server (NTRS)

    Lee, Youn-Hyung; Dsouza, Lisa; Fox, George E.

    1993-01-01

    Modern rRNAs are the historic consequence of an ongoing evolutionary exploration of a sequence space. These extant sequences belong to a special subset of the sequence space that is comprised only of those primary sequences that can validly perform the biological function(s) required of the particular RNA. If it were possible to readily identify all such valid sequences, stochastic predictions could be made about the relative likelihood of various evolutionary pathways available to an RNA. Herein an experimental system which can assess whether a particular sequence is likely to have validity as a eubacterial 5S rRNA is described. A total of ten naturally occurring, and hence known to be valid, sequences and two point mutants of unknown validity were used to test the usefulness of the approach. Nine of the ten valid sequences tested positive whereas both mutants tested as clearly defective. The tenth valid sequence gave results that would be interpreted as reflecting a borderline status were the answer not known. These results demonstrate that it is possible to experimentally determine which sequences in local regions of the sequence space are potentially valid 5S rRNAs.

  19. Experimental investigation of an RNA sequence space

    NASA Technical Reports Server (NTRS)

    Lee, Youn-Hyung; Dsouza, Lisa; Fox, George E.

    1993-01-01

    Modern rRNAs are the historic consequence of an ongoing evolutionary exploration of a sequence space. These extant sequences belong to a special subset of the sequence space that is comprised only of those primary sequences that can validly perform the biological function(s) required of the particular RNA. If it were possible to readily identify all such valid sequences, stochastic predictions could be made about the relative likelihood of various evolutionary pathways available to an RNA. Herein an experimental system which can assess whether a particular sequence is likely to have validity as a eubacterial 5S rRNA is described. A total of ten naturally occurring, and hence known to be valid, sequences and two point mutants of unknown validity were used to test the usefulness of the approach. Nine of the ten valid sequences tested positive whereas both mutants tested as clearly defective. The tenth valid sequence gave results that would be interpreted as reflecting a borderline status were the answer not known. These results demonstrate that it is possible to experimentally determine which sequences in local regions of the sequence space are potentially valid 5S rRNAs.

  20. Analysis of Pteridium ribosomal RNA sequences by rapid direct sequencing.

    PubMed

    Tan, M K

    1991-08-01

    A total of 864 bases from 5 regions interspersed in the 18S and 26S rRNA molecules from various clones of Pteridium covering the general geographical distribution of the genus was analysed using a rapid rRNA sequencing technique. No base difference has been detected amongst the three major lineages, two of which apparently separated before the breakup of the ancient supercontinent, Pangaea. These regions of the rRNA sequences have thus been conserved for at least 160 million years and are here compared with other eukaryotic, especially plant rRNAs.

  1. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    PubMed Central

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  2. Blind SELEX Approach Identifies RNA Aptamer that Regulate EMT and Inhibit Metastasis.

    PubMed

    Yoon, Sorah; Armstrong, Brian; Habib, Nagy; Rossi, John J

    2017-04-10

    Identifying targets that are exposed on the plasma membrane of tumor cells, but expressed internally in normal cells, is a fundamental issue for improving the specificity and efficacy of anticancer therpeutics. Using blind cell SELEX (Systemic Evolution of Ligands by EXponetial enrichment) which is untargeted SELEX, we have identified an aptamer, P15, which specifically bound to the human pancreatic adenocarcinoma cells. To identify the aptamer binding plasma membrane protein, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used. The results of this unbiased proteomic mass spectrometry approach identified the target of P15 as the intermediate filament vimentin, biomarker of epithelial mesenchymal transition (EMT), which is an intracellular protein but is specifically expressed on the plasma membrane of cancer cells. As EMT plays a pivotal role to transit cancer cells to invasive cells, tumor cell metastasis assays were performed in vitro. P15 treated pancreatic cancer cells showed the significant inhibition of tumor metastasis. To investigate the downstream effects of P15, EMT related gene expression analysis was performed to identify differently expressed genes (DEGs). Among five DEGs, P15 treated cells showed the down-regulated expression of matrix metallopeptidase 3 (MMP3), which is involved in cancer invasion. These results, for the first time, demonstrate that P15 binding to cell surface vimentin inhibits the tumor cell invasion and is associated with reduced MMP3 expression. Thus, suggesting that P15 has potential as an anti-metastatic therapy in pancreatic cancer.

  3. Discovering New Biology through Sequencing of RNA.

    PubMed

    Weber, Andreas P M

    2015-11-01

    Sequencing of RNA (RNA-Seq) was invented approximately 1 decade ago and has since revolutionized biological research. This update provides a brief historic perspective on the development of RNA-Seq and then focuses on the application of RNA-Seq in qualitative and quantitative analyses of transcriptomes. Particular emphasis is given to aspects of data analysis. Since the wet-lab and data analysis aspects of RNA-Seq are still rapidly evolving and novel applications are continuously reported, a printed review will be rapidly outdated and can only serve to provide some examples and general guidelines for planning and conducting RNA-Seq studies. Hence, selected references to frequently update online resources are given. © 2015 American Society of Plant Biologists. All Rights Reserved.

  4. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome.

    PubMed

    Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

    2017-02-07

    The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.

  5. Comparative crystallization and preliminary X-ray diffraction studies of locked nucleic acid and RNA stems of a tenascin C-binding aptamer

    PubMed Central

    Förster, Charlotte; Brauer, Arnd B. E.; Brode, Svenja; Schmidt, Kathrin S.; Perbandt, Markus; Meyer, Arne; Rypniewski, Wojciech; Betzel, Christian; Kurreck, Jens; Fürste, Jens P.; Erdmann, Volker A.

    2006-01-01

    The pharmacokinetic properties of an aptamer against the tumour-marker protein tenascin-C have recently been successfully improved by the introduction of locked nucleic acids (LNAs) into the terminal stem of the aptamer. Since it is believed that this post-SELEX optimization is likely to provide a more general route to enhance the in vitro and in vivo stability of aptamers, elucidation of the structural basis of this improvement was embarked upon. Here, the crystallographic and X-ray diffraction data of the isolated aptamer stem encompassed in a six-base-pair duplex both with and without the LNA modification are presented. The obtained all-LNA crystals belong to space group P41212 or P43212, with unit-cell parameters a = b = 52.80, c = 62.83 Å; the all-RNA crystals belong to space group R32, with unit-cell parameters a = b = 45.21, c = 186.97 Å, γ = 120.00°. PMID:16820689

  6. Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2

    PubMed Central

    Oberthür, Dominik; Achenbach, John; Gabdulkhakov, Azat; Buchner, Klaus; Maasch, Christian; Falke, Sven; Rehders, Dirk; Klussmann, Sven; Betzel, Christian

    2015-01-01

    We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine's activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide·L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamer's intramolecular contacts and permits a detailed analysis of its structure–function relationship. Furthermore, the analysis of the intermolecular drug–target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines. PMID:25901662

  7. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

    PubMed Central

    Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

    2017-01-01

    The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type–specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs. PMID:28030843

  8. Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2

    NASA Astrophysics Data System (ADS)

    Oberthür, Dominik; Achenbach, John; Gabdulkhakov, Azat; Buchner, Klaus; Maasch, Christian; Falke, Sven; Rehders, Dirk; Klussmann, Sven; Betzel, Christian

    2015-04-01

    We report the crystal structure of a 40mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine's activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide.L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamer's intramolecular contacts and permits a detailed analysis of its structure-function relationship. Furthermore, the analysis of the intermolecular drug-target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.

  9. Aptamer/Au nanoparticles/cobalt sulfide nanosheets biosensor for 17β-estradiol detection using a guanine-rich complementary DNA sequence for signal amplification.

    PubMed

    Huang, Ke-Jing; Liu, Yu-Jie; Zhang, Ji-Zong; Cao, Jun-Tao; Liu, Yan-Ming

    2015-05-15

    We have developed a sensitive sensing platform for 17β-estradiol by combining the aptamer probe and hybridization reaction. In this assay, 2-dimensional cobalt sulfide nanosheet (CoS) was synthesized by a simple hydrothermal method with L-cysteine as sulfur donor. An electrochemical aptamer biosensor was constructed by assembling a thiol group tagged 17β-estradiol aptamer on CoS and gold nanoparticles (AuNPs) modified electrode. Methylene blue was applied as a tracer and a guanine-rich complementary DNA sequence was designed to bind with the unbound 17β-estradiol aptamer for signal amplification. The binding of guanine-rich DNA to the aptamer was inhibited when the aptamer captured 17β-estradiol. Using guanine-rich DNA in the assay greatly amplified the redox signal of methylene blue bound to the detection probe. The CoS/AuNPs film formed on the biosensor surface appeared to be a good conductor for accelerating the electron transfer. The method demonstrated a high sensitivity of detection with the dynamic concentration range spanning from 1.0×10(-9) to 1.0×10(-12) M and a detection limit of 7.0×10(-13) M. Besides, the fabricated biosensor exhibited good selectivity toward 17β-estradiol even when interferents were presented at 100-fold concentrations. Our attempt will extend the application of the CoS nanosheet and this signal amplification assay to biosensing areas. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Alternative applications for distinct RNA sequencing strategies

    PubMed Central

    Han, Leng; Vickers, Kasey C.; Samuels, David C.

    2015-01-01

    Recent advances in RNA library preparation methods, platform accessibility and cost efficiency have allowed high-throughput RNA sequencing (RNAseq) to replace conventional hybridization microarray platforms as the method of choice for mRNA profiling and transcriptome analyses. RNAseq is a powerful technique to profile both long and short RNA expression, and the depth of information gained from distinct RNAseq methods is striking and facilitates discovery. In addition to expression analysis, distinct RNAseq approaches also allow investigators the ability to assess transcriptional elongation, DNA variance and exogenous RNA content. Here we review the current state of the art in transcriptome sequencing and address epigenetic regulation, quantification of transcription activation, RNAseq output and a diverse set of applications for RNAseq data. We detail how RNAseq can be used to identify allele-specific expression, single-nucleotide polymorphisms and somatic mutations and discuss the benefits and limitations of using RNAseq to monitor DNA characteristics. Moreover, we highlight the power of combining RNA- and DNAseq methods for genomic analysis. In summary, RNAseq provides the opportunity to gain greater insight into transcriptional regulation and output than simply miRNA and mRNA profiling. PMID:25246237

  11. Sequence Fingerprints of MicroRNA Conservation

    PubMed Central

    Shi, Bing; Gao, Wei; Wang, Juan

    2012-01-01

    It is known that the conservation of protein-coding genes is associated with their sequences both various species, such as animals and plants. However, the association between microRNA (miRNA) conservation and their sequences in various species remains unexplored. Here we report the association of miRNA conservation with its sequence features, such as base content and cleavage sites, suggesting that miRNA sequences contain the fingerprints for miRNA conservation. More interestingly, different species show different and even opposite patterns between miRNA conservation and sequence features. For example, mammalian miRNAs show a positive/negative correlation between conservation and AU/GC content, whereas plant miRNAs show a negative/positive correlation between conservation and AU/GC content. Further analysis puts forward the hypothesis that the introns of protein-coding genes may be a main driving force for the origin and evolution of mammalian miRNAs. At the 5′ end, conserved miRNAs have a preference for base U, while less-conserved miRNAs have a preference for a non-U base in mammals. This difference does not exist in insects and plants, in which both conserved miRNAs and less-conserved miRNAs have a preference for base U at the 5′ end. We further revealed that the non-U preference at the 5′ end of less-conserved mammalian miRNAs is associated with miRNA function diversity, which may have evolved from the pressure of a highly sophisticated environmental stimulus the mammals encountered during evolution. These results indicated that miRNA sequences contain the fingerprints for conservation, and these fingerprints vary according to species. More importantly, the results suggest that although species share common mechanisms by which miRNAs originate and evolve, mammals may develop a novel mechanism for miRNA origin and evolution. In addition, the fingerprint found in this study can be predictor of miRNA conservation, and the findings are helpful in achieving a

  12. Epitranscriptome sequencing technologies: decoding RNA modifications.

    PubMed

    Li, Xiaoyu; Xiong, Xushen; Yi, Chengqi

    2016-12-29

    In recent years, major breakthroughs in RNA-modification-mediated regulation of gene expression have been made, leading to the emerging field of epitranscriptomics.Our understanding of the distribution, regulation and function of these dynamic RNA modifications is based on sequencing technologies. In this Review, we focus on the major mRNA modifications in the transcriptome of eukaryotic cells: N6-methyladenosine, N6, 2'-O-dimethyladenosine, 5-methylcytidine, 5-hydroxylmethylcytidine, inosine, pseudouridine and N(1)-methyladenosine. We discuss the sequencing technologies used to profile these epitranscriptomic marks, including scale, resolution, quantitative feature, pre-enrichment capability and the corresponding bioinformatics tools. We also discuss the challenges of epitranscriptome profiling and highlight the prospect of future detection tools. We aim to guide the choice of different detection methods and inspire new ideas in RNA biology.

  13. Synergistic inhibition of lung cancer cell invasion, tumor growth and angiogenesis using aptamer-siRNA chimeras.

    PubMed

    Lai, Wei-Yun; Wang, Wei-Ya; Chang, Yi-Chung; Chang, Cheng-Ju; Yang, Pan-Chyr; Peck, Konan

    2014-03-01

    Early metastasis is one of the major causes of mortality among patient with lung cancer. The process of tumor metastasis involves a cascade of events, including epithelial-mesenchymal transition, tumor cell migration and invasion, and angiogenesis. To specifically suppress tumor invasion and angiogenesis, two nucleolin aptamer-siRNA chimeras (aptNCL-SLUGsiR and aptNCL-NRP1siR) were used to block key signaling pathways involved in lung cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Through nucleolin-mediated endocytosis, the aptNCL-siRNA chimeras specifically and significantly knocked down the expressions of SLUG and NRP1 in nucleolin-expressing cancer cells. Furthermore, simultaneous suppression of SLUG and NRP1 expressions by the chimeras synergistically retarded cancer cell motility and invasive ability. The synergistic effect was also observed in a xenograft mouse model, wherein the combined treatment using two chimeras suppressed tumor growth, the invasiveness, circulating tumor cell amount, and angiogenesis in tumor tissue without affecting liver and kidney functions. This study demonstrates that combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR can synergistically suppress lung cancer cell invasion, tumor growth and angiogenesis by cancer-specific targeting combined with gene-specific silencing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. RNA-RNA interaction prediction based on multiple sequence alignments.

    PubMed

    Li, Andrew X; Marz, Manja; Qin, Jing; Reidys, Christian M

    2011-02-15

    Many computerized methods for RNA-RNA interaction structure prediction have been developed. Recently, O(N(6)) time and O(N(4)) space dynamic programming algorithms have become available that compute the partition function of RNA-RNA interaction complexes. However, few of these methods incorporate the knowledge concerning related sequences, thus relevant evolutionary information is often neglected from the structure determination. Therefore, it is of considerable practical interest to introduce a method taking into consideration both: thermodynamic stability as well as sequence/structure covariation. We present the a priori folding algorithm ripalign, whose input consists of two (given) multiple sequence alignments (MSA). ripalign outputs (i) the partition function, (ii) base pairing probabilities, (iii) hybrid probabilities and (iv) a set of Boltzmann-sampled suboptimal structures consisting of canonical joint structures that are compatible to the alignments. Compared to the single sequence-pair folding algorithm rip, ripalign requires negligible additional memory resource but offers much better sensitivity and specificity, once alignments of suitable quality are given. ripalign additionally allows to incorporate structure constraints as input parameters. The algorithm described here is implemented in C as part of the rip package.

  15. Probabilistic error correction for RNA sequencing.

    PubMed

    Le, Hai-Son; Schulz, Marcel H; McCauley, Brenna M; Hinman, Veronica F; Bar-Joseph, Ziv

    2013-05-01

    Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not available. Current read error correction methods, developed for DNA sequence data, cannot handle the overlapping effects of non-uniform abundance, polymorphisms and alternative splicing. Here we present SEquencing Error CorrEction in Rna-seq data (SEECER), a hidden Markov Model (HMM)-based method, which is the first to successfully address these problems. SEECER efficiently learns hundreds of thousands of HMMs and uses these to correct sequencing errors. Using human RNA-Seq data, we show that SEECER greatly improves on previous methods in terms of quality of read alignment to the genome and assembly accuracy. To illustrate the usefulness of SEECER for de novo transcriptome studies, we generated new RNA-Seq data to study the development of the sea cucumber Parastichopus parvimensis. Our corrected assembled transcripts shed new light on two important stages in sea cucumber development. Comparison of the assembled transcripts to known transcripts in other species has also revealed novel transcripts that are unique to sea cucumber, some of which we have experimentally validated. Supporting website: http://sb.cs.cmu.edu/seecer/.

  16. Probabilistic error correction for RNA sequencing

    PubMed Central

    Le, Hai-Son; Schulz, Marcel H.; McCauley, Brenna M.; Hinman, Veronica F.; Bar-Joseph, Ziv

    2013-01-01

    Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not available. Current read error correction methods, developed for DNA sequence data, cannot handle the overlapping effects of non-uniform abundance, polymorphisms and alternative splicing. Here we present SEquencing Error CorrEction in Rna-seq data (SEECER), a hidden Markov Model (HMM)–based method, which is the first to successfully address these problems. SEECER efficiently learns hundreds of thousands of HMMs and uses these to correct sequencing errors. Using human RNA-Seq data, we show that SEECER greatly improves on previous methods in terms of quality of read alignment to the genome and assembly accuracy. To illustrate the usefulness of SEECER for de novo transcriptome studies, we generated new RNA-Seq data to study the development of the sea cucumber Parastichopus parvimensis. Our corrected assembled transcripts shed new light on two important stages in sea cucumber development. Comparison of the assembled transcripts to known transcripts in other species has also revealed novel transcripts that are unique to sea cucumber, some of which we have experimentally validated. Supporting website: http://sb.cs.cmu.edu/seecer/. PMID:23558750

  17. Detection theory in identification of RNA-DNA sequence differences using RNA-sequencing.

    PubMed

    Toung, Jonathan M; Lahens, Nicholas; Hogenesch, John B; Grant, Gregory

    2014-01-01

    Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters.

  18. Quantitative selection and parallel characterization of aptamers

    PubMed Central

    Cho, Minseon; Soo Oh, Seung; Nie, Jeff; Stewart, Ron; Eisenstein, Michael; Chambers, James; Marth, Jamey D.; Walker, Faye; Thomson, James A.; Soh, H. Tom

    2013-01-01

    Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and next-generation sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (Kd) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with Kd as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure−function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding. PMID:24167271

  19. Aptamer Sensors

    PubMed Central

    Marrazza, Giovanna

    2017-01-01

    In the last years, great progress has been accomplished in the development of aptamer sensors with different transducers. In order to improve the sensitivity of these biosensors, several methodologies have been employed. In this Special Issue, the state of art and the future trends in the field of aptamer sensors have been explored. PMID:28054983

  20. Targeted inhibition of {alpha}v{beta}3 integrin with an RNA aptamer impairs endothelial cell growth and survival

    SciTech Connect

    Mi Jing; Zhang Xiuwu; Giangrande, Paloma H.; McNamara, James O.; Nimjee, Shahid M.; Sarraf-Yazdi, Shiva; Sullenger, Bruce A.; Clary, Bryan M. . E-mail: mi001@duke.edu

    2005-12-16

    {alpha}v{beta}3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. {alpha}v{beta}3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of {alpha}v{beta}3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-{alpha}v{beta}3 that binds recombinant {alpha}v{beta}3 integrin, for its ability to bind endogenous {alpha}v{beta}3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-{alpha}v{beta}3 binds {alpha}v{beta}3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-{alpha}v{beta}3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-{alpha}v{beta}3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-{alpha}v{beta}3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

  1. Calculation of absolute free energy of binding for theophylline and its analogs to RNA aptamer using nonequilibrium work values

    NASA Astrophysics Data System (ADS)

    Tanida, Yoshiaki; Ito, Masakatsu; Fujitani, Hideaki

    2007-08-01

    The massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed [H. Fujitani, Y. Tanida, M. Ito, G. Jayachandran, C.D. Snow, M.R. Shirts, E.J. Sorin, V.S. Pande, J. Chem. Phys. 123 (2005) 084108]. As an application, we perform the binding affinity calculations of six theophylline-related ligands with RNA aptamer. Basically, our method is applicable when using many compute nodes to accelerate simulations, thus a parallel computing system is also developed. To further reduce the computational cost, the adequate non-uniform intervals of coupling constant λ, connecting two equilibrium states, namely bound and unbound, are determined. The absolute binding energies Δ G thus obtained have effective linear relation between the computed and experimental values. If the results of two other different methods are compared, thermodynamic integration (TI) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) by the paper of Gouda et al. [H. Gouda, I.D. Kuntz, D.A. Case, P.A. Kollman, Biopolymers 68 (2003) 16], the predictive accuracy of the relative values ΔΔ G is almost comparable to that of TI: the correlation coefficients ( R) obtained are 0.99 (this work), 0.97 (TI), and 0.78 (MM-PBSA). On absolute binding energies meanwhile, a constant energy shift of ˜-7 kcal/mol against the experimental values is evident. To solve this problem, several presumable reasons are investigated.

  2. Predicting pseudoknotted structures across two RNA sequences

    PubMed Central

    Sperschneider, Jana; Datta, Amitava; Wise, Michael J.

    2012-01-01

    Motivation: Laboratory RNA structure determination is demanding and costly and thus, computational structure prediction is an important task. Single sequence methods for RNA secondary structure prediction are limited by the accuracy of the underlying folding model, if a structure is supported by a family of evolutionarily related sequences, one can be more confident that the prediction is accurate. RNA pseudoknots are functional elements, which have highly conserved structures. However, few comparative structure prediction methods can handle pseudoknots due to the computational complexity. Results: A comparative pseudoknot prediction method called DotKnot-PW is introduced based on structural comparison of secondary structure elements and H-type pseudoknot candidates. DotKnot-PW outperforms other methods from the literature on a hand-curated test set of RNA structures with experimental support. Availability: DotKnot-PW and the RNA structure test set are available at the web site http://dotknot.csse.uwa.edu.au/pw. Contact: janaspe@csse.uwa.edu.au Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23044552

  3. Ribosomal RNA sequence suggest microsporidia are extremely ancient eukaryotes

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Maddox, J. V.; Friedman, S.; Debrunner-Vossbrinck, B. A.; Woese, C. R.

    1987-01-01

    A comparative sequence analysis of the 18S small subunit ribosomal RNA (rRNA) of the microsporidium Vairimorpha necatrix is presented. The results show that this rRNA sequence is more unlike those of other eukaryotes than any known eukaryote rRNA sequence. It is concluded that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

  4. Ribosomal RNA sequence suggest microsporidia are extremely ancient eukaryotes

    NASA Technical Reports Server (NTRS)

    Vossbrinck, C. R.; Maddox, J. V.; Friedman, S.; Debrunner-Vossbrinck, B. A.; Woese, C. R.

    1987-01-01

    A comparative sequence analysis of the 18S small subunit ribosomal RNA (rRNA) of the microsporidium Vairimorpha necatrix is presented. The results show that this rRNA sequence is more unlike those of other eukaryotes than any known eukaryote rRNA sequence. It is concluded that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

  5. RiboaptDB: A Comprehensive Database of Ribozymes and Aptamers

    PubMed Central

    Thodima, Venkata; Pirooznia, Mehdi; Deng, Youping

    2006-01-01

    Background Catalytic RNA molecules are called ribozymes. The aptamers are DNA or RNA molecules that have been selected from vast populations of random sequences, through a combinatorial approach known as SELEX. The selected oligo-nucleotide sequences (~200 bp in length) have the ability to recognize a broad range of specific ligands by forming binding pockets. These novel aptamer sequences can bind to nucleic acids, proteins or small organic and inorganic chemical compounds and have many potential uses in medicine and technology. Results The comprehensive sequence information on aptamers and ribozymes that have been generated by in vitro selection methods are included in this RiboaptDB database. Such types of unnatural data generated by in vitro methods are not available in the public 'natural' sequence databases such as GenBank and EMBL. The amount of sequence data generated by in vitro selection experiments has been accumulating exponentially. There are 370 artificial ribozyme sequences and 3842 aptamer sequences in the total 4212 sequences from 423 citations in this RiboaptDB. We included general search feature, and individual feature wise search, user submission form for new data through online and also local BLAST search. Conclusion This database, besides serving as a storehouse of sequences that may have diagnostic or therapeutic utility in medicine, provides valuable information for computational and theoretical biologists. The RiboaptDB is extremely useful for garnering information about in vitro selection experiments as a whole and for better understanding the distribution of functional nucleic acids in sequence space. The database is updated regularly and is publicly available at . PMID:17118149

  6. An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries

    PubMed Central

    Wiesmayr, Anna; Fournier, Pierre; Jäschke, Andres

    2012-01-01

    Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer binding sites, which can be limiting for certain applications, like the screening of on-bead libraries. Here, we report a method to amplify and sequence on-bead RNA libraries that requires not more than five known nucleotides. A key element is the attachment of the starting nucleoside to the synthesis resin via the nucleobase, which leaves the 3′-OH group accessible to subsequent enzymatic manipulations. After split-and-mix synthesis of the oligonucleotide library and deprotection, a poly(A)-tail can be efficiently added to this free 3′-hydroxyl terminus by Escherichia coli poly(A) polymerase that serves as an anchored primer binding site for reverse transcription. The cDNA is joined to a DNA adapter by T4 DNA ligase. PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads. The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination. PMID:22298510

  7. In-situ Spatial Complementation of Aptamer-mediated Recognition Enables Live-cell Imaging of Native RNA Transcripts in Real Time.

    PubMed

    Wang, Zejun; Luo, Yao; Xie, Xiaodong; Hu, Xingjie; Song, Haiyun; Zhao, Yun; Shi, Jiye; Wang, Lihua; Glinsky, Gennadi; Chen, Nan; Lal, Ratnesh; Fan, Chunhai

    2017-10-09

    Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their functions and reveals novel mechanisms at the single-cell resolution. Here, we report the development of an aptamer-initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, nascent mRNA molecules recruited split Broccoli and brought the two fragments to the spatial proximity, which in-situ formed a fluorophore-binding site and turned on fluorescence. Significantly, we demonstrated the use of AiFC for real-time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single-cell visualization and mechanistic analyses of macromolecular interactions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

    PubMed Central

    Hong, Ka Lok; Sooter, Letha J.

    2015-01-01

    Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

  9. Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications.

    PubMed

    Hong, Ka Lok; Sooter, Letha J

    2015-01-01

    Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed.

  10. Advanced Applications of RNA Sequencing and Challenges

    PubMed Central

    Han, Yixing; Gao, Shouguo; Muegge, Kathrin; Zhang, Wei; Zhou, Bing

    2015-01-01

    Next-generation sequencing technologies have revolutionarily advanced sequence-based research with the advantages of high-throughput, high-sensitivity, and high-speed. RNA-seq is now being used widely for uncovering multiple facets of transcriptome to facilitate the biological applications. However, the large-scale data analyses associated with RNA-seq harbors challenges. In this study, we present a detailed overview of the applications of this technology and the challenges that need to be addressed, including data preprocessing, differential gene expression analysis, alternative splicing analysis, variants detection and allele-specific expression, pathway analysis, co-expression network analysis, and applications combining various experimental procedures beyond the achievements that have been made. Specifically, we discuss essential principles of computational methods that are required to meet the key challenges of the RNA-seq data analyses, development of various bioinformatics tools, challenges associated with the RNA-seq applications, and examples that represent the advances made so far in the characterization of the transcriptome. PMID:26609224

  11. Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer.

    PubMed

    Anthony, Peter C; Perez, Christian F; García-García, Cuauhtémoc; Block, Steven M

    2012-01-31

    Riboswitches are motifs in the untranslated regions (UTRs) of RNA transcripts that sense metabolite levels and modulate the expression of the corresponding genes for metabolite import, export, synthesis, or degradation. All riboswitches contain an aptamer: an RNA structure that, upon binding ligand, folds to expose or sequester regulatory elements in the adjacent sequence through alternative nucleotide pairing. The coupling between ligand binding and aptamer folding is central to the regulatory mechanisms of thiamine pyrophosphate (TPP) riboswitches and has not been fully characterized. Here, we show that TPP aptamer folding can be decomposed into ligand-independent and -dependent steps that correspond to the formation of secondary and tertiary structures, respectively. We reconstructed the full energy landscape for folding of the wild-type (WT) aptamer and measured perturbations of this landscape arising from mutations or ligand binding. We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family.

  12. Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

    PubMed Central

    Ausländer, Simon; Fuchs, David; Hürlemann, Samuel; Ausländer, David; Fussenegger, Martin

    2016-01-01

    Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes. PMID:26939886

  13. Detection Theory in Identification of RNA-DNA Sequence Differences Using RNA-Sequencing

    PubMed Central

    Toung, Jonathan M.; Lahens, Nicholas; Hogenesch, John B.; Grant, Gregory

    2014-01-01

    Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters. PMID:25396741

  14. A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats

    PubMed Central

    Curtis, Edward A; Liu, David R

    2014-01-01

    Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the “CA motif.” The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides. Several active variants of the CA motif aptamer lack the ability to form consecutive Watson-Crick base pairs in any register, while others consist of repeats containing only cytidine and adenosine residues, indicating that noncanonical interactions play important roles in its structure. The circular dichroism spectrum of the CA motif aptamer is distinct from that of A-form RNA and other major classes of nucleic acid structures. Bioinformatic searches indicate that the CA motif is absent from most archaeal and bacterial genomes, but occurs in at least 70 percent of approximately 400 eukaryotic genomes examined. These searches also uncovered several phylogenetically conserved examples of the CA motif in rodent (mouse and rat) genomes. Together, these results reveal the existence of a second class of naturally occurring GTP aptamer whose sequence requirements, like that of the G motif, are not consistent with those of a canonical secondary structure. They also indicate a new and unexpected potential biochemical activity of certain naturally occurring tandem repeats. PMID:24824832

  15. Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor

    PubMed Central

    Rowe, Aaron A.; Miller, Erin A.; Plaxco, Kevin W.

    2011-01-01

    Biosensors built using ribonucleic acid (RNA) aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degradation when deployed in clinical samples. To explore methods for protecting RNA-based biosensors from such degradation we have constructed and characterized an electrochemical, aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2′-hydroxyl groups outside of the aptamer’s antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degradation-resistant deoxyribonucleic acid (DNA) equivalents. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-molecular-weight-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clinically relevant concentrations using the original RNA-based sensor. PMID:20687587

  16. Development of Aptamer Beacons for Antemortem Diagnosis of Chronic Wasting Disease

    DTIC Science & Technology

    2007-05-01

    leaving bound species and the protein target within the membrane. Aliquots of this electrodialysed solution inside the dialysis membrane are then used...Mercey, R., et al., Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns. Arch Virol, 2006. 151(11

  17. Aptamer-assembled nanomaterials for biosensing and biomedical applications.

    PubMed

    Kong, Rong-Mei; Zhang, Xiao-Bing; Chen, Zhuo; Tan, Weihong

    2011-09-05

    Aptamers represent a class of single-stranded DNA or RNA oligonucleotides that play important roles in biosensing and biomedical applications. However, aptamers can gain more flexibility as molecular recognition tools by taking advantage of the unique chemical and physical properties provided by nanomaterials. Such aptamer-nanomaterial conjugates are having an increasing impact in the fields of biosensing, bioimaging, and therapy. The recent advances and limitations of aptamer-assembled nanomaterials in biosensing and biomedical applications are briefly introduced and discussed.

  18. De novo assembly of a bell pepper endornavirus genome sequence using RNA sequencing data.

    PubMed

    Jo, Yeonhwa; Choi, Hoseng; Cho, Won Kyong

    2015-03-19

    The genus Endornavirus is a double-stranded RNA virus that infects a wide range of hosts. In this study, we report on the de novo assembly of a bell pepper endornavirus genome sequence by RNA sequencing (RNA-Seq). Our result demonstrates the successful application of RNA-Seq to obtain a complete viral genome sequence from the transcriptome data.

  19. Functional In Vivo Delivery of Multiplexed Anti-HIV-1 siRNAs via a Chemically Synthesized Aptamer With a Sticky Bridge

    PubMed Central

    Zhou, Jiehua; Neff, C Preston; Swiderski, Piotr; Li, Haitang; Smith, David D; Aboellail, Tawfik; Remling-Mulder, Leila; Akkina, Ramesh; Rossi, John J

    2013-01-01

    One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3′ 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2′ OMe/2′ Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo. PMID:23164935

  20. Studies of RNA Sequence and Structure Using Nanopores

    PubMed Central

    Henley, Robert Y.; Carson, Spencer; Wanunu, Meni

    2016-01-01

    Nanopores are powerful single-molecule sensors with nanometer scale dimensions suitable for detection, quantification, and characterization of nucleic acids and proteins. Beyond sequencing applications, both biological and solid-state nanopores hold great promise as tools for studying the biophysical properties of RNA. In this review, we highlight selected landmark nanopore studies with regards to RNA sequencing, microRNA detection, RNA/ligand interactions, and RNA structural/conformational analysis. PMID:26970191

  1. Studies of RNA Sequence and Structure Using Nanopores.

    PubMed

    Henley, Robert Y; Carson, Spencer; Wanunu, Meni

    2016-01-01

    Nanopores are powerful single-molecule sensors with nanometer scale dimensions suitable for detection, quantification, and characterization of nucleic acids and proteins. Beyond sequencing applications, both biological and solid-state nanopores hold great promise as tools for studying the biophysical properties of RNA. In this review, we highlight selected landmark nanopore studies with regards to RNA sequencing, microRNA detection, RNA/ligand interactions, and RNA structural/conformational analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Nucleic acid aptamers: clinical applications and promising new horizons

    PubMed Central

    Ni, Xiaohua; Castanares, Mark; Mukherjee, Amarnath; Lupold, Shawn E.

    2011-01-01

    Aptamers are a special class of nucleic acid molecules that are beginning to be investigated for clinical use. These small RNA/DNA molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets; they are essentially a chemical equivalent of antibodies. Aptamers have the advantage of being highly specific, relatively small in size, and non-immunogenic. Since the discovery of aptamers in the early 1990s, great efforts have been made to make them clinically relevant for diseases like cancer, HIV, and macular degeneration. In the last two decades, many aptamers have been clinically developed as inhibitors for targets such as vascular endothelial growth factor (VEGF) and thrombin. The first aptamer based therapeutic was FDA approved in 2004 for the treatment of age-related macular degeneration and several other aptamers are currently being evaluated in clinical trials. With advances in targeted-therapy, imaging, and nanotechnology, aptamers are readily considered as potential targeting ligands because of their chemical synthesis and ease of modification for conjugation. Preclinical studies using aptamer-siRNA chimeras and aptamer targeted nanoparticle therapeutics have been very successful in mouse models of cancer and HIV. In summary aptamers are in several stages of development, from pre-clinical studies to clinical trials and even as FDA approved therapeutics. In this review, we will discuss the current state of aptamers in clinical trials as well as some promising aptamers in pre-clinical development. PMID:21838685

  3. Aptamer-Based Fluorescent Biosensors

    PubMed Central

    Wang, Rongsheng E.; Zhang, Yin; Cai, Jianfeng; Cai, Weibo; Gao, Ting

    2011-01-01

    Selected from random pools of DNA or RNA molecules through systematic evolution of ligands by exponential enrichment (SELEX), aptamers can bind to target molecules with high affinity and specificity, which makes them ideal recognition elements in the development of biosensors. To date, aptamer-based biosensors have used a wide variety of detection techniques, which are briefly summarized in this article. The focus of this review is on the development of aptamer-based fluorescent biosensors, with emphasis on their design as well as properties such as sensitivity and specificity. These biosensors can be broadly divided into two categories: those using fluorescently-labeled aptamers and others that employ label-free aptamers. Within each category, they can be further divided into “signal-on” and “signal-off” sensors. A number of these aptamer-based fluorescent biosensors have shown promising results in biological samples such as urine and serum, suggesting their potential applications in biomedical research and disease diagnostics. PMID:21838688

  4. RNA-sequencing from single nuclei.

    PubMed

    Grindberg, Rashel V; Yee-Greenbaum, Joyclyn L; McConnell, Michael J; Novotny, Mark; O'Shaughnessy, Andy L; Lambert, Georgina M; Araúzo-Bravo, Marcos J; Lee, Jun; Fishman, Max; Robbins, Gillian E; Lin, Xiaoying; Venepally, Pratap; Badger, Jonathan H; Galbraith, David W; Gage, Fred H; Lasken, Roger S

    2013-12-03

    It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.

  5. RNA-sequencing from single nuclei

    PubMed Central

    Grindberg, Rashel V.; Yee-Greenbaum, Joyclyn L.; McConnell, Michael J.; Novotny, Mark; O’Shaughnessy, Andy L.; Lambert, Georgina M.; Araúzo-Bravo, Marcos J.; Lee, Jun; Fishman, Max; Robbins, Gillian E.; Lin, Xiaoying; Venepally, Pratap; Badger, Jonathan H.; Galbraith, David W.; Gage, Fred H.; Lasken, Roger S.

    2013-01-01

    It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues. PMID:24248345

  6. NMR monitoring of the SELEX process to confirm enrichment of structured RNA.

    PubMed

    Amano, Ryo; Aoki, Kazuteru; Miyakawa, Shin; Nakamura, Yoshikazu; Kozu, Tomoko; Kawai, Gota; Sakamoto, Taiichi

    2017-03-21

    RNA aptamers are RNA molecules that bind to a target molecule with high affinity and specificity using uniquely-folded tertiary structures. RNA aptamers are selected from an RNA pool typically comprising up to 10(15) different sequences generated by iterative steps of selection and amplification known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Over several rounds of SELEX, the diversity of the RNA pool decreases and the aptamers are enriched. Hence, monitoring of the enrichment of these RNA pools is critical for the successful selection of aptamers, and several methods for monitoring them have been developed. In this study, we measured one-dimensional imino proton NMR spectra of RNA pools during SELEX. The spectrum of the initial RNA pool indicates that the RNAs adopt tertiary structures. The structural diversity of the RNA pools was shown to depend highly on the design of the primer-binding sequence. Furthermore, we demonstrate that enrichment of RNA aptamers can be monitored using NMR. The RNA pools can be recovered from the NMR tube after measurement of NMR spectra. We also can monitor target binding in the NMR tubes. Thus, we propose using NMR to monitor the enrichment of structured aptamers during the SELEX process.

  7. ABCs of DNA aptamer and related assay development.

    PubMed

    Sharma, Tarun Kumar; Bruno, John G; Dhiman, Abhijeet

    This review is intended to guide the novice in aptamer research and development to understand virtually all of the aptamer development options and currently available assay modalities. Aptamer development topics range from discussions of basic and advanced versions of Systematic Evolution of Ligands by EXponential Enrichment (SELEX) and SELEX variations involving incorporation of exotic unnatural nucleotides to expand library diversity for even greater aptamer affinity and specificity to improved next generation methods of DNA sequencing, screening and tracking aptamer development throughout the SELEX process and characterization of lead aptamer candidates. Aptamer assay development topics include descriptions of various colorimetric and fluorescent assays in microplates or on membranes including homogeneous beacon and multiplexed Fluorescence Resonance Energy Transfer (FRET) assays. Finally, a discussion of the potential for marketing successful aptamer-based assays or test kits is included.

  8. Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

    PubMed Central

    Gillum, A M; Hecker, L I; Silberklang, M; Schwartzbach, S D; RajBhandary, U L; Barnett, W E

    1977-01-01

    Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs. Images PMID:146192

  9. Short RNA indicator sequences are not completely degraded by autoclaving

    PubMed Central

    Unnithan, Veena V.; Unc, Adrian; Joe, Valerisa; Smith, Geoffrey B.

    2014-01-01

    Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification. Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses. If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving. PMID:24518856

  10. Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer

    NASA Astrophysics Data System (ADS)

    Yatime, Laure; Maasch, Christian; Hoehlig, Kai; Klussmann, Sven; Andersen, Gregers R.; Vater, Axel

    2015-04-01

    L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca2+ ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.

  11. Concentrations of individual RNA sequences in polyadenylated nuclear and cytoplasmic RNA populations of Drosophila cells.

    PubMed Central

    Biessmann, H

    1980-01-01

    Steady state concentrations of individual RNA sequences in poly(A) nuclear and cytoplasmic RNA populations of Drosophila Kc cells were determined using cloned cDNA fragments. These cDNAs represent poly(A) RNA sequences of different abundance in the cytoplasm of Kc cells, but their steady state concentrations in poly(A) hnRNA was always lower. Of ten different sequences analysed, eight showed some four-fold lower concentration in hnRNA mRNA, two were underrepresented in hnRNA relative to the others. The obvious clustering of mRNA/hnRNA ratios is discussed in relation to sequence complexity and turnover rates of these RNA populations. Images PMID:6162158

  12. Potential uses of G-quadruplex-forming aptamers.

    PubMed

    Viglasky, Viktor; Hianik, Tibor

    2013-06-01

    Guanine quadruplex (G-quadruplex) structures are one of a number of structures which are capable of adopting aptamers. G-rich DNA or RNA has an increased propensity to form quadruplex structures which have unusual biophysical and biological properties. G-rich aptamers which form G-quadruplexes have several advantages over unstructured sequences: G-quadruplexes are non-immunogenic, thermodynamically and chemically stable and they have both higher resistance to various serum nucleases and an enhanced cellular uptake. These advantages have led to a number of synthetic oligonucleotides being studied for their potential use as therapeutic agents for cancer therapy and in the treatment of various other diseases. In addition to their suitability in the fields of medicine and biotechnology, these, highly specified, aptameric G-quadruplexes also have great potential in the further development of nano-devices; e.g. basic components in microarrays, microfluidics, sandwich assays and electrochemical biosensors. This review summarizes the biophysical properties of G-quadruplexes and highlights the importance of the stability and recognition properties of aptamers. Examples of the application of aptamers in medical therapy and in biosensors are also discussed.

  13. Depletion of Ribosomal RNA Sequences from Single-Cell RNA-Sequencing Library.

    PubMed

    Fang, Nan; Akinci-Tolun, Rumeysa

    2016-07-01

    Recent advances in single-cell RNA sequencing technologies have revealed high heterogeneity of gene expression profiles in individual cells. However, most current single-cell RNA-seq methods use oligo-dT priming in the reverse transcription steps and detect only polyA-positive for more accuracy, since there are also polyA-positive non-coding RNAs transcripts, not other important RNA species, such as polyA-negative noncoding RNA. Reverse transcription using random oligos enables detection of not only the noncoding RNA species without polyA tails, but also ribosomal RNA (rRNA). rRNA comprises more than 90% of the total RNA and should be depleted from the RNA-seq library to ensure efficient usage of the sequencing capacity. Commonly used hybridization-based rRNA depletion methods can preserve noncoding RNA in the standard RNA-seq library. However, such rRNA depletion methods require high input amounts of total RNA and do not work at the single-cell level or with limited input DNA. This unit describes a novel procedure for RNA-seq library construction from single cells or a minimal amount of RNA. A thermostable duplex-specific nuclease is used in this method to effectively remove ribosomal RNA sequences following whole-transcriptome amplification and sequencing library construction. © 2016 by John Wiley & Sons, Inc.

  14. Approaching marine bioprospecting in hexacorals by RNA deep sequencing.

    PubMed

    Johansen, Steinar D; Emblem, Ase; Karlsen, Bård Ove; Okkenhaug, Siri; Hansen, Hilde; Moum, Truls; Coucheron, Dag H; Seternes, Ole Morten

    2010-07-31

    RNA deep sequencing represents a new complementary approach in marine bioprospecting. Next-generation sequencing platforms have recently been developed for de novo whole transcriptome analysis, small RNA discovery and gene expression profiling. Deep sequencing transcriptomics (sequencing the complete set of cellular transcripts at a specific stage or condition) leads to sequential identification of all expressed genes in a sample. When combined to high-throughput bioinformatics and protein synthesis, RNA deep sequencing represents a new powerful approach in gene product discovery and bioprospecting. Here we summarize recent progress in the analyses of hexacoral transcriptomes with the focus on cold-water sea anemones and related organisms.

  15. Empirical insights into the stochasticity of small RNA sequencing

    NASA Astrophysics Data System (ADS)

    Qin, Li-Xuan; Tuschl, Thomas; Singer, Samuel

    2016-04-01

    The choice of stochasticity distribution for modeling the noise distribution is a fundamental assumption for the analysis of sequencing data and consequently is critical for the accurate assessment of biological heterogeneity and differential expression. The stochasticity of RNA sequencing has been assumed to follow Poisson distributions. We collected microRNA sequencing data and observed that its stochasticity is better approximated by gamma distributions, likely because of the stochastic nature of exponential PCR amplification. We validated our findings with two independent datasets, one for microRNA sequencing and another for RNA sequencing. Motivated by the gamma distributed stochasticity, we provided a simple method for the analysis of RNA sequencing data and showed its superiority to three existing methods for differential expression analysis using three data examples of technical replicate data and biological replicate data.

  16. Empirical insights into the stochasticity of small RNA sequencing.

    PubMed

    Qin, Li-Xuan; Tuschl, Thomas; Singer, Samuel

    2016-04-07

    The choice of stochasticity distribution for modeling the noise distribution is a fundamental assumption for the analysis of sequencing data and consequently is critical for the accurate assessment of biological heterogeneity and differential expression. The stochasticity of RNA sequencing has been assumed to follow Poisson distributions. We collected microRNA sequencing data and observed that its stochasticity is better approximated by gamma distributions, likely because of the stochastic nature of exponential PCR amplification. We validated our findings with two independent datasets, one for microRNA sequencing and another for RNA sequencing. Motivated by the gamma distributed stochasticity, we provided a simple method for the analysis of RNA sequencing data and showed its superiority to three existing methods for differential expression analysis using three data examples of technical replicate data and biological replicate data.

  17. BS-RNA: An efficient mapping and annotation tool for RNA bisulfite sequencing data.

    PubMed

    Liang, Fang; Hao, Lili; Wang, Jinyue; Shi, Shuo; Xiao, Jingfa; Li, Rujiao

    2016-12-01

    Cytosine methylation is one of the most important RNA epigenetic modifications. With the development of experimental technology, scientists attach more importance to RNA cytosine methylation and find bisulfite sequencing is an effective experimental method for RNA cytosine methylation study. However, there are only a few tools can directly deal with RNA bisulfite sequencing data efficiently. Herein, we developed a specialized tool BS-RNA, which can analyze cytosine methylation of RNA based on bisulfite sequencing data and support both paired-end and single-end sequencing reads from directional bisulfite libraries. For paired-end reads, simply removing the biased positions from the 5' end may result in "dovetailing" reads, where one or both reads seem to extend past the start of the mate read. BS-RNA could map "dovetailing" reads successfully. The annotation result of BS-RNA is exported in BED (.bed) format, including locations, sequence context types (CG/CHG/CHH, H=A,T, or C), reference sequencing depths, cytosine sequencing depths, and methylation levels of covered cytosine sites on both Watson and Crick strands. BS-RNA is an efficient, specialized and highly automated mapping and annotation tool for RNA bisulfite sequencing data. It performs better than the existing program in terms of accuracy and efficiency. BS-RNA is developed by Perl language and the source code of this tool is freely available from the website: http://bs-rna.big.ac.cn. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Construction of an Aptamer-SiRNA Chimera-Modified Tissue-Engineered Blood Vessel for Cell-Type-Specific Capture and Delivery.

    PubMed

    Chen, Wen; Zeng, Wen; Sun, Jiansen; Yang, Mingcan; Li, Li; Zhou, Jingting; Wu, Yangxiao; Sun, Jun; Liu, Ge; Tang, Rui; Tan, Ju; Zhu, Chuhong

    2015-06-23

    The application of tissue-engineered blood vessels (TEBVs) is the main developmental direction of vascular replacement therapy. Due to few and/or dysfunctional endothelial progenitor cells (EPCs), it is difficult to successfully construct EPC capture TEBVs in diabetes. RNA has a potential application in cell protection and diabetes treatment, but poor specificity and low efficiency of RNA transfection in vivo limit the application of RNA. On the basis of an acellular vascular matrix, we propose an aptamer-siRNA chimera-modified TEBV that can maintain a satisfactory patency in diabetes. This TEBV consists of two parts, CD133-adenosine kinase (ADK) chimeras and a TEBV scaffold. Our results showed that CD133-ADK chimeras could selectively capture the CD133-positive cells in vivo, and then captured cells can internalize the bound chimeras to achieve RNA self-transfection. Subsequently, CD133-ADK chimeras were cut into ADK siRNA by a dicer, resulting in depletion of ADK. An ADK-deficient cell may act as a bioreactor that sustainably releases adenosine. To reduce nonspecific RNA transfection, we increased the proportion of HAuCl4 during the material preparation, through which the transfection capacity of polyethylenimine (PEI)/polyethylene glycol (PEG)-capped gold nanoparticles (PEI/PEG-AuNPs) was significantly decreased and the ability of TEBV to resist tensile and liquid shear stress was greatly enhanced. PEG and 2'-O-methyl modification was used to enhance the in vivo stability of RNA chimeras. At day 30 postgrafting, the patency rate of CD133-ADK chimera-modified TEBVs reached 90% in diabetic rats and good endothelialization was observed.

  19. RNAcentral: a comprehensive database of non-coding RNA sequences

    PubMed Central

    2017-01-01

    RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/. PMID:27794554

  20. miRBase: the microRNA sequence database.

    PubMed

    Griffiths-Jones, Sam

    2006-01-01

    The miRBase Sequence database is the primary repository for published microRNA (miRNA) sequence and annotation data. miRBase provides a user-friendly web interface for miRNA data, allowing the user to search using key words or sequences, trace links to the primary literature referencing the miRNA discoveries, analyze genomic coordinates and context, and mine relationships between miRNA sequences. miRBase also provides a confidential gene-naming service, assigning official miRNA names to novel genes before their publication. The methods outlined in this chapter describe these functions. miRBase is freely available to all at http://microrna.sanger.ac.uk/.

  1. RNAcentral: A comprehensive database of non-coding RNA sequences

    DOE PAGES

    Williams, Kelly Porter; Lau, Britney Yan

    2016-10-28

    RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. Furthermore, the website has been subject to continuous improvements focusing on text and sequence similaritymore » searches as well as genome browsing functionality.« less

  2. RNAcentral: A comprehensive database of non-coding RNA sequences

    SciTech Connect

    Williams, Kelly Porter; Lau, Britney Yan

    2016-10-28

    RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. Furthermore, the website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality.

  3. Novel Approach to Analyzing MFE of Noncoding RNA Sequences

    PubMed Central

    George, Tina P.; Thomas, Tessamma

    2016-01-01

    Genomic studies have become noncoding RNA (ncRNA) centric after the study of different genomes provided enormous information on ncRNA over the past decades. The function of ncRNA is decided by its secondary structure, and across organisms, the secondary structure is more conserved than the sequence itself. In this study, the optimal secondary structure or the minimum free energy (MFE) structure of ncRNA was found based on the thermodynamic nearest neighbor model. MFE of over 2600 ncRNA sequences was analyzed in view of its signal properties. Mathematical models linking MFE to the signal properties were found for each of the four classes of ncRNA analyzed. MFE values computed with the proposed models were in concordance with those obtained with the standard web servers. A total of 95% of the sequences analyzed had deviation of MFE values within ±15% relative to those obtained from standard web servers. PMID:27695341

  4. Aptamers as Valuable Molecular Tools in Neurosciences.

    PubMed

    Wolter, Olga; Mayer, Günter

    2017-03-08

    Aptamers are short nucleic acids that interact with a variety of targets with high affinity and specificity. They have been shown to inhibit biological functions of cognate target proteins, and they are identifiable by an in vitro selection process, also termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). Being nucleic acids, aptamers can be synthesized chemically or enzymatically. The latter renders RNA aptamers compatible with the cell's own transcription machinery and, thus, expressable inside cells. The synthesis of aptamers by chemical approaches opens up the possibility of producing aptamers on a large scale and enables a straightforward access to introduce modifications in a site-specific manner (e.g., fluorophores or photo-labile groups). These characteristics make aptamers broadly applicable (e.g., as an analytical, diagnostic, or separation tool). In this TechSight, we provide a brief overview on aptamer technology and the potential of aptamers as valuable research tools in neurosciences. Copyright © 2017 the authors 0270-6474/17/372517-07$15.00/0.

  5. A DNA Bubble-Mediated Gene Regulation System Based on Thrombin-Bound DNA Aptamers.

    PubMed

    Wang, Jing; Yang, Le; Cui, Xun; Zhang, Zhe; Dong, Lichun; Guan, Ningzi

    2017-05-19

    We describe here a novel approach to enhance the transcription of a target gene in cell-free systems by symmetrically introducing duplex aptamers upstream to a T7 promoter in both the sense and antisense strands of double-stranded plasmids, which leads to the formation of a DNA bubble due to the none-complementary state of the ssDNA region harboring the aptamer sequences. With the presence of thrombins, the DNA bubble would be enlarged due to the binding of aptamers with thrombins. Consequently, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNA polymerases, and the separation efficiency of the unwinding region can also be significantly improved, leading to the enhanced expression of the target gene at the transcriptional level. The effectiveness of the proposed gene regulation system was demonstrated by enhancing the expression of gfp and ecaA genes in cell-free systems.

  6. Aptamer-based technology for food analysis.

    PubMed

    Liu, Xiaofei; Zhang, Xuewu

    2015-01-01

    Aptamers are short and functional single-stranded oligonucleotide sequences selected from systematic evolution of ligands by exponential enrichment (SELEX) process, which have the capacity to recognize various classes of target molecules with high affinity and specificity. Various analytical aptamers acquired by SELEX are widely used in many research fields, such as medicine, biology, and chemistry. However, the application of this innovative and emerging technology to food safety is just in infant stage. Food safety plays a very important role in our daily lives because varieties of poisonous and harmful substances in food affect human health. Aptamer technique is promising, which can overcome many disadvantages of existing detection methods in food safety, such as long detection time, low sensitivity, difficult, and expensive antibody preparation. This review provides an overview of various aptamer screening technologies and summarizes the recent applications of aptamers in food safety, and future prospects are also discussed.

  7. DSAP: deep-sequencing small RNA analysis pipeline.

    PubMed

    Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

    2010-07-01

    DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

  8. Accurate multiple sequence-structure alignment of RNA sequences using combinatorial optimization.

    PubMed

    Bauer, Markus; Klau, Gunnar W; Reinert, Knut

    2007-07-27

    The discovery of functional non-coding RNA sequences has led to an increasing interest in algorithms related to RNA analysis. Traditional sequence alignment algorithms, however, fail at computing reliable alignments of low-homology RNA sequences. The spatial conformation of RNA sequences largely determines their function, and therefore RNA alignment algorithms have to take structural information into account. We present a graph-based representation for sequence-structure alignments, which we model as an integer linear program (ILP). We sketch how we compute an optimal or near-optimal solution to the ILP using methods from combinatorial optimization, and present results on a recently published benchmark set for RNA alignments. The implementation of our algorithm yields better alignments in terms of two published scores than the other programs that we tested: This is especially the case with an increasing number of input sequences. Our program LARA is freely available for academic purposes from http://www.planet-lisa.net.

  9. Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

    PubMed Central

    Perez-Gonzalez, Cibran; Lafontaine, Daniel A.; Penedo, J. Carlos

    2016-01-01

    In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

  10. Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

    NASA Astrophysics Data System (ADS)

    Perez-Gonzalez, Cibran; Lafontaine, Daniel; Penedo, J.

    2016-08-01

    In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

  11. RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences.

    PubMed

    Derksen, Merel; Mertens, Vicky; Pruijn, Ger J M

    2015-11-09

    The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.

  12. RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences

    PubMed Central

    Derksen, Merel; Mertens, Vicky; Pruijn, Ger J.M.

    2015-01-01

    The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels. PMID:26569326

  13. Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

    PubMed Central

    Matranga, Christian B.; Gladden-Young, Adrianne; Qu, James; Winnicki, Sarah; Nosamiefan, Dolo; Levin, Joshua Z.; Sabeti, Pardis C.

    2016-01-01

    Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA — including poly(rA) carrier and ribosomal RNA — from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based 'tagmentation' step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies. PMID:27403729

  14. Mapping the affinity landscape of Thrombin-binding aptamers on 2'F-ANA/DNA chimeric G-Quadruplex microarrays.

    PubMed

    Lietard, Jory; Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos; Somoza, Mark M; Damha, Masad J

    2017-01-18

    In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2'F-ANA and 2'F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2'F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2'F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2'F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2'F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

  15. Noncoding RNA gene detection using comparative sequence analysis

    PubMed Central

    Rivas, Elena; Eddy, Sean R

    2001-01-01

    Background Noncoding RNA genes produce transcripts that exert their function without ever producing proteins. Noncoding RNA gene sequences do not have strong statistical signals, unlike protein coding genes. A reliable general purpose computational genefinder for noncoding RNA genes has been elusive. Results We describe a comparative sequence analysis algorithm for detecting novel structural RNA genes. The key idea is to test the pattern of substitutions observed in a pairwise alignment of two homologous sequences. A conserved coding region tends to show a pattern of synonymous substitutions, whereas a conserved structural RNA tends to show a pattern of compensatory mutations consistent with some base-paired secondary structure. We formalize this intuition using three probabilistic "pair-grammars": a pair stochastic context free grammar modeling alignments constrained by structural RNA evolution, a pair hidden Markov model modeling alignments constrained by coding sequence evolution, and a pair hidden Markov model modeling a null hypothesis of position-independent evolution. Given an input pairwise sequence alignment (e.g. from a BLASTN comparison of two related genomes) we classify the alignment into the coding, RNA, or null class according to the posterior probability of each class. Conclusions We have implemented this approach as a program, QRNA, which we consider to be a prototype structural noncoding RNA genefinder. Tests suggest that this approach detects noncoding RNA genes with a fair degree of reliability. PMID:11801179

  16. Simulations Using Random-Generated DNA and RNA Sequences

    ERIC Educational Resources Information Center

    Bryce, C. F. A.

    1977-01-01

    Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students use these sequences to predict complementary sequences and translational products, evaluate base compositions, determine frequencies of particular triplet codons, and suggest possible secondary structures.…

  17. Aptamer-functionalized peptide H3CR5C as a novel nanovehicle for codelivery of fasudil and miRNA-195 targeting hepatocellular carcinoma

    PubMed Central

    Liu, Ying; Wu, Xin; Gao, Yuan; Zhang, Jigang; Zhang, Dandan; Gu, Shengying; Zhu, Guanhua; Liu, Gaolin; Li, Xiaoyu

    2016-01-01

    Liver cancer is the fifth most commonly diagnosed malignancy, of which hepatocellular carcinoma (HCC) represents the dominating histological subtype. Antiangiogenic therapy aimed at vascular endothelial growth factor (VEGF) has shown promising but deficient clinical prospects on account of vasculogenic mimicry, a highly patterned vascular channel distinguished from the endothelium-dependent blood vessel, which may function as blood supply networks occurring in aggressive tumors including HCC. In this study, we used a new cationic peptide, disulfide cross-linked stearylated polyarginine peptide modified with histidine (H3R5), as a reducible vector, cell penetrating peptide-modified aptamer (ST21) with specific binding to HCC cells to conjugate to peptide H3R5 as the targeting probe, miRNA-195 (miR195) as a powerful gene drug to inhibit VEGF, and fasudil to suppress vasculogenic mimicry by blocking ROCK2, all of which were simultaneously encapsulated in the same nanoparticles. Fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient and miR195 was condensed through electrostatic interaction. ST21-H3R5-polyethylene glycol (PEG) exhibited excellent loading capacities for both fasudil and miR195 with adjustable dosing ratios. Western blot analysis showed that FasudilST21-H3R5-PEGmiR195 had strong silencing activity of ROCK2 and VEGF, as compared with FasudilH3R5-PEGmiR195. In vitro and in vivo experiments confirmed that ST21-modified nanoparticles showed significantly higher cellular uptake and therapeutic efficacy in tumor cells or tumor tissues than the unmodified counterparts. These findings suggest that aptamer-conjugated peptide holds great promise for delivering chemical drugs and gene drugs simultaneously to overcome HCC. PMID:27574422

  18. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

    PubMed

    Zhang, Rui; Li, Xin; Ramaswami, Gokul; Smith, Kevin S; Turecki, Gustavo; Montgomery, Stephen B; Li, Jin Billy

    2014-01-01

    We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

  19. RNAcentral: an international database of ncRNA sequences

    DOE PAGES

    Williams, Kelly Porter

    2014-10-28

    The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

  20. RNAcentral: an international database of ncRNA sequences

    SciTech Connect

    Williams, Kelly Porter

    2014-10-28

    The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

  1. RNAcentral: an international database of ncRNA sequences

    PubMed Central

    2015-01-01

    The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species. PMID:25352543

  2. Nucleic acid aptamers as stabilizers of proteins: the stability of tetanus toxoid.

    PubMed

    Jain, Nishant Kumar; Jetani, Hardik C; Roy, Ipsita

    2013-07-01

    Exposure of tetanus toxoid to moisture leads to its aggregation and reduction of potency. The aim of this work was to use SELEX (systematic evolution of ligands by exponential enrichment) protocol and select aptamers which recognize tetanus toxoid (Mr ~150 kDa) with high affinity. Colyophilized preparations of tetanus toxoid and specific aptamers were encapsulated in PLGA microspheres and sustained release of the antigen was observed up to 55 days using different techniques. The total protein released was between 40-55% (24-45% residual antigenicity) in the presence of the aptamers as compared to 25% (11% residual antigenicity) for the antigen alone. We show that instead of inhibiting absorption of moisture, the aptamers blocked the protein unfolding upon absorption of moisture, inhibiting the initiation of aggregation. When exposed to accelerated storage conditions, some of the RNA sequences were able to inhibit moisture-induced aggregation in vitro and retain antigenicity of tetanus toxoid. Nucleic acid aptamers represent a novel class of protein stabilizers which stabilize the protein by interacting directly with it. This mechanism is unlike that of small molecules which alter the medium properties and hence depend on the stress condition a protein is exposed to.

  3. Selective inhibitory DNA aptamers of the human RNase H1.

    PubMed

    Pileur, Frédéric; Andreola, Marie-Line; Dausse, Eric; Michel, Justine; Moreau, Serge; Yamada, Hirofumi; Gaidamakov, Sergei A; Crouch, Robert J; Toulmé, Jean-Jacques; Cazenave, Christian

    2003-10-01

    Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.

  4. Efficient delivery of micro RNA to bone-metastatic prostate tumors by using aptamer-conjugated atelocollagen in vitro and in vivo.

    PubMed

    Hao, Zhao; Fan, Wei; Hao, Jian; Wu, Xin; Zeng, Guo Qing; Zhang, Li Juan; Nie, Sui Feng; Wang, Xu Dong

    2016-01-01

    Bone is the primary site of skeletal metastasis in prostate cancer (PCa). Atelocollagen (ATE)-mediated siRNA delivery system can be used to silence endogenous genes involved in PCa metastatic tumor cell growth. However, we hope that the delivery system can target PCa cells to reduce damage to the bone tissue and improve the therapeutic effect. RNA aptamer (APT) A10-3.2 has been used as a ligand to target PCa cells that express prostate-specific membrane antigen (PSMA). APT was investigated as a PSMA-targeting ligand in the design of an ATE-based microRNA (miRNA; miR-15a and miR-16-1) vector to PCa bone metastasis. To observe the targeted delivery and transfection efficiency of ATE-APT in PSMA-overexpressing cells, luciferase activity and biodistribution of nanoparticles in Balb/c mice was analyzed. The anticancer effect of nanoparticles in vivo was investigated using the survival times of human PCa bone metastasis mice model. Luciferase assays of pGL-3 expression against PC3 (PSMA(-)) and LNCaP (PSMA(+)) cells showed that the transfection efficiency of the synthesized DNA/ATE-APT complex was higher than that of the DNA/ATE complex. The anticancer efficacy of miRNA/ATE-APT was superior to those of other treatments in vivo. This PSMA-targeted system may prove useful in widening the therapeutic window and allow for selective killing of PCa cells in bone metastatic foci.

  5. Nucleotide sequence of a human tRNA gene heterocluster

    SciTech Connect

    Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

    1986-05-01

    Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both (3'-/sup 32/P)-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these ..gamma..-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues.

  6. Compilation of 5S rRNA and 5S rRNA gene sequences

    PubMed Central

    Specht, Thomas; Wolters, Jörn; Erdmann, Volker A.

    1990-01-01

    The BERLIN RNA DATABANK as of Dezember 31, 1989, contains a total of 667 sequences of 5S rRNAs or their genes, which is an increase of 114 new sequence entries over the last compilation (1). It covers sequences from 44 archaebacteria, 267 eubacteria, 20 plastids, 6 mitochondria, 319 eukaryotes and 11 eukaryotic pseudogenes. The hardcopy shows only the list (Table 1) of those organisms whose sequences have been determined. The BERLIN RNA DATABANK uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information. PMID:1692116

  7. Translating RNA sequencing into clinical diagnostics: opportunities and challenges.

    PubMed

    Byron, Sara A; Van Keuren-Jensen, Kendall R; Engelthaler, David M; Carpten, John D; Craig, David W

    2016-05-01

    With the emergence of RNA sequencing (RNA-seq) technologies, RNA-based biomolecules hold expanded promise for their diagnostic, prognostic and therapeutic applicability in various diseases, including cancers and infectious diseases. Detection of gene fusions and differential expression of known disease-causing transcripts by RNA-seq represent some of the most immediate opportunities. However, it is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease. Ongoing efforts towards the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.

  8. A Detailed Protocol for Subcellular RNA Sequencing (subRNA-seq).

    PubMed

    Mayer, Andreas; Churchman, L Stirling

    2017-10-02

    In eukaryotic cells, RNAs at various maturation and processing levels are distributed across cellular compartments. The standard approach to determine transcript abundance and identity in vivo is RNA sequencing (RNA-seq). RNA-seq relies on RNA isolation from whole-cell lysates and thus mainly captures fully processed, stable, and more abundant cytoplasmic RNAs over nascent, unstable, and nuclear RNAs. Here, we provide a step-by-step protocol for subcellular RNA-seq (subRNA-seq). subRNA-seq allows the quantitative measurement of RNA polymerase II-generated RNAs from the chromatin, nucleoplasm, and cytoplasm of mammalian cells. This approach relies on cell fractionation prior to RNA isolation and sequencing library preparation. High-throughput sequencing of the subcellular RNAs can then be used to reveal the identity, abundance, and subcellular distribution of transcripts, thus providing insights into RNA processing and maturation. Deep sequencing of the chromatin-associated RNAs further offers the opportunity to study nascent RNAs. Subcellular RNA-seq libraries are obtained within 5 days. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  9. Tuning RNA Flexibility with Helix Length and Junction Sequence.

    PubMed

    Sutton, Julie L; Pollack, Lois

    2015-12-15

    The increasing awareness of RNA's central role in biology calls for a new understanding of how RNAs, like proteins, recognize biological partners. Because RNA is inherently flexible, it assumes a variety of conformations. This conformational flexibility can be a critical aspect of how RNA attracts and binds molecular partners. Structurally, RNA consists of rigid basepaired duplexes, separated by flexible non-basepaired regions. Here, using an RNA system consisting of two short helices, connected by a single-stranded (non-basepaired) junction, we explore the role of helix length and junction sequence in determining the range of conformations available to a model RNA. Single-molecule Förster resonance energy transfer reports on the RNA conformation as a function of either mono- or divalent ion concentration. Electrostatic repulsion between helices dominates at low salt concentration, whereas junction sequence effects determine the conformations at high salt concentration. Near physiological salt concentrations, RNA conformation is sensitive to both helix length and junction sequence, suggesting a means for sensitively tuning RNA conformations. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

    NASA Astrophysics Data System (ADS)

    Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

    2017-04-01

    Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

  11. Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection.

    PubMed

    Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

    2017-04-03

    Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5' and 3' termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

  12. Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

    PubMed Central

    Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

    2017-01-01

    Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5′ and 3′ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses. PMID:28367958

  13. FLDS: A Comprehensive dsRNA Sequencing Method for Intracellular RNA Virus Surveillance

    PubMed Central

    Urayama, Syun-ichi; Takaki, Yoshihiro; Nunoura, Takuro

    2016-01-01

    Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein constructed an effective metagenomic method for RNA viruses by targeting long double-stranded (ds)RNA in cellular organisms, which is a hallmark of infection, or the replication of dsRNA and single-stranded (ss)RNA viruses, except for retroviruses. This novel dsRNA targeting metagenomic method is characterized by an extremely high recovery rate of viral RNA sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting RNA viruses. This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. Our approach will be a powerful tool for cataloging RNA viruses associated with organisms of interest. PMID:26877136

  14. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  15. The chemical structure of DNA sequence signals for RNA transcription

    NASA Technical Reports Server (NTRS)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  16. TARDIS, a targeted RNA directional sequencing method for rare RNA discovery.

    PubMed

    Portal, Maximiliano M; Pavet, Valeria; Erb, Cathie; Gronemeyer, Hinrich

    2015-12-01

    High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap-based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes ∼10 d to implement.

  17. Aptamer-mediated cancer gene therapy.

    PubMed

    Xiang, Dongxi; Shigdar, Sarah; Qiao, Greg; Zhou, Shu-Feng; Li, Yong; Wei, Ming Q; Qiao, Liang; Shamaileh, Hadi Al; Zhu, Yimin; Zheng, Conglong; Pu, Chunwen; Duan, Wei

    2015-01-01

    Cancer as a genetic disorder is one of the leading causes of death worldwide. Conventional anticancer options such as chemo- and/or radio-therapy have their own drawbacks and could not provide a cure in most cases at present. More effective therapeutic strategies with less side effects are urgently needed. Aptamers, also known as chemical antibodies, are single strand DNA or RNA molecules that can bind to their target molecules with high affinity and specificity. Such site-specific binding ability of aptamers facilitates the delivery and interaction of exogenous nucleic acids with diseased genes. Thus, aptamer-guided gene therapy has emerged as a promising anticancer strategy in addition to the classic treatment regimen. Aptamers can directly deliver anti-cancer nucleic acids, e.g. small interfering RNA, micro RNA, antimicroRNA and small hairpin RNA, to cancer cells or function as a targeting ligand to guide nanoparticles containing therapeutic nucleic acids. This review focuses on recent progress in aptamer-mediated gene therapy for the treatment of hepatocellular carcinoma and other types of cancers, shedding light on the potential of this novel approach of targeted cancer gene therapy.

  18. Isolation of peptide aptamers that inhibit intracellular processes

    PubMed Central

    Blum, Jonathan H.; Dove, Simon L.; Hochschild, Ann; Mekalanos, John J.

    2000-01-01

    We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible PBAD promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolated by this method resulted in a ThyA− phenotype that was suppressed by simultaneous overexpression of ThyA. Two-hybrid analysis showed that this aptamer is likely to interact with ThyA in vivo. The library also was screened for aptamers that inhibited growth of bacteria expressing them, and five such aptamers were characterized. Four aptamers were bacteriostatic when expressed, whereas one showed a bactericidal effect. Introduction of translational stop codons into various aptamers blocked their activity, suggesting that their biological effects were likely to be due to protein aptamer rather than RNA. Combinatorial aptamers provide a new genetic and biochemical tool for identifying targets for antibacterial drug development. PMID:10688899

  19. RNA sequencing analysis of the developing chicken retina

    PubMed Central

    Langouet-Astrie, Christophe J.; Meinsen, Annamarie L.; Grunwald, Emily R.; Turner, Stephen D.; Enke, Raymond A.

    2016-01-01

    RNA sequencing transcriptome analysis using massively parallel next generation sequencing technology provides the capability to understand global changes in gene expression throughout a range of tissue samples. Development of the vertebrate retina requires complex temporal orchestration of transcriptional activation and repression. The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology and retinogenesis. Existing retinal transcriptome projects have been critical to the vision research community for studying aspects of murine and human retinogenesis, however, there are currently no publicly available data sets describing the developing chicken retinal transcriptome. Here we used Illumina RNA sequencing (RNA-seq) analysis to characterize the mRNA transcriptome of the developing chicken retina in an effort to identify genes critical for retinal development in this important model organism. These data will be valuable to the vision research community for characterizing global changes in gene expression between ocular tissues and critical developmental time points during retinogenesis in the chicken retina. PMID:27996968

  20. Tuning RNA Flexibility with Helix Length and Junction Sequence

    PubMed Central

    Sutton, Julie L.; Pollack, Lois

    2015-01-01

    The increasing awareness of RNA’s central role in biology calls for a new understanding of how RNAs, like proteins, recognize biological partners. Because RNA is inherently flexible, it assumes a variety of conformations. This conformational flexibility can be a critical aspect of how RNA attracts and binds molecular partners. Structurally, RNA consists of rigid basepaired duplexes, separated by flexible non-basepaired regions. Here, using an RNA system consisting of two short helices, connected by a single-stranded (non-basepaired) junction, we explore the role of helix length and junction sequence in determining the range of conformations available to a model RNA. Single-molecule Förster resonance energy transfer reports on the RNA conformation as a function of either mono- or divalent ion concentration. Electrostatic repulsion between helices dominates at low salt concentration, whereas junction sequence effects determine the conformations at high salt concentration. Near physiological salt concentrations, RNA conformation is sensitive to both helix length and junction sequence, suggesting a means for sensitively tuning RNA conformations. PMID:26682821

  1. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    PubMed

    Mitchell, Jennifer A; Clay, Ieuan; Umlauf, David; Chen, Chih-Yu; Moir, Catherine A; Eskiw, Christopher H; Schoenfelder, Stefan; Chakalova, Lyubomira; Nagano, Takashi; Fraser, Peter

    2012-01-01

    In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  2. Identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity.

    PubMed

    Cui, Wei; Liu, Jiaojiao; Su, Donghua; Hu, Danyang; Hou, Shuai; Hu, Tongnan; Yang, Jiyong; Luo, Yanping; Xi, Qing; Chu, Bingfeng; Wang, Chenglong

    2016-06-01

    Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is considered to be a major etiological factor for dental caries. In this study, plaques from dental enamel surfaces of caries-active and caries-free individuals were obtained and cultivated for S. mutans isolation. Morphology examination, biochemical characterization, and polymerase chain reaction were performed to identify S. mutans The cariogenicity of S. mutans strains isolated from clinical specimens was evaluated by testing the acidogenicity, aciduricity, extracellular polysaccharide production, and adhesion ability of the bacteria. Finally, subtractive SELEX (systematic evolution of ligands by exponential enrichment) technology targeting whole intact cells was used to screen for ssDNA aptamers specific to the strains with high cariogenicity. After nine rounds of subtractive SELEX, sufficient pool enrichment was achieved as shown by radioactive isotope analysis. The enriched pool was cloned and sequenced randomly, followed by MEME online and RNA structure software analysis of the sequences. Results from the flow cytometry indicated that aptamers H1, H16, H4, L1, L10, and H19 could discriminate highly cariogenic S. mutans strains from poorly cariogenic strains. Among these, Aptamer H19 had the strongest binding capacity with cariogenic S. mutans strains with a dissociation constant of 69.45 ± 38.53 nM. In conclusion, ssDNA aptamers specific to highly cariogenic clinical S. mutans strains were successfully obtained. These ssDNA aptamers might be used for the early diagnosis and treatment of dental caries.

  3. Identifying novel sequence variants of RNA 3D motifs

    PubMed Central

    Zirbel, Craig L.; Roll, James; Sweeney, Blake A.; Petrov, Anton I.; Pirrung, Meg; Leontis, Neocles B.

    2015-01-01

    Predicting RNA 3D structure from sequence is a major challenge in biophysics. An important sub-goal is accurately identifying recurrent 3D motifs from RNA internal and hairpin loop sequences extracted from secondary structure (2D) diagrams. We have developed and validated new probabilistic models for 3D motif sequences based on hybrid Stochastic Context-Free Grammars and Markov Random Fields (SCFG/MRF). The SCFG/MRF models are constructed using atomic-resolution RNA 3D structures. To parameterize each model, we use all instances of each motif found in the RNA 3D Motif Atlas and annotations of pairwise nucleotide interactions generated by the FR3D software. Isostericity relations between non-Watson–Crick basepairs are used in scoring sequence variants. SCFG techniques model nested pairs and insertions, while MRF ideas handle crossing interactions and base triples. We use test sets of randomly-generated sequences to set acceptance and rejection thresholds for each motif group and thus control the false positive rate. Validation was carried out by comparing results for four motif groups to RMDetect. The software developed for sequence scoring (JAR3D) is structured to automatically incorporate new motifs as they accumulate in the RNA 3D Motif Atlas when new structures are solved and is available free for download. PMID:26130723

  4. IVT-seq reveals extreme bias in RNA sequencing

    PubMed Central

    2014-01-01

    Background RNA-seq is a powerful technique for identifying and quantifying transcription and splicing events, both known and novel. However, given its recent development and the proliferation of library construction methods, understanding the bias it introduces is incomplete but critical to realizing its value. Results We present a method, in vitro transcription sequencing (IVT-seq), for identifying and assessing the technical biases in RNA-seq library generation and sequencing at scale. We created a pool of over 1,000 in vitro transcribed RNAs from a full-length human cDNA library and sequenced them with polyA and total RNA-seq, the most common protocols. Because each cDNA is full length, and we show in vitro transcription is incredibly processive, each base in each transcript should be equivalently represented. However, with common RNA-seq applications and platforms, we find 50% of transcripts have more than two-fold and 10% have more than 10-fold differences in within-transcript sequence coverage. We also find greater than 6% of transcripts have regions of dramatically unpredictable sequencing coverage between samples, confounding accurate determination of their expression. We use a combination of experimental and computational approaches to show rRNA depletion is responsible for the most significant variability in coverage, and several sequence determinants also strongly influence representation. Conclusions These results show the utility of IVT-seq for promoting better understanding of bias introduced by RNA-seq. We find rRNA depletion is responsible for substantial, unappreciated biases in coverage introduced during library preparation. These biases suggest exon-level expression analysis may be inadvisable, and we recommend caution when interpreting RNA-seq results. PMID:24981968

  5. Library preparation for highly accurate population sequencing of RNA viruses

    PubMed Central

    Acevedo, Ashley; Andino, Raul

    2015-01-01

    Circular resequencing (CirSeq) is a novel technique for efficient and highly accurate next-generation sequencing (NGS) of RNA virus populations. The foundation of this approach is the circularization of fragmented viral RNAs, which are then redundantly encoded into tandem repeats by ‘rolling-circle’ reverse transcription. When sequenced, the redundant copies within each read are aligned to derive a consensus sequence of their initial RNA template. This process yields sequencing data with error rates far below the variant frequencies observed for RNA viruses, facilitating ultra-rare variant detection and accurate measurement of low-frequency variants. Although library preparation takes ~5 d, the high-quality data generated by CirSeq simplifies downstream data analysis, making this approach substantially more tractable for experimentalists. PMID:24967624

  6. Recent Advances in Aptamers Targeting Immune System.

    PubMed

    Hu, Piao-Ping

    2017-02-01

    The immune system plays important role in protecting the organism by recognizing non-self molecules from pathogen such as bacteria, parasitic worms, and viruses. When the balance of the host defense system is disturbed, immunodeficiency, autoimmunity, and inflammation occur. Nucleic acid aptamers are short single-stranded DNA (ssDNA) or RNA ligands that interact with complementary molecules with high specificity and affinity. Aptamers that target the molecules involved in immune system to modulate their function have great potential to be explored as new diagnostic and therapeutic agents for immune disorders. This review summarizes recent advances in the development of aptamers targeting immune system. The selection of aptamers with superior chemical and biological characteristics will facilitate their application in the diagnosis and treatment of immune disorders.

  7. Current progress on aptamer-targeted oligonucleotide therapeutics

    PubMed Central

    Dassie, Justin P; Giangrande, Paloma H

    2014-01-01

    Exploiting the power of the RNAi pathway through the use of therapeutic siRNA drugs has remarkable potential for treating a vast array of human disease conditions. However, difficulties in delivery of these and similar nucleic acid-based pharmacological agents to appropriate organs or tissues, remains a major impediment to their broad clinical application. Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery vehicles for therapeutic oligonucleotides, including siRNAs. In this review, we summarize recent attractive developments in creatively employing cell-internalizing aptamers to deliver therapeutic oligonucleotides (e.g., siRNAs, miRNAs, anti-miRs and antisense oligos) to target cells. We also discuss advancements in aptamer-siRNA chimera technology, as well as, aptamer-functionalized nanoparticles for siRNA delivery. In addition, the challenges and future prospects of aptamer-targeted oligonucleotide drugs for clinical translation are further highlighted. PMID:24304250

  8. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications

    PubMed Central

    Herzog, Michel; Maroteaux, Luc

    1986-01-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage. PMID:16578795

  9. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications.

    PubMed

    Herzog, M; Maroteaux, L

    1986-11-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage.

  10. Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish

    USDA-ARS?s Scientific Manuscript database

    RNA sequencing (RNA-Seq) is becoming the standard for transcriptome analysis. Removal of contaminating ribosomal RNA (rRNA) is a priority in the preparation of libraries suitable for sequencing. rRNAs are commonly removed from total RNA via either mRNA selection or rRNA depletion. These methods have...

  11. Nucleotide sequence of papaya mosaic virus RNA.

    PubMed

    Sit, T L; Abouhaidar, M G; Holy, S

    1989-09-01

    The RNA genome of papaya mosaic virus is 6656 nucleotides long [excluding the poly(A) tail] with six open reading frames (ORFs) more than 200 nucleotides long. The four nearest the 5' end each overlap with adjacent ORFs and could code for proteins with Mr 176307, 26248, 11949 and 7224 (ORFs 1 to 4). The fifth ORF produces the capsid protein of Mr 23043 and the sixth ORF, located completely within ORF1, could code for a protein with Mr 14113. The translation products of ORFs 1 to 3 show strong similarity with those of other potexviruses but the ORF 4 protein has only limited similarity with the other potexvirus ORF 4 proteins of 7K to 11K.

  12. Boron-containing aptamers to ATP

    PubMed Central

    Lato, Susan M.; Ozerova, Nicole D. S.; He, Kaizhang; Sergueeva, Zinaida; Shaw, Barbara Ramsay; Burke, Donald H.

    2002-01-01

    Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5′-(α-P-borano) triphosphate (bG) for GTP or uridine 5′-(α-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the ζ-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some ζ-fold aptamers, while the bG selection yielded none of this type. Non-ζ aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT. PMID:11884639

  13. Discovering New Biology through Sequencing of RNA1

    PubMed Central

    Weber, Andreas P.M.

    2015-01-01

    Sequencing of RNA (RNA-Seq) was invented approximately 1 decade ago and has since revolutionized biological research. This update provides a brief historic perspective on the development of RNA-Seq and then focuses on the application of RNA-Seq in qualitative and quantitative analyses of transcriptomes. Particular emphasis is given to aspects of data analysis. Since the wet-lab and data analysis aspects of RNA-Seq are still rapidly evolving and novel applications are continuously reported, a printed review will be rapidly outdated and can only serve to provide some examples and general guidelines for planning and conducting RNA-Seq studies. Hence, selected references to frequently update online resources are given. PMID:26353759

  14. Cell growth inhibition by sequence-specific RNA minihelices.

    PubMed

    Hipps, D; Schimmel, P

    1995-08-15

    RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.

  15. Nucleotide sequences important for translation initiation of enterovirus RNA.

    PubMed Central

    Iizuka, N; Yonekawa, H; Nomoto, A

    1991-01-01

    An infectious cDNA clone was constructed from the genome of coxsackievirus B1 strain. A number of RNA transcripts that have mutations in the 5' noncoding region were synthesized in vitro from the modified cDNA clones and examined for their abilities to act as mRNAs in a cell-free translation system prepared from HeLa S3 cells. RNAs that lack nucleotide sequences at positions 568 to 726 and 565 to 726 were found to be less efficient and inactive mRNAs, respectively. To understand the biological significance of this region of RNA, small deletions and point mutations were introduced in the nucleotide sequence between positions 538 and 601. Except for a nucleotide substitution at 592 (U----C) within the 7-base conserved sequence, mutations introduced in the sequence downstream of position 568 did not affect much, if any, of the ability of RNA to act as mRNA. Except for a point mutation at 558 (C----U), mutations upstream of position 567 appeared to inactivate the mRNA. In the upstream region, a sequence consisting of 21 nucleotides at positions 546 to 566 is perfectly conserved in the 5' noncoding regions of enterovirus and rhinovirus genomes. These results suggest that the 7-base conserved sequence functions to maintain the efficiency of translation initiation and that the nucleotide sequence upstream of position 567, including the 21-base conserved sequence, plays essential roles in translation initiation. A deletion mutant whose genome lacks the nucleotide sequence at positions 568 to 726 showed a small-plaque phenotype and less virulence against suckling mice than the wild-type virus. Thus, reduction of the efficiency of translation initiation may result in the construction of enteroviruses with the lower-virulence phenotype. Images PMID:1651409

  16. Aptamer-based liposomes improve specific drug loading and release.

    PubMed

    Plourde, Kevin; Derbali, Rabeb Mouna; Desrosiers, Arnaud; Dubath, Céline; Vallée-Bélisle, Alexis; Leblond, Jeanne

    2017-04-10

    Aptamer technology has shown much promise in cancer therapeutics for its targeting abilities. However, its potential to improve drug loading and release from nanocarriers has not been thoroughly explored. In this study, we employed drug-binding aptamers to actively load drugs into liposomes. We designed a series of DNA aptamer sequences specific to doxorubicin, displaying multiple binding sites and various binding affinities. The binding ability of aptamers was preserved when incorporated into cationic liposomes, binding up to 15equivalents of doxorubicin per aptamer, therefore drawing the drug into liposomes. Optimization of the charge and drug/aptamer ratios resulted in ≥80% encapsulation efficiency of doxorubicin, ten times higher than classical passively-encapsulating liposomal formulations and similar to a pH-gradient active loading strategy. In addition, kinetic release profiles and cytotoxicity assay on HeLa cells demonstrated that the release and therapeutic efficacy of liposomal doxorubicin could be controlled by the aptamer's structure. Our results suggest that the aptamer exhibiting a specific intermediate affinity is the best suited to achieve high drug loading while maintaining efficient drug release and therapeutic activity. This strategy was successfully applied to tobramycin, a hydrophilic drug suffering from low encapsulation into liposomes, where its loading was improved six-fold using aptamers. Overall, we demonstrate that aptamers could act, in addition to their targeting properties, as multifunctional excipients for liposomal formulations.

  17. Selective inhibitory DNA aptamers of the human RNase H1

    PubMed Central

    Pileur, Frédéric; Andreola, Marie-Line; Dausse, Eric; Michel, Justine; Moreau, Serge; Yamada, Hirofumi; Gaidamakov, Sergei A.; Crouch, Robert J.; Toulmé, Jean-Jacques; Cazenave, Christian

    2003-01-01

    Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA–DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (Kd values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5′ and 3′ tails that form a stem of six base pairs. Base pairing between the 5′ and 3′ tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM. PMID:14500841

  18. Rattus norvegicus BN/SHR liver and heart left ventricle ribosomal RNA depleted directional RNA sequencing.

    PubMed

    Wyler, Emanuel; van Heesch, Sebastiaan; Adami, Eleonora; Hubner, Norbert; Landthaler, Markus

    2017-08-11

    The spontaneously hypertensive rat strain is a frequently used disease model. In a previous study, we measured translational efficiency from this strain and BN-Lx animals. Here, we describe long RNA sequencing reads from ribosomal RNA depleted samples from the same animals. This data can be used to investigate splicing-related events. RNA was extracted from rat liver and heart left ventricle from BN-Lx and SHR/Ola rats in biological replicates. Ribosomal RNA was removed and the samples subjected to directional high-throughput RNA-sequencing. Read and alignment statistics indicate high quality of the data. The raw sequencing reads are freely available on the NCBI short read archive and can be used for further research on tissue and strain differences, or analysed together with other published high-throughput data from the same animals.

  19. Local sequence and sequencing depth dependent accuracy of RNA-seq reads.

    PubMed

    Cai, Guoshuai; Liang, Shoudan; Zheng, Xiaofeng; Xiao, Feifei

    2017-08-09

    Many biases and spurious effects are inherent in RNA-seq technology, resulting in a non-uniform distribution of sequencing read counts for each base position in a gene. Therefore, a base-level strategy is required to model the non-uniformity. Also, the properties of sequencing read counts can be leveraged to achieve a more precise estimation of the mean and variance of measurement. In this study, we aimed to unveil the effects on RNA-seq accuracy from multiple factors and develop accurate modeling of RNA-seq reads in comparison. We found that the overdispersion rate decreased when sequencing depth increased on the base level. Moreover, the influence of local sequence(s) on the overdispersion rate was notable but no longer significant after adjusting the effect from sequencing depth. Based on these findings, we propose a desirable beta-binomial model with a dynamic overdispersion rate on the base-level proportion of sequencing read counts from two samples. The current study provides thorough insights into the impact of overdispersion at the position level and especially into its relationship with sequencing depth, local sequence, and preparation protocol. These properties of RNA-seq will aid in improvement of the quality control procedure and development of statistical methods for RNA-seq downstream analyses.

  20. RNAcentral: A vision for an international database of RNA sequences

    PubMed Central

    Bateman, Alex; Agrawal, Shipra; Birney, Ewan; Bruford, Elspeth A.; Bujnicki, Janusz M.; Cochrane, Guy; Cole, James R.; Dinger, Marcel E.; Enright, Anton J.; Gardner, Paul P.; Gautheret, Daniel; Griffiths-Jones, Sam; Harrow, Jen; Herrero, Javier; Holmes, Ian H.; Huang, Hsien-Da; Kelly, Krystyna A.; Kersey, Paul; Kozomara, Ana; Lowe, Todd M.; Marz, Manja; Moxon, Simon; Pruitt, Kim D.; Samuelsson, Tore; Stadler, Peter F.; Vilella, Albert J.; Vogel, Jan-Hinnerk; Williams, Kelly P.; Wright, Mathew W.; Zwieb, Christian

    2011-01-01

    During the last decade there has been a great increase in the number of noncoding RNA genes identified, including new classes such as microRNAs and piRNAs. There is also a large growth in the amount of experimental characterization of these RNA components. Despite this growth in information, it is still difficult for researchers to access RNA data, because key data resources for noncoding RNAs have not yet been created. The most pressing omission is the lack of a comprehensive RNA sequence database, much like UniProt, which provides a comprehensive set of protein knowledge. In this article we propose the creation of a new open public resource that we term RNAcentral, which will contain a comprehensive collection of RNA sequences and fill an important gap in the provision of biomedical databases. We envision RNA researchers from all over the world joining a federated RNAcentral network, contributing specialized knowledge and databases. RNAcentral would centralize key data that are currently held across a variety of databases, allowing researchers instant access to a single, unified resource. This resource would facilitate the next generation of RNA research and help drive further discoveries, including those that improve food production and human and animal health. We encourage additional RNA database resources and research groups to join this effort. We aim to obtain international network funding to further this endeavor. PMID:21940779

  1. RNAcentral: A vision for an international database of RNA sequences.

    PubMed

    Bateman, Alex; Agrawal, Shipra; Birney, Ewan; Bruford, Elspeth A; Bujnicki, Janusz M; Cochrane, Guy; Cole, James R; Dinger, Marcel E; Enright, Anton J; Gardner, Paul P; Gautheret, Daniel; Griffiths-Jones, Sam; Harrow, Jen; Herrero, Javier; Holmes, Ian H; Huang, Hsien-Da; Kelly, Krystyna A; Kersey, Paul; Kozomara, Ana; Lowe, Todd M; Marz, Manja; Moxon, Simon; Pruitt, Kim D; Samuelsson, Tore; Stadler, Peter F; Vilella, Albert J; Vogel, Jan-Hinnerk; Williams, Kelly P; Wright, Mathew W; Zwieb, Christian

    2011-11-01

    During the last decade there has been a great increase in the number of noncoding RNA genes identified, including new classes such as microRNAs and piRNAs. There is also a large growth in the amount of experimental characterization of these RNA components. Despite this growth in information, it is still difficult for researchers to access RNA data, because key data resources for noncoding RNAs have not yet been created. The most pressing omission is the lack of a comprehensive RNA sequence database, much like UniProt, which provides a comprehensive set of protein knowledge. In this article we propose the creation of a new open public resource that we term RNAcentral, which will contain a comprehensive collection of RNA sequences and fill an important gap in the provision of biomedical databases. We envision RNA researchers from all over the world joining a federated RNAcentral network, contributing specialized knowledge and databases. RNAcentral would centralize key data that are currently held across a variety of databases, allowing researchers instant access to a single, unified resource. This resource would facilitate the next generation of RNA research and help drive further discoveries, including those that improve food production and human and animal health. We encourage additional RNA database resources and research groups to join this effort. We aim to obtain international network funding to further this endeavor.

  2. Folding versus charge: understanding selective target recognition by the thrombin aptamers.

    PubMed

    Marson, Giuseppe; Palumbo, Manlio; Sissi, Claudia

    2012-01-01

    The use of nucleic acids as drugs represents a consistently growing approach. Different therapeutical strategies take advantage of the biological and biophysical properties of DNA and RNA to properly modulate activity of selected targets. A peculiar characteristic of these molecules is their structural flexibility which allows them to assume distinct foldings depending upon their sequence and/or environment. During the last twenty years this has led to the theoretical and experimental development of oligonucleotide aptamers, short sequences which can recognize a target with specificity and affinity comparable to antibodies. A leading example is represented by the Thrombin aptamer (15fTBA), a 15-mer DNA selected by its high affinity for the exosite I (fibrinogen binding site) of the coagulation factor. The very stable protein-DNA complex formation is the result of complementarities between the two macromolecules promoted by the aptamer sequence and folding as well as of electrostatic interactions generated by the charge balance at the binding site/s. Here, we investigated the relative role of these contributions and their involvement in defining the biological properties of the resulting complex. Thus we compared the Thrombin binding and inhibition properties of TBA to those of unrelated single stranded oligonucleotides. Additionally, the differences between the two protein exosites were assessed by using 29hTBA, a longer (29-mer) aptamer known to bind exosite II (heparin binding site). A subtle balance of aptamer folding and sequence is shown to cooperate with charge density for effective and selective recognition of exosite I or exosite II by TBAs.

  3. Integrated bioinformatics analysis of chromatin regulator EZH2 in regulating mRNA and lncRNA expression by ChIP sequencing and RNA sequencing

    PubMed Central

    Li, Yuan; Luo, Mei; Shi, Xuejiao; Lu, Zhiliang; Sun, Shouguo; Huang, Jianbing; Chen, Zhaoli; He, Jie

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2), a dynamic chromatin regulator in cancer, represents a potential therapeutic target showing early signs of promise in clinical trials. EZH2 ChIP sequencing data in 19 cell lines and RNA sequencing data in ten cancer types were downloaded from GEO and TCGA, respectively. Integrated ChIP sequencing analysis and co-expressing analysis were conducted and both mRNA and long noncoding RNA (lncRNA) targets were detected. We detected a median of 4,672 mRNA targets and 4,024 lncRNA targets regulated by EZH2 in 19 cell lines. 20 mRNA targets and 27 lncRNA targets were found in all 19 cell lines. These mRNA targets were enriched in pathways in cancer, Hippo, Wnt, MAPK and PI3K-Akt pathways. Co-expression analysis confirmed numerous targets, mRNA genes (RRAS, TGFBR2, NUF2 and PRC1) and lncRNA genes (lncRNA LINC00261, DIO3OS, RP11-307C12.11 and RP11-98D18.9) were potential targets and were significantly correlated with EZH2. We predicted genome-wide potential targets and the role of EZH2 in regulating as a transcriptional suppressor or activator which could pave the way for mechanism studies and the targeted therapy of EZH2 in cancer. PMID:27835578

  4. Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA.

    PubMed

    Shanker, Savita; Paulson, Ariel; Edenberg, Howard J; Peak, Allison; Perera, Anoja; Alekseyev, Yuriy O; Beckloff, Nicholas; Bivens, Nathan J; Donnelly, Robert; Gillaspy, Allison F; Grove, Deborah; Gu, Weikuan; Jafari, Nadereh; Kerley-Hamilton, Joanna S; Lyons, Robert H; Tepper, Clifford; Nicolet, Charles M

    2015-04-01

    This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.

  5. Nucleic acid-based aptamers: applications, development and clinical trials.

    PubMed

    Kanwar, Jagat R; Roy, Kislay; Maremanda, Nihal G; Subramanian, Krishnakumar; Veedu, Rakesh N; Bawa, Raj; Kanwar, Rupinder K

    2015-01-01

    Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easier manufacturing protocol, freedom to introduce chemical modifications for further improvement, etc. Reiterative selection process of aptamers over 10-15 cycles starting from a large initial pool of random nucleotide sequences renders them with high binding affinity, thereby making them extremely specific for their targets. Aptamer-based detection systems are well investigated and likely to displace primitive detection systems. Aptamer chimeras (combination of aptamers with another aptamer or biomacromolecule or chemical moiety) have the potential activity of both the parent molecules, and thus hold the capability to perform diverse functions at the same time. Owing to their extremely high specificity and lack of immunogenicity or pathogenicity, a number of other aptamers have recently entered clinical trials and have garnered favorable attention from pharmaceutical companies. Promising results from the clinical trials provide new hope to change the conventional style of therapy. Aptamers have attained high therapeutic relevance in a short time as compared to synthetic drugs and/or other modes of therapy. This review follows the various trends in aptamer technology including production, selection, modifications and success in clinical fields. It focusses largely on the various applications of aptamers which mainly depend upon their selection procedures. The review also sheds light on various modifications and chimerizations that have been implemented in order to improve the stability and functioning of the aptamers, including introduction of locked nucleic acids (LNAs). The application of various aptamers in detection systems has been discussed elaborately in order to stress on their role as efficient

  6. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  7. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Moyzis, Robert K.; Ratliff, Robert L.; Shera, E. Brooks; Stewart, Carleton C.

    1990-01-01

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

  8. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  9. Complete nucleotide sequence of tobacco streak virus RNA 3.

    PubMed Central

    Cornelissen, B J; Janssen, H; Zuidema, D; Bol, J F

    1984-01-01

    Double-stranded cDNA of in vitro polyadenylated tobacco streak virus (TSV) RNA 3 has been cloned and sequenced. The complete primary structure of 2,205 nucleotides reveals two open reading frames flanked by a leader sequence of 210 bases, an intercistronic region of 123 nucleotides and a 3'-extracistronic sequence of 288 nucleotides. The 5'-terminal open reading frame codes for a Mr 31,742 protein, which probably corresponds to the only in vitro translation product of TSV RNA 3. The 3'-terminal coding region predicts a Mr 26,346 protein, probably the viral coat protein, which is the translation product of the subgenomic messenger, RNA 4. Although the coat proteins of alfalfa mosaic virus (A1MV) and TSV are functionally equivalent in activating their own and each others genomes, no homology between the primary structures of those two proteins is detectable. PMID:6546793

  10. Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison.

    PubMed

    Johnson, A M; Fielke, R; Lumb, R; Baverstock, P R

    1990-04-01

    Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa. The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis. The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups. The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa.

  11. Normalizing single-cell RNA sequencing data: Challenges and opportunities

    PubMed Central

    Dudoit, Sandrine; Marioni, John C.

    2017-01-01

    Single-cell transcriptomics is becoming an important component of the molecular biologist’s toolkit. A critical step when analyzing this type of data is normalization. However, normalization is typically performed using methods developed for bulk RNA sequencing or even microarray data, whose suitability for single-cell transcriptomics has not been assessed. In this perspective, we discuss commonly used normalization approaches and illustrate how these can lead to misleading results. Finally, we present alternative approaches and provide recommendations for single-cell RNA sequencing users. PMID:28504683

  12. Multiple structural alignment and clustering of RNA sequences.

    PubMed

    Torarinsson, Elfar; Havgaard, Jakob H; Gorodkin, Jan

    2007-04-15

    An apparent paradox in computational RNA structure prediction is that many methods, in advance, require a multiple alignment of a set of related sequences, when searching for a common structure between them. However, such a multiple alignment is hard to obtain even for few sequences with low sequence similarity without simultaneously folding and aligning them. Furthermore, it is of interest to conduct a multiple alignment of RNA sequence candidates found from searching as few as two genomic sequences. Here, based on the PMcomp program, we present a global multiple alignment program, foldalignM, which performs especially well on few sequences with low sequence similarity, and is comparable in performance with state of the art programs in general. In addition, it can cluster sequences based on sequence and structure similarity and output a multiple alignment for each cluster. Furthermore, preliminary results with local datasets indicate that the program is useful for post processing foldalign pairwise scans. The program foldalignM is implemented in JAVA and is, along with some accompanying PERL scripts, available at http://foldalign.ku.dk/

  13. Splatter: simulation of single-cell RNA sequencing data.

    PubMed

    Zappia, Luke; Phipson, Belinda; Oshlack, Alicia

    2017-09-12

    As single-cell RNA sequencing (scRNA-seq) technologies have rapidly developed, so have analysis methods. Many methods have been tested, developed, and validated using simulated datasets. Unfortunately, current simulations are often poorly documented, their similarity to real data is not demonstrated, or reproducible code is not available. Here, we present the Splatter Bioconductor package for simple, reproducible, and well-documented simulation of scRNA-seq data. Splatter provides an interface to multiple simulation methods including Splat, our own simulation, based on a gamma-Poisson distribution. Splat can simulate single populations of cells, populations with multiple cell types, or differentiation paths.

  14. Single-cell sequencing of the small-RNA transcriptome.

    PubMed

    Faridani, Omid R; Abdullayev, Ilgar; Hagemann-Jensen, Michael; Schell, John P; Lanner, Fredrik; Sandberg, Rickard

    2016-12-01

    Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.

  15. Probing dimensionality beyond the linear sequence of mRNA.

    PubMed

    Del Campo, Cristian; Ignatova, Zoya

    2016-05-01

    mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions.

  16. Comparative Analysis of Single-Cell RNA Sequencing Methods.

    PubMed

    Ziegenhain, Christoph; Vieth, Beate; Parekh, Swati; Reinius, Björn; Guillaumet-Adkins, Amy; Smets, Martha; Leonhardt, Heinrich; Heyn, Holger; Hellmann, Ines; Enard, Wolfgang

    2017-02-16

    Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols.

  17. Effect of Chemical Modifications on Aptamer Stability in Serum.

    PubMed

    Kratschmer, Christina; Levy, Matthew

    2017-09-25

    There is increasing interest in the use of aptamers for the development of therapeutics. However, as oligonucleotides, aptamers are susceptible to nuclease degradation; poor serum stability is likely to negatively affect in vivo function. Modified nucleotides have been used to thwart nuclease degradation. However, few studies report the serum stability of selected aptamers. In this study, we examined the effect of various chemical modifications (2'-deoxy, 2'-hydroxyl, 2'-fluoro, and 2'-O-methyl) on the stability of a control oligonucleotide sequence following incubation in frozen human, fresh mouse, and fresh human serum. We also assessed the effect of the 3' inverted dT cap on stability. Surprisingly, we found that fYrR (2'-fluoro RNA) is only roughly as stable as DNA (2'-deoxy). Interestingly, the inclusion of a 3' inverted dT cap had only a modest effect on serum stability, if any. In one instance, the addition of a 3' inverted dT cap rendered a molecule composed of DNA more stable than its fYrR counterpart. By far, fully modified oligonucleotides (100% 2-O-Methyl or 2'-O-methyl A, C, and U in combination with 2'-fluoro G, termed fGmH) had the longest half-lives. These compositions demonstrated little degradation in human serum even after prolonged incubation. Together these results support the need for using fully modified aptamers for in vivo applications and should encourage those in the field to exploit newer polymerase variants capable of directly generating such polymers.

  18. Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

    PubMed

    Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

    2017-10-06

    Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

  19. MicroRNA target prediction using thermodynamic and sequence curves.

    PubMed

    Ghoshal, Asish; Shankar, Raghavendran; Bagchi, Saurabh; Grama, Ananth; Chaterji, Somali

    2015-11-25

    MicroRNAs (miRNAs) are small regulatory RNA that mediate RNA interference by binding to various mRNA target regions. There have been several computational methods for the identification of target mRNAs for miRNAs. However, these have considered all contributory features as scalar representations, primarily, as thermodynamic or sequence-based features. Further, a majority of these methods solely target canonical sites, which are sites with "seed" complementarity. Here, we present a machine-learning classification scheme, titled Avishkar, which captures the spatial profile of miRNA-mRNA interactions via smooth B-spline curves, separately for various input features, such as thermodynamic and sequence features. Further, we use a principled approach to uniformly model canonical and non-canonical seed matches, using a novel seed enrichment metric. We demonstrate that large number of seed-match patterns have high enrichment values, conserved across species, and that majority of miRNA binding sites involve non-canonical matches, corroborating recent findings. Using spatial curves and popular categorical features, such as target site length and location, we train a linear SVM model, utilizing experimental CLIP-seq data. Our model significantly outperforms all established methods, for both canonical and non-canonical sites. We achieve this while using a much larger candidate miRNA-mRNA interaction set than prior work. We have developed an efficient SVM-based model for miRNA target prediction using recent CLIP-seq data, demonstrating superior performance, evaluated using ROC curves, specifically about 20% better than the state-of-the-art, for different species (human or mouse), or different target types (canonical or non-canonical). To the best of our knowledge we provide the first distributed framework for microRNA target prediction based on Apache Hadoop and Spark. All source code and data is publicly available at https://bitbucket.org/cellsandmachines/avishkar.

  20. Aptamers: A Feasible Technology in Cancer Immunotherapy

    PubMed Central

    Villanueva, H.; Pastor, F.

    2016-01-01

    Aptamers are single-chained RNA or DNA oligonucleotides (ODNs) with three-dimensional folding structures which allow them to bind to their targets with high specificity. Aptamers normally show affinities comparable to or higher than that of antibodies. They are chemically synthesized and therefore less expensive to manufacture and produce. A variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors. Some of them have been conjugated to other molecules to target the immune system and reduce off-target side effects. Despite the success of first-line treatments against cancer, the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer. The advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect. Here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer-based immunotherapy could provide either alone or combined with first-line treatments in cancer therapy. PMID:27413756

  1. General approach for engineering small-molecule-binding DNA split aptamers.

    PubMed

    Kent, Alexandra D; Spiropulos, Nicholas G; Heemstra, Jennifer M

    2013-10-15

    Here we report a general method for engineering three-way junction DNA aptamers into split aptamers. Split aptamers show significant potential for use as recognition elements in biosensing applications, but reliable methods for generating these sequences are currently lacking. We hypothesize that the three-way junction is a "privileged architecture" for the elaboration of aptamers into split aptamers, as it provides two potential splitting sites that are distal from the target binding pocket. We propose a general method for split aptamer engineering that involves removing one loop region, then systematically modifying the number of base pairs in the remaining stem regions in order to achieve selective assembly only in the presence of the target small molecule. We screen putative split aptamer sequence pairs using split aptamer proximity ligation (StAPL) technology developed by our laboratory, but we validate that the results obtained using StAPL translate directly to systems in which the aptamer fragments are assembling noncovalently. We introduce four new split aptamer sequences, which triples the number of small-molecule-binding DNA split aptamers reported to date, and the methods described herein provide a reliable route for the engineering of additional split aptamers, dramatically advancing the potential substrate scope of DNA assembly based biosensors.

  2. Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence.

    PubMed

    Kim, Hui-Bae; Kim, Do-Yeong; Cho, Tae-Ju

    2014-06-01

    Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3'-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected.

  3. (99m)Tc-MAG3-aptamer for imaging human tumors associated with high level of matrix metalloprotease-9.

    PubMed

    Da Rocha Gomes, Sonia; Miguel, Julie; Azéma, Laurent; Eimer, Sandrine; Ries, Colette; Dausse, Eric; Loiseau, Hugues; Allard, Michèle; Toulmé, Jean-Jacques

    2012-11-21

    The human matrix metalloprotease 9 (hMMP-9) is involved in many physiological processes such as tissue remodeling. Its overexpression in tumors promotes the release of cancer cells thus contributing to tumor metastasis. It is a relevant marker of malignant tumors. We selected an RNA aptamer containing 2'-fluoro, pyrimidine ribonucleosides, that exhibits a strong affinity for hMMP-9 (K(d) = 20 nM) and that discriminates other human MMPs: no binding was detected to either hMMP-2 or -7. Investigating the binding properties of different MMP-9 aptamer variants by surface plasmon resonance allowed the determination of recognition elements. As a result, a truncated aptamer, 36 nucleotides long, was made fully resistant to nuclease following the substitution of every purine ribonucleoside residue by 2'-O-methyl analogues and was conjugated to S-acetylmercaptoacetyltriglycine for imaging purposes. The resulting modified aptamer retained the binding properties of the originally selected sequence. Following (99m)Tc labeling, this aptamer was used for ex vivo imaging slices of human brain tumors. We were able to specifically detect the presence of hMMP-9 in such tissues.

  4. KnotSeeker: heuristic pseudoknot detection in long RNA sequences.

    PubMed

    Sperschneider, Jana; Datta, Amitava

    2008-04-01

    Pseudoknots are folded structures in RNA molecules that perform essential functions as part of cellular transcription machinery and regulatory processes. The prediction of these structures in RNA molecules has important implications in antiviral drug design. It has been shown that the prediction of pseudoknots is an NP-complete problem. Practical structure prediction algorithms based on free energy minimization employ a restricted problem class and dynamic programming. However, these algorithms are computationally very expensive, and their accuracy deteriorates if the input sequence containing the pseudoknot is too long. Heuristic methods can be more efficient, but do not guarantee an optimal solution in regards to the minimum free energy model. We present KnotSeeker, a new heuristic algorithm for the detection of pseudoknots in RNA sequences as a preliminary step for structure prediction. Our method uses a hybrid sequence matching and free energy minimization approach to perform a screening of the primary sequence. We select short sequence fragments as possible candidates that may contain pseudoknots and verify them by using an existing dynamic programming algorithm and a minimum weight independent set calculation. KnotSeeker is significantly more accurate in detecting pseudoknots compared to other common methods as reported in the literature. It is very efficient and therefore a practical tool, especially for long sequences. The algorithm has been implemented in Python and it also uses C/C++ code from several other known techniques. The code is available from http://www.csse.uwa.edu.au/~datta/pseudoknot.

  5. Studying RNA homology and conservation with Infernal: from single sequences to RNA families

    PubMed Central

    Barquist, Lars; Burge, Sarah W.; Gardner, Paul P.

    2016-01-01

    Emerging high-throughput technologies have led to a deluge of putative non-coding RNA (ncRNA) sequences identified in a wide variety of organisms. Systematic characterization of these transcripts will be a tremendous challenge. Homology detection is critical to making maximal use of functional information gathered about ncRNAs: identifying homologous sequence allows us to transfer information gathered in one organism to another quickly and with a high degree of confidence. ncRNA presents a challenge for homology detection, as the primary sequence is often poorly conserved and de novo secondary structure prediction and search remains difficult. This protocol introduces methods developed by the Rfam database for identifying “families” of homologous ncRNAs starting from single “seed” sequences using manually curated sequence alignments to build powerful statistical models of sequence and structure conservation known as covariance models (CMs), implemented in the Infernal software package. We provide a step-by-step iterative protocol for identifying ncRNA homologs, then constructing an alignment and corresponding CM. We also work through an example for the bacterial small RNA MicA, discovering a previously unreported family of divergent MicA homologs in genus Xenorhabdus in the process. PMID:27322404

  6. Studying RNA Homology and Conservation with Infernal: From Single Sequences to RNA Families.

    PubMed

    Barquist, Lars; Burge, Sarah W; Gardner, Paul P

    2016-06-20

    Emerging high-throughput technologies have led to a deluge of putative non-coding RNA (ncRNA) sequences identified in a wide variety of organisms. Systematic characterization of these transcripts will be a tremendous challenge. Homology detection is critical to making maximal use of functional information gathered about ncRNAs: identifying homologous sequence allows us to transfer information gathered in one organism to another quickly and with a high degree of confidence. ncRNA presents a challenge for homology detection, as the primary sequence is often poorly conserved and de novo secondary structure prediction and search remain difficult. This unit introduces methods developed by the Rfam database for identifying "families" of homologous ncRNAs starting from single "seed" sequences, using manually curated sequence alignments to build powerful statistical models of sequence and structure conservation known as covariance models (CMs), implemented in the Infernal software package. We provide a step-by-step iterative protocol for identifying ncRNA homologs and then constructing an alignment and corresponding CM. We also work through an example for the bacterial small RNA MicA, discovering a previously unreported family of divergent MicA homologs in genus Xenorhabdus in the process. © 2016 by John Wiley & Sons, Inc.

  7. Specific alignment of structured RNA: stochastic grammars and sequence annealing.

    PubMed

    Bradley, Robert K; Pachter, Lior; Holmes, Ian

    2008-12-01

    Whole-genome screens suggest that eukaryotic genomes are dense with non-coding RNAs (ncRNAs). We introduce a novel approach to RNA multiple alignment which couples a generative probabilistic model of sequence and structure with an efficient sequence annealing approach for exploring the space of multiple alignments. This leads to a new software program, Stemloc-AMA, that is both accurate and specific in the alignment of multiple related RNA sequences. When tested on the benchmark datasets BRalibase II and BRalibase 2.1, Stemloc-AMA has comparable sensitivity to and better specificity than the best competing methods. We use a large-scale random sequence experiment to show that while most alignment programs maximize sensitivity at the expense of specificity, even to the point of giving complete alignments of non-homologous sequences, Stemloc-AMA aligns only sequences with detectable homology and leaves unrelated sequences largely unaligned. Such accurate and specific alignments are crucial for comparative-genomics analysis, from inferring phylogeny to estimating substitution rates across different lineages. Stemloc-AMA is available from http://biowiki.org/StemLocAMA as part of the dart software package for sequence analysis.

  8. Discovery of Aptamer Ligands for Hepatic Stellate Cells Using SELEX.

    PubMed

    Chen, Zhijin; Liu, Hao; Jain, Akshay; Zhang, Li; Liu, Chang; Cheng, Kun

    2017-01-01

    Insulin like growth factor II receptor (IGFIIR) is a transmembrane protein overexpressed in activated hepatic stellate cells (HSCs), which are the major target for the treatment of liver fibrosis. In this study, we aim to discover an IGFIIR-specific aptamer that can be potentially used as a targeting ligand for the treatment and diagnosis of liver fibrosis. Systematic evolution of ligands by exponential enrichment (SELEX) was conducted on recombinant human IGFIIR to identify IGFIIR-specific aptamers. The binding affinity and specificity of the discovered aptamers to IGFIIR and hepatic stellate cells were studied using flow cytometry and Surface Plasmon Resonance (SPR). Aptamer-20 showed the highest affinity to recombinant human IGFIIR protein with a Kd of 35.5 nM, as determined by SPR. Aptamer-20 also has a high affinity (apparent Kd 45.12 nM) to LX-2 human hepatic stellate cells. Binding of aptamer-20 to hepatic stellate cells could be inhibited by knockdown of IGFIIR using siRNA, indicating a high specificity of the aptamer. The aptamer formed a chimera with an anti-fibrotic PCBP2 siRNA and delivered the siRNA to HSC-T6 cells to trigger silencing activity. In Vivo biodistribution study of the siRNA-aptamer chimera also demonstrated a high and specific uptake in the liver of the rats with CCl4-induced liver fibrosis. These data suggest that aptamer-20 is a high-affinity ligand for antifibrotic and diagnostic agents for liver fibrosis.

  9. New anti-HIV aptamers based on tetra-end-linked DNA G-quadruplexes: effect of the base sequence on anti-HIV activity.

    PubMed

    D'Atri, Valentina; Oliviero, Giorgia; Amato, Jussara; Borbone, Nicola; D'Errico, Stefano; Mayol, Luciano; Piccialli, Vincenzo; Haider, Shozeb; Hoorelbeke, Bart; Balzarini, Jan; Piccialli, Gennaro

    2012-10-04

    This communication reports on the synthesis and biophysical, biological and SAR studies of a small library of new anti-HIV aptamers based on the tetra-end-linked G-quadruplex structure. The new aptamers showed EC(50) values against HIV-1 in the range of 0.04-0.15 μM as well as affinities for the HIV-1 gp120 envelope in the same order of magnitude.

  10. RNA-DNA sequence differences spell genetic code ambiguities

    PubMed Central

    Nielsen, Michael L.

    2011-01-01

    A recent paper in Science by Li et al. 20111 reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized.  PMID:22567189

  11. Sequence and structural conservation in RNA ribose zippers

    SciTech Connect

    Tamura, Makio; Holbrook, Stephen R.

    2002-03-01

    The ribose zipper, an important element of RNA tertiary structure, is characterized by consecutive hydrogen-bonding interactions between ribose 20-hydroxyls from different regions of an RNA chain or between RNA chains. These tertiary contacts have previously been observed to also involve base backbone and base base interactions (A-minor type). We searched for ribose zipper tertiary interactions in the crystal structures of the large ribosomal subunit RNAs of Haloarcula marismortui and Deinococcus radiodurans, and the small ribosomal subunit RNA of Thermus thermophilus and identified a total of 97 ribose zippers. Of these, 20 were found in T. thermophilus 16 S rRNA, 44 in H. marismortui 23 S rRNA (plus 2 bridging 5 S and 23 S rRNAs) and 30 in D. radiodurans 23 S rRNA (plus 1 bridging 5 S and 23 S rRNAs). These were analyzed in terms of sequence conservation, structural conservation and stability, location in secondary structure, and phylogenetic conservation. Eleven types of ribose zippers were defined based on ribose base interactions. Of these 11, seven were observed in the ribosomal RNAs. The most common of these is the canonical ribose zipper, originally observed in the P4 P6 group I intron fragment. All ribose zippers were formed by antiparallel chain interactions and only a single example extended beyond two residues, forming an overlapping ribose zipper of three consecutive residues near the small subunit A-site. Almost all ribose zippers link stem (Watson Crick duplex) or stem-like (base-paired), with loop (external, internal, or junction) chain segments. About two-thirds of the observed ribose zippers interact with ribosomal proteins. Most of these ribosomal proteins bridge the ribose zipper chain segments with basic amino acid residues hydrogen bonding to the RNA backbone. Proteins involved in crucial ribosome function and in early stages of ribosomal assembly also stabilize ribose zipper interactions. All ribose zippers show strong sequence conservation

  12. High Throughput Sequencing of Extracellular RNA from Human Plasma

    PubMed Central

    Danielson, Kirsty M.; Rubio, Renee; Abderazzaq, Fieda; Das, Saumya; Wang, Yaoyu E.

    2017-01-01

    The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms. PMID:28060806

  13. Single Molecule Electrical Sequencing of DNA and RNA

    NASA Astrophysics Data System (ADS)

    Taniguchi, Masateru

    2013-03-01

    Gating nanopore devices are composed of nanopores with embedded nanoelectrodes, and they are expected to be one of the core devices used to realize label-free, low-cost DNA sequencing, subsequently leading to 1000-genome sequencing technologies. The operating principle of these nanodevices is based on identifying single base molecules of single DNA passing through a nanopore using a tunneling current between nanoelectrodes. We successfully identified single base molecules of DNA and RNA using tunneling currents. To make gating nanopore devices fit for practical use, core technologies should be integrated on one device chip. One core technology is the identification of single DNA and RNA composed of many base molecules using tunneling currents. We have succeeded in the single-molecule electrical sequencing of DNA and RNA formed by 3 and 7 base molecules, respectively, using a hybrid method of identifying single base molecules via a tunnelling current and random sequencing. A method that controls the speed of a single DNA passing through a nanopore is one core technology that determines the speed and accuracy of sequencing. We successfully developed a method that controls the translocation speed of a single DNA by three orders of magnitude using a voltage between nanoelectrodes.

  14. Learning to Predict miRNA-mRNA Interactions from AGO CLIP Sequencing and CLASH Data

    PubMed Central

    Lu, Yuheng; Leslie, Christina S.

    2016-01-01

    Recent technologies like AGO CLIP sequencing and CLASH enable direct transcriptome-wide identification of AGO binding and miRNA target sites, but the most widely used miRNA target prediction algorithms do not exploit these data. Here we use discriminative learning on AGO CLIP and CLASH interactions to train a novel miRNA target prediction model. Our method combines two SVM classifiers, one to predict miRNA-mRNA duplexes and a second to learn a binding model of AGO’s local UTR sequence preferences and positional bias in 3’UTR isoforms. The duplex SVM model enables the prediction of non-canonical target sites and more accurately resolves miRNA interactions from AGO CLIP data than previous methods. The binding model is trained using a multi-task strategy to learn context-specific and common AGO sequence preferences. The duplex and common AGO binding models together outperform existing miRNA target prediction algorithms on held-out binding data. Open source code is available at https://bitbucket.org/leslielab/chimiric. PMID:27438777

  15. rnaSeqMap: a Bioconductor package for RNA sequencing data exploration

    PubMed Central

    2011-01-01

    Background The throughput of commercially available sequencers has recently significantly increased. It has reached the point where measuring the RNA expression by the depth of coverage has become feasible even for largest genomes. The development of software tools is constantly following the progress of biological hardware. In particular, as RNA sequencing software can be regarded genome browsers, exon junction tools and statistical tools operating on counts of reads in predefined regions. The library rnaSeqMap, freely available via Bioconductor, is an RNA sequencing software which is independent of any biological hardware platform. It is based upon standard Bioconductor infrastructure for sequencing data and includes several novel features focused on deeper understanding of coverage expression profiles and discovery of novel transcription regions. Results rnaSeqMap is a toolbox for analyses that may be performed with the use of gene annotations or alternatively, in an unsupervised mode, on any genomic region to find novel or non-standard transcripts. The data back-end may be a MySQL database or a set of files in standard BAM format. The processing in R can be run on a machine without any particular hardware requirements, and scales linearly with the number of genomic loci and number of samples analyzed. The main features of rnaSeqMap include coverage operations, discovering irreducible regions of high expression, significance search and splicing analyses with nucleotide granularity. Conclusions This software may be used for a range of applications related to RNA sequencing by building customized analysis pipelines. The applicability and precision is expected to increase in parallel with the progress of the genome coverage in sequencers. PMID:21612622

  16. Prediction and prioritization of neoantigens: integration of RNA sequencing data with whole-exome sequencing.

    PubMed

    Karasaki, Takahiro; Nagayama, Kazuhiro; Kuwano, Hideki; Nitadori, Jun-Ichi; Sato, Masaaki; Anraku, Masaki; Hosoi, Akihiro; Matsushita, Hirokazu; Takazawa, Masaki; Ohara, Osamu; Nakajima, Jun; Kakimi, Kazuhiro

    2017-02-01

    The importance of neoantigens for cancer immunity is now well-acknowledged. However, there are diverse strategies for predicting and prioritizing candidate neoantigens, and thus reported neoantigen loads vary a great deal. To clarify this issue, we compared the numbers of neoantigen candidates predicted by four currently utilized strategies. Whole-exome sequencing and RNA sequencing (RNA-Seq) of four non-small-cell lung cancer patients was carried out. We identified 361 somatic missense mutations from which 224 candidate neoantigens were predicted using MHC class I binding affinity prediction software (strategy I). Of these, 207 exceeded the set threshold of gene expression (fragments per kilobase of transcript per million fragments mapped ≥1), resulting in 124 candidate neoantigens (strategy II). To verify mutant mRNA expression, sequencing of amplicons from tumor cDNA including each mutation was undertaken; 204 of the 207 mutations were successfully sequenced, yielding 121 mutant mRNA sequences, resulting in 75 candidate neoantigens (strategy III). Sequence information was extracted from RNA-Seq to confirm the presence of mutated mRNA. Variant allele frequencies ≥0.04 in RNA-Seq were found for 117 of the 207 mutations and regarded as expressed in the tumor, and finally, 72 candidate neoantigens were predicted (strategy IV). Without additional amplicon sequencing of cDNA, strategy IV was comparable to strategy III. We therefore propose strategy IV as a practical and appropriate strategy to predict candidate neoantigens fully utilizing currently available information. It is of note that different neoantigen loads were deduced from the same tumors depending on the strategies applied.

  17. siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

    PubMed

    Galka-Marciniak, Paulina; Olejniczak, Marta; Starega-Roslan, Julia; Szczesniak, Michal W; Makalowska, Izabela; Krzyzosiak, Wlodzimierz J

    2016-04-01

    shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data.

    PubMed

    An, Jiyuan; Lai, John; Lehman, Melanie L; Nelson, Colleen C

    2013-01-01

    miRDeep and its varieties are widely used to quantify known and novel micro RNA (miRNA) from small RNA sequencing (RNAseq). This article describes miRDeep*, our integrated miRNA identification tool, which is modeled off miRDeep, but the precision of detecting novel miRNAs is improved by introducing new strategies to identify precursor miRNAs. miRDeep* has a user-friendly graphic interface and accepts raw data in FastQ and Sequence Alignment Map (SAM) or the binary equivalent (BAM) format. Known and novel miRNA expression levels, as measured by the number of reads, are displayed in an interface, which shows each RNAseq read relative to the pre-miRNA hairpin. The secondary pre-miRNA structure and read locations for each predicted miRNA are shown and kept in a separate figure file. Moreover, the target genes of known and novel miRNAs are predicted using the TargetScan algorithm, and the targets are ranked according to the confidence score. miRDeep* is an integrated standalone application where sequence alignment, pre-miRNA secondary structure calculation and graphical display are purely Java coded. This application tool can be executed using a normal personal computer with 1.5 GB of memory. Further, we show that miRDeep* outperformed existing miRNA prediction tools using our LNCaP and other small RNAseq datasets. miRDeep* is freely available online at http://www.australianprostatecentre.org/research/software/mirdeep-star.

  19. Maize Gene Atlas Developed by RNA Sequencing and Comparative Evaluation of Transcriptomes Based on RNA Sequencing and Microarrays

    PubMed Central

    Sekhon, Rajandeep S.; Briskine, Roman; Hirsch, Candice N.; Myers, Chad L.; Springer, Nathan M.; Buell, C. Robin; de Leon, Natalia; Kaeppler, Shawn M.

    2013-01-01

    Transcriptome analysis is a valuable tool for identification and characterization of genes and pathways underlying plant growth and development. We previously published a microarray-based maize gene atlas from the analysis of 60 unique spatially and temporally separated tissues from 11 maize organs [1]. To enhance the coverage and resolution of the maize gene atlas, we have analyzed 18 selected tissues representing five organs using RNA sequencing (RNA-Seq). For a direct comparison of the two methodologies, the same RNA samples originally used for our microarray-based atlas were evaluated using RNA-Seq. Both technologies produced similar transcriptome profiles as evident from high Pearson's correlation statistics ranging from 0.70 to 0.83, and from nearly identical clustering of the tissues. RNA-Seq provided enhanced coverage of the transcriptome, with 82.1% of the filtered maize genes detected as expressed in at least one tissue by RNA-Seq compared to only 56.5% detected by microarrays. Further, from the set of 465 maize genes that have been historically well characterized by mutant analysis, 427 show significant expression in at least one tissue by RNA-Seq compared to 390 by microarray analysis. RNA-Seq provided higher resolution for identifying tissue-specific expression as well as for distinguishing the expression profiles of closely related paralogs as compared to microarray-derived profiles. Co-expression analysis derived from the microarray and RNA-Seq data revealed that broadly similar networks result from both platforms, and that co-expression estimates are stable even when constructed from mixed data including both RNA-Seq and microarray expression data. The RNA-Seq information provides a useful complement to the microarray-based maize gene atlas and helps to further understand the dynamics of transcription during maize development. PMID:23637782

  20. Aptamer-Conjugated Nanoparticles for Cancer Cell Detection

    PubMed Central

    Medley, Colin D.; Bamrungsap, Suwussa; Tan, Weihong; Smith, Joshua E.

    2011-01-01

    Aptamer-conjugated nanoparticles (ACNPs) have been used for a variety of applications, particularly dual nanoparticles for magnetic extraction and fluorescent labeling. In this type of assay, silica-coated magnetic and fluorophore-doped silica nanoparticles are conjugated to highly selective aptamers to detect and extract targeted cells in a variety of matrices. However, considerable improvements are required in order to increase the selectivity and sensitivity of this two-particle assay to be useful in a clinical setting. To accomplish this, several parameters were investigated, including nanoparticle size, conjugation chemistry, use of multiple aptamer sequences on the nanoparticles, and use of multiple nanoparticles with different aptamer sequences. After identifying the best-performing elements, the improvements made to this assay’s conditional parameters were combined to illustrate the overall enhanced sensitivity and selectivity of the two particle assay using an innovative multiple aptamer approach, signifying a critical feature in the advancement of this technique. PMID:21218774

  1. Structurally complex and highly active RNA ligases derived from random RNA sequences

    NASA Technical Reports Server (NTRS)

    Ekland, E. H.; Szostak, J. W.; Bartel, D. P.

    1995-01-01

    Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

  2. Simultaneous rapid sequencing of multiple RNA virus genomes.

    PubMed

    Neill, John D; Bayles, Darrell O; Ridpath, Julia F

    2014-06-01

    Comparing sequences of archived viruses collected over many years to the present allows the study of viral evolution and contributes to the design of new vaccines. However, the difficulty, time and expense of generating full-length sequences individually from each archived sample have hampered these studies. Next generation sequencing technologies have been utilized for analysis of clinical and environmental samples to identify viral pathogens that may be present. This has led to the discovery of many new, uncharacterized viruses from a number of viral families. Use of these sequencing technologies would be advantageous in examining viral evolution. In this study, a sequencing procedure was used to sequence simultaneously and rapidly multiple archived samples using a single standard protocol. This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3'-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. This conferred sequence independence by random priming both first and second strand cDNA synthesis. Viral stocks were treated with a nuclease cocktail to reduce the presence of host nucleic acids. Viral RNA was extracted, followed by single tube random-primed double-stranded cDNA synthesis. The resultant cDNAs were amplified by primer-specific PCR, pooled, size fractionated and sequenced on the Ion Torrent PGM platform. The individual virus genomes were readily assembled by both de novo and template-assisted assembly methods. This procedure consistently resulted in near full length, if not full-length, genomic sequences and was used to sequence multiple bovine pestivirus and coronavirus isolates simultaneously.

  3. Using Small RNA Deep Sequencing Data to Detect Human Viruses.

    PubMed

    Wang, Fang; Sun, Yu; Ruan, Jishou; Chen, Rui; Chen, Xin; Chen, Chengjie; Kreuze, Jan F; Fei, ZhangJun; Zhu, Xiao; Gao, Shan

    2016-01-01

    Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans.

  4. Using Small RNA Deep Sequencing Data to Detect Human Viruses

    PubMed Central

    Wang, Fang; Sun, Yu; Ruan, Jishou; Chen, Rui; Chen, Xin; Chen, Chengjie; Kreuze, Jan F.; Fei, ZhangJun; Zhu, Xiao

    2016-01-01

    Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans. PMID:27066498

  5. Cell growth inhibition by sequence-specific RNA minihelices.

    PubMed Central

    Hipps, D; Schimmel, P

    1995-01-01

    RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose. Images PMID:7664744

  6. Clinical applications of nucleic acid aptamers in cancer.

    PubMed

    Pei, Xiaoyu; Zhang, Jun; Liu, Jie

    2014-05-01

    Nucleic acid aptamers are small single-stranded DNA or RNA oligonucleotide segments, which bind to their targets with high affinity and specificity via unique three-dimensional structures. Aptamers are generated by an iterative in vitro selection process, termed as systematic evolution of ligands by exponential enrichment. Owing to their specificity, non-immunogenicity, non-toxicity, easily modified chemical structure and wide range of targets, aptamers appear to be ideal candidates for various clinical applications (diagnosis or treatment), such as cell detection, target diagnosis, molecular imaging and drug delivery. Several aptamers have entered the clinical pipeline for applications in diseases such as macular degeneration, coronary artery bypass graft surgery and various types of cancer. The aim of this review was to summarize and highlight the clinical applications of aptamers in cancer diagnosis and treatment.

  7. Aptamers Against Immunologic Targets: Diagnostic and Therapeutic Prospects.

    PubMed

    Vorobyeva, Mariya; Timoshenko, Valentina; Vorobjev, Pavel; Venyaminova, Alya

    2016-02-01

    The concept of in vitro selection of nucleic acid aptamers emerged 25 years ago, and since then tremendous progress has been achieved in the development of different aptamers and their applications for various bioanalytical and therapeutic purposes. Among other protein targets of aptamers, immune system proteins are of particular interest both as diagnostic markers and therapeutic targets. The present review summarizes up-to-date articles concerning the selection and design of DNA and RNA aptamers against immunologic targets such as antibodies, cytokines, and T-cell and B-cell receptors. We also discuss the prospects of employing aptamers as recognizing modules of diagnostic aptasensors, potential therapeutic candidates for the treatment of autoimmune diseases and cancer, and specific tools for functional studies of immune system proteins.

  8. Preparation of modified long-mer RNAs and analysis of FMN binding to the ypaA aptamer from B. subtilis.

    PubMed

    Frommer, Jennifer; Hieronymus, Robert; Selvi Arunachalam, Tamil; Heeren, Sabine; Jenckel, Maria; Strahl, Anne; Appel, Bettina; Müller, Sabine

    2014-01-01

    In recent years, RNA has been shown to fulfil a number of cellular functions. This has led to much interest in elucidation of the structure of functional RNA molecules, and thus, in the preparation of suitably functionalized RNAs. The chemical synthesis of RNAs allows for the site-specific modification; however, is limited to sequences of about 60-70 nucleotides in length. At the example of the flavine mononucleotide (FMN) responsive aptamer of the ypaA riboswitch from B. subtilis, we demonstrate the highly efficient preparation of site-specifically modified long-mer RNAs. Our strategy consists of the chemical synthesis of fragments followed by enzymatic or chemical ligation. Splint ligation with T4 RNA ligase turned out to be most successful among the enyzymatic protocols. Highly efficient chemical ligation was performed by azide-alkyne cycloaddition of suitably modified RNA fragments. Wild-type and 2-aminopurine (2-AP)-modified variants of the ypaA aptamer were prepared. FMN binding to all synthesized ypaA aptamer variants is demonstrated. However, dissociation of FMN from its binding site by reduction of the isoalloxazin unit as demonstrated before for a small-hairpin-derived aptazyme could not be shown. This implies that either FMN is less accessible to reduction when it is bound to its natural aptamer; that reduced FMN remains bound to the aptamer; or that FMN upon reduction indeed is released from its binding site, without the aptamer folding back in the natural ligand-free state. The results of this study are of general interest to the preparation of site-specifically modified RNAs for investigation into structure and function.

  9. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  10. Diversified sequences of peptide epitope for same-RNA recognition.

    PubMed Central

    Kim, S; Ribas de Pouplana, L; Schimmel, P

    1993-01-01

    We replaced an essential RNA-binding, 30-amino acid helix-loop in an Escherichia coli tRNA synthetase with an inactive and simplified "generic" sequence having 23 of the 30 amino acids as alanine and serine. Wild-type residues were restored in random combinations to generate a library with a sequence complexity of about 1.9 x 10(7). Active molecules were obtained by genetic selection at a frequency of approximately 1% and contained variants with as many as 11 alanine/serine replacements and a total of 17 alanine/serine residues. These variants have activities which are thermodynamically competitive with that of the native protein and therefore are functionally and, most likely, conformationally equivalent. Images Fig. 2 Fig. 4 PMID:7694278

  11. Power analysis of single-cell RNA-sequencing experiments.

    PubMed

    Svensson, Valentine; Natarajan, Kedar Nath; Ly, Lam-Ha; Miragaia, Ricardo J; Labalette, Charlotte; Macaulay, Iain C; Cvejic, Ana; Teichmann, Sarah A

    2017-04-01

    Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.

  12. Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments

    PubMed Central

    2011-01-01

    Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the biology, diversity and abundance of sRNA populations. In this review, we discuss issues involved in the design of sRNA sequencing experiments, including choosing a sequencing platform, inherent biases that affect sRNA measurements and replication. We outline the steps involved in preprocessing sRNA sequencing data and review both the principles behind and the current options for normalization. Finally, we discuss differential expression analysis in the absence and presence of biological replicates. While our focus is on sRNA sequencing experiments, many of the principles discussed are applicable to the sequencing of other RNA populations. PMID:21356093

  13. Toward Rare Blood Cell Preservation for RNA Sequencing.

    PubMed

    Vickovic, Sanja; Ahmadian, Afshin; Lewensohn, Rolf; Lundeberg, Joakim

    2015-07-01

    Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues-a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of limitations in low blood-input volume and RNA stability over the whole gene body. Therefore, we have developed a robust protocol for blood preservation and long-term storage while maintaining RNA integrity. Furthermore, we explored the possibility of using the protocol for preserving rare cell samples, such as circulating tumor cells. The results of our study confirmed that gene expression was not impacted by the preservation procedure (r(2) > 0.88) or by long-term storage (r(2) = 0.95), with RNA integrity number values averaging over 8. Similarly, cell surface antigens were still available for antibody selection (r(2) = 0.95). Lastly, data mining for fusion events showed that it was possible to detect rare tumor cells among a background of other cells present in blood irrespective of fixation. Thus, the developed protocol would be suitable for rare blood cell preservation followed by RNA sequencing analysis. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  14. The distribution of RNA motifs in natural sequences.

    PubMed

    Bourdeau, V; Ferbeyre, G; Pageau, M; Paquin, B; Cedergren, R

    1999-11-15

    Functional analysis of genome sequences has largely ignored RNA genes and their structures. We introduce here the notion of 'ribonomics' to describe the search for the distribution of and eventually the determination of the physiological roles of these RNA structures found in the sequence databases. The utility of this approach is illustrated here by the identification in the GenBank database of RNA motifs having known binding or chemical activity. The frequency of these motifs indicates that most have originated from evolutionary drift and are selectively neutral. On the other hand, their distribution among species and their location within genes suggest that the destiny of these motifs may be more elaborate. For example, the hammerhead motif has a skewed organismal presence, is phylogenetically stable and recent work on a schistosome version confirms its in vivo biological activity. The under-representation of the valine-binding motif and the Rev-binding element in GenBank hints at a detrimental effect on cell growth or viability. Data on the presence and the location of these motifs may provide critical guidance in the design of experiments directed towards the understanding and the manipulation of RNA complexes and activities in vivo.

  15. RNA sequencing of archived neonatal dried blood spots.

    PubMed

    Bybjerg-Grauholm, Jonas; Hagen, Christian Munch; Khoo, Sok Kean; Johannesen, Maria Louise; Hansen, Christine Søholm; Bækvad-Hansen, Marie; Christiansen, Michael; Hougaard, David Michael; Hollegaard, Mads V

    2017-03-01

    Neonatal dried blood spots (DBS) are routinely collected on standard Guthrie cards for all-comprising national newborn screening programs for inborn errors of metabolism, hypothyroidism and other diseases. In Denmark, the Guthrie cards are stored at - 20 °C in the Danish Neonatal Screening Biobank and each sample is linked to elaborate social and medical registries. This provides a unique biospecimen repository to enable large population research at a perinatal level. Here, we demonstrate the feasibility to obtain gene expression data from DBS using next-generation RNA sequencing (RNA-seq). RNA-seq was performed on five males and five females. Sequencing results have an average of > 30 million reads per sample. 26,799 annotated features can be identified with 64% features detectable without fragments per kilobase of transcript per million mapped reads (FPKM) cutoff; number of detectable features dropped to 18% when FPKM ≥ 1. Sex can be discriminated using blood-based sex-specific gene set identified by the Genotype-Tissue Expression consortium. Here, we demonstrate the feasibility to acquire biologically-relevant gene expression from DBS using RNA-seq which provide a new avenue to investigate perinatal diseases in a high throughput manner.

  16. Development of HGF-binding aptamers with the combination of G4 promoter-derived aptamer selection and in silico maturation.

    PubMed

    Yokoyama, Tomomi; Tsukakoshi, Kaori; Yoshida, Wataru; Saito, Taiki; Teramoto, Kentaro; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2017-10-01

    We describe the selection of aptamers based on bioinformatics-based approaches without Systematic Evolution of Ligands by EXponential enrichment (SELEX). SELEX is a potent method; however, it is time intensive and the PCR-amplification step, which is essential step for SELEX, leads to the loss of good aptamers. We have developed an aptamer-screening method, G4 promoter-derived aptamer selection (G4PAS), and an aptamer-improving method, in silico maturation (ISM). They are based on in silico sequence selection and computer assisted directed evolution, respectively. In this study, we succeeded in identifying new aptamers against hepatocyte growth factor (HGF) by G4PAS as well as improving the specificity of the HGF aptamers by ISM. Using ISM improved the specificity of the aptamer for HGF by up to 45-fold in comparison with the original aptamer. These methods enable easy and efficient identification of good aptamers, and the combination of G4PAS with ISM can thus serve as a potent approach for aptamer identification. Biotechnol. Bioeng. 2017;114: 2196-2203. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Statistical mechanics of secondary structures formed by random RNA sequences.

    PubMed

    Bundschuh, R; Hwa, T

    2002-03-01

    The formation of secondary structures by a random RNA sequence is studied as a model system for the sequence-structure problem omnipresent in biopolymers. Several toy energy models are introduced to allow detailed analytical and numerical studies. First, a two-replica calculation is performed. By mapping the two-replica problem to the denaturation of a single homogeneous RNA molecule in six-dimensional embedding space, we show that sequence disorder is perturbatively irrelevant, i.e., an RNA molecule with weak sequence disorder is in a molten phase where many secondary structures with comparable total energy coexist. A numerical study of various models at high temperature reproduces behaviors characteristic of the molten phase. On the other hand, a scaling argument based on the external statistics of rare regions can be constructed to show that the low-temperature phase is unstable to sequence disorder. We performed a detailed numerical study of the low-temperature phase using the droplet theory as a guide, and characterized the statistics of large-scale, low-energy excitations of the secondary structures from the ground state structure. We find the excitation energy to grow very slowly (i.e., logarithmically) with the length scale of the excitation, suggesting the existence of a marginal glass phase. The transition between the low-temperature glass phase and the high-temperature molten phase is also characterized numerically. It is revealed by a change in the coefficient of the logarithmic excitation energy, from being disorder dominated to being entropy dominated.

  18. Computational Approaches to RNA Structure Prediction, Analysis and Design

    PubMed Central

    Laing, Christian; Schlick, Tamar

    2011-01-01

    RNA molecules are important cellular components involved in many fundamental biological processes. Understanding the mechanisms behind their functions requires RNA tertiary structure knowledge. While modeling approaches for the study of RNA structures and dynamics lag behind efforts in protein folding, much progress has been achieved in the past two years. Here, we review recent advances in RNA folding algorithms, RNA tertiary motif discovery, applications of graph theory approaches to RNA structure and function, and in silico generation of RNA sequence pools for aptamer design. Advances within each area can be combined to impact many problems in RNA structure and function. PMID:21514143

  19. Bacteriorhodopsin: partial sequence of mRNA provides amino acid sequence in the precursor region.

    PubMed Central

    Chang, S H; Majumdar, A; Dunn, R; Makabe, O; RajBhandary, U L; Khorana, H G; Ohtsuka, E; Tanaka, T; Taniyama, Y O; Ikehara, M

    1981-01-01

    mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis. Images PMID:6943548

  20. Aptamer-targeted RNAi for HIV-1 therapy.

    PubMed

    Zhou, Jiehua; Rossi, John J

    2011-01-01

    The highly specific mechanism of RNA (RNAi) that inhibits the expression of disease genes is increasingly being harnessed to develop a new class of therapeutics for a wide variety of human maladies. The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Herein, we demonstrate novel cell type-specific dual inhibitory function anti-gp120 aptamer-siRNA delivery systems for HIV-1 therapy, in which both the aptamer and the siRNA portions have potent anti-HIV activities. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. The Dicer substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells. Our results provide a set of novel aptamer-targeted RNAi therapeutics to combat HIV and further validate the use of anti-gp120 aptamers for delivery of Dicer substrate siRNAs.

  1. Ultrasensitive Label-Free Sensing of IL-6 Based on PASE Functionalized Carbon Nanotube Micro-Arrays with RNA-Aptamers as Molecular Recognition Elements.

    PubMed

    Khosravi, Farhad; Loeian, Seyed Masoud; Panchapakesan, Balaji

    2017-04-17

    This study demonstrates the rapid and label-free detection of Interleukin-6 (IL-6) using carbon nanotube micro-arrays with aptamer as the molecular recognition element. Single wall carbon nanotubes micro-arrays biosensors were manufactured using photo-lithography, metal deposition, and etching techniques. Nanotube biosensors were functionalized with 1-Pyrenebutanoic Acid Succinimidyl Ester (PASE) conjugated IL-6 aptamers. Real time response of the sensor conductance was monitored with increasing concentration of IL-6 (1 pg/mL to 10 ng/mL), exposure to the sensing surface in buffer solution, and clinically relevant spiked blood samples. Non-specific Bovine Serum Albumin (BSA), PBS samples, and anti-IgG functionalized devices gave similar signatures in the real time conductance versus time experiments with no significant change in sensor signal. Exposure of the aptamer functionalized nanotube surface to IL-6 decreased the conductance with increasing concentration of IL-6. Experiments based on field effect transistor arrays suggested shift in drain current versus gate voltage for 1 pg and 1 ng of IL-6 exposure. Non-specific BSA did not produce any appreciable shift in the Ids versus Vg suggesting specific interactions of IL-6 on PASE conjugated aptamer surface gave rise to the change in electrical signal. Both Z axis and phase image in an Atomic Force Microscope (AFM) suggested unambiguous molecular interaction of the IL-6 on the nanotube-aptamer surface at 1 pg/mL concentration. The concentration of 1 pg falls below the diagnostic gray zone for cancer (2.3 pg-4 ng/mL), which is an indicator of early stage cancer. Thus, nanotube micro-arrays could potentially be developed for creating multiplexed assays involving cancer biomarker proteins and possibly circulating tumor cells all in a single assay using PASE functionalization protocol.

  2. Bias detection and correction in RNA-Sequencing data.

    PubMed

    Zheng, Wei; Chung, Lisa M; Zhao, Hongyu

    2011-07-19

    High throughput sequencing technology provides us unprecedented opportunities to study transcriptome dynamics. Compared to microarray-based gene expression profiling, RNA-Seq has many advantages, such as high resolution, low background, and ability to identify novel transcripts. Moreover, for genes with multiple isoforms, expression of each isoform may be estimated from RNA-Seq data. Despite these advantages, recent work revealed that base level read counts from RNA-Seq data may not be randomly distributed and can be affected by local nucleotide composition. It was not clear though how the base level read count bias may affect gene level expression estimates. In this paper, by using five published RNA-Seq data sets from different biological sources and with different data preprocessing schemes, we showed that commonly used estimates of gene expression levels from RNA-Seq data, such as reads per kilobase of gene length per million reads (RPKM), are biased in terms of gene length, GC content and dinucleotide frequencies. We directly examined the biases at the gene-level, and proposed a simple generalized-additive-model based approach to correct different sources of biases simultaneously. Compared to previously proposed base level correction methods, our method reduces bias in gene-level expression estimates more effectively. Our method identifies and corrects different sources of biases in gene-level expression measures from RNA-Seq data, and provides more accurate estimates of gene expression levels from RNA-Seq. This method should prove useful in meta-analysis of gene expression levels using different platforms or experimental protocols.

  3. Aptamer and its applications in neurodegenerative diseases.

    PubMed

    Qu, Jing; Yu, Shuqing; Zheng, Yuan; Zheng, Yan; Yang, Hui; Zhang, Jianliang

    2017-02-01

    Aptamers are small single-stranded DNA or RNA oligonucleotide fragments or small peptides, which can bind to targets by high affinity and specificity. Because aptamers are specific, non-immunogenic and non-toxic, they are ideal materials for clinical applications. Neurodegenerative disorders are ravaging the lives of patients. Even though the mechanism of these diseases is still elusive, they are mainly characterized by the accumulation of misfolded proteins in the central nervous system. So it is essential to develop potential measures to slow down or prevent the onset of these diseases. With the advancements of the technologies, aptamers have opened up new areas in this research field. Aptamers could bind with these related target proteins to interrupt their accumulation, subsequently blocking or preventing the process of neurodegenerative diseases. This review presents recent advances in the aptamer generation and its merits and limitations, with emphasis on its applications in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathy, Huntington's disease and multiple sclerosis.

  4. Aptamers against prion proteins and prions.

    PubMed

    Gilch, Sabine; Schätzl, Hermann M

    2009-08-01

    Prion diseases are fatal neurodegenerative and infectious disorders of humans and animals, characterized by structural transition of the host-encoded cellular prion protein (PrP(c)) into the aberrantly folded pathologic isoform PrP(Sc). RNA, DNA or peptide aptamers are classes of molecules which can be selected from complex combinatorial libraries for high affinity and specific binding to prion proteins and which might therefore be useful in diagnosis and therapy of prion diseases. Nucleic acid aptamers, which can be chemically synthesized, stabilized and immobilized, appear more suitable for diagnostic purposes, allowing use of PrP(Sc) as selection target. Peptide aptamers facilitate appropriate intracellular expression, targeting and re-routing without losing their binding properties to PrP, a requirement for potential therapeutic gene transfer experiments in vivo. Elucidation of structural properties of peptide aptamers might be used as basis for rational drug design, providing another attractive application of peptide aptamers in the search for effective anti-prion strategies.

  5. In Vitro Selection of DNA Aptamers to Glioblastoma Multiforme

    PubMed Central

    2011-01-01

    Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (Kd) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas. PMID:21892384

  6. High affinity truncated DNA aptamers for the development of fluorescence based progesterone biosensors.

    PubMed

    Alhadrami, Hani A; Chinnappan, Raja; Eissa, Shimaa; Rahamn, Anas Abdel; Zourob, Mohammed

    2017-02-24

    Aptamers have shown a number of potential applications in sensing and therapeutic due to the high affinity and specificity towards their target molecules. Not all the nucleotides in the full length aptamers are involved in the binding with their targets. The non-binding domain of the aptamer may affect the binding affinity of the aptamer-target complex. Mapping the aptamer binding region could increase the affinity and the specificity. In this paper, we designed aptamer-based fluorescence sensors from a truncated progesterone (P4) aptamer. Then, fluorescein and quencher labelled aptamer complementary oligonucleotide sequences were hybridized to the truncated aptamer at different sites to form duplex structures. We used fluorescence-quencher pair displacement assay upon progesterone binding for the determination of P4. One of the truncated sequences has shown high binding affinity with 16 fold increase in the dissociation constant, Kd (2.1 nM) compared to the original aptamer. The aptasensor was highly selective for P4 against similar compounds such as 17-β estradiol, bisphenol-A and vitamin D. The sensor has been applied for the detection of P4 in spiked tap water and in urine samples showing good recovery. This new developed aptamer-based fluorescence biosensor can be applied in food, pharmaceutical, and clinical industries.

  7. Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA).

    PubMed

    Soldevilla, Mario Martínez; Hervas, Sandra; Villanueva, Helena; Lozano, Teresa; Rabal, Obdulia; Oyarzabal, Julen; Lasarte, Juan José; Bendandi, Maurizio; Inoges, Susana; López-Díaz de Cerio, Ascensión; Pastor, Fernando

    2017-01-01

    LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

  8. tRNA-Related Sequences Trigger Systemic mRNA Transport in Plants[OPEN

    PubMed Central

    Zhang, Wenna; Kollwig, Gregor; Apelt, Federico; Walther, Dirk

    2016-01-01

    In plants, protein-coding mRNAs can move via the phloem vasculature to distant tissues, where they may act as non-cell-autonomous signals. Emerging work has identified many phloem-mobile mRNAs, but little is known regarding RNA motifs triggering mobility, the extent of mRNA transport, and the potential of transported mRNAs to be translated into functional proteins after transport. To address these aspects, we produced reporter transcripts harboring tRNA-like structures (TLSs) that were found to be enriched in the phloem stream and in mRNAs moving over chimeric graft junctions. Phenotypic and enzymatic assays on grafted plants indicated that mRNAs harboring a distinctive TLS can move from transgenic roots into wild-type leaves and from transgenic leaves into wild-type flowers or roots; these mRNAs can also be translated into proteins after transport. In addition, we provide evidence that dicistronic mRNA:tRNA transcripts are frequently produced in Arabidopsis thaliana and are enriched in the population of graft-mobile mRNAs. Our results suggest that tRNA-derived sequences with predicted stem-bulge-stem-loop structures are sufficient to mediate mRNA transport and seem to be necessary for the mobility of a large number of endogenous transcripts that can move through graft junctions. PMID:27268430

  9. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. © 2015 Lama and Ryan; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. An aptamer beacon responsive to botulinum toxins.

    PubMed

    Bruno, John G; Richarte, Alicia M; Carrillo, Maria P; Edge, Allison

    2012-01-15

    Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs.

  11. Screening of aptamers on microfluidic systems for clinical applications.

    PubMed

    Weng, Chen-Hsun; Huang, Chao-Jyun; Lee, Gwo-Bin

    2012-01-01

    The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications.

  12. Aptamers: Active Targeting Ligands for Cancer Diagnosis and Therapy

    PubMed Central

    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment. PMID:25699094

  13. Aptamers: active targeting ligands for cancer diagnosis and therapy.

    PubMed

    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment.

  14. Sequence and expression of ferredoxin mRNA in barley

    SciTech Connect

    Zielinski, R.; Funder, P.M.; Ling, V. )

    1990-05-01

    We have isolated and structurally characterized a full-length cDNA clone encoding ferredoxin from a {lambda}gt10 cDNA library prepared from barley leaf mRNA. The ferredoxin clone (pBFD-1) was fused head-to-head with a partial-length cDNA clone encoding calmodulin, and was fortuitously isolated by screening the library with a calmodulin-specific oligonucleotide probe. The mRNA sequence from which pBFD-1 was derived is expressed exclusively in the leaf tissues of 7-d old barley seedlings. Barley pre-ferredoxin has a predicted size of 15.3 kDal, of which 4.6 kDal are accounted for by the transit peptide. The polypeptide encoded by pBFD-1 is identical to wheat ferredoxin, and shares slightly more amino acid sequence similarity with spinach ferredoxin I than with ferredoxin II. Ferredoxin mRNA levels are rapidly increased 10-fold by white light in etiolated barley leaves.

  15. RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction.

    PubMed

    Dubey, Ashok K; Baker, Carol S; Romeo, Tony; Babitzke, Paul

    2005-10-01

    The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and the GU sequence being present in all but one ligand. The majority (51/55) of the RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition.

  16. Total RNA Sequencing Analysis of DCIS Progressing to Invasive Breast Cancer

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0080 TITLE: Total RNA Sequencing Analysis of DCIS Progressing to Invasive Breast Cancer. PRINCIPAL INVESTIGATOR...Aug 2015 4. TITLE AND SUBTITLE Total RNA Sequencing Analysis of DCIS Progressing to Invasive Breast Cancer. 5a. CONTRACT NUMBER 5b. GRANT...analysis of expression data on the entire range of informative RNA categories, including mRNA, miRNA, and lncRNA, as well as their splice variants

  17. Aptamer oligonucleotides as potential therapeutics in hematologic diseases.

    PubMed

    Li, Weibin; Zhao, Meng; Wang, Kaiyu; Yan, Huihui; Lan, XIaopeng

    2017-10-02

    Aptamers are single-stranded DNA or RNA oligonucleotides generated by a novel in vitro selection technique termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). During the past two decades, various aptamer drugs have been developed and many of them have entered into clinical trials. In the present review, we focus on aptamers as potential therapeutics for hematological diseases, including anemia of chronic inflammation (ACI) and anemia of chronic disease (ACD), hemophilia, thrombotic thrombocytopenic purpura (TTP) or VWD type-2B, and sickle cell disease (SCD), in particular, those that have entered into clinical trials. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer

    PubMed Central

    Gijs, Marlies; Penner, Gregory; Blackler, Garth B.; Impens, Nathalie R.E.N.; Baatout, Sarah; Luxen, André; Aerts, An M.

    2016-01-01

    Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer. PMID:27213406

  19. How to analyze gene expression using RNA-sequencing data.

    PubMed

    Ramsköld, Daniel; Kavak, Ersen; Sandberg, Rickard

    2012-01-01

    RNA-Seq is arising as a powerful method for transcriptome analyses that will eventually make microarrays obsolete for gene expression analyses. Improvements in high-throughput sequencing and efficient sample barcoding are now enabling tens of samples to be run in a cost-effective manner, competing with microarrays in price, excelling in performance. Still, most studies use microarrays, partly due to the ease of data analyses using programs and modules that quickly turn raw microarray data into spreadsheets of gene expression values and significant differentially expressed genes. Instead RNA-Seq data analyses are still in its infancy and the researchers are facing new challenges and have to combine different tools to carry out an analysis. In this chapter, we provide a tutorial on RNA-Seq data analysis to enable researchers to quantify gene expression, identify splice junctions, and find novel transcripts using publicly available software. We focus on the analyses performed in organisms where a reference genome is available and discuss issues with current methodology that have to be solved before RNA-Seq data can utilize its full potential.

  20. Building an RNA Sequencing Transcriptome of the Central Nervous System

    PubMed Central

    Dong, Xiaomin; You, Yanan; Wu, Jia Qian

    2015-01-01

    The composition and function of the central nervous system (CNS) is extremely complex. In addition to hundreds of subtypes of neurons, other cell types, including glia (astrocytes, oligodendrocytes, and microglia) and vascular cells (endothelial cells and pericytes) also play important roles in CNS function. Such heterogeneity makes the study of gene transcription in CNS challenging. Transcriptomic studies, namely the analyses of the expression levels and structures of all genes, are essential for interpreting the functional elements and understanding the molecular constituents of the CNS. Microarray has been a predominant method for large-scale gene expression profiling in the past. However, RNA-sequencing (RNA-Seq) technology developed in recent years has many advantages over microarrays, and has enabled building more quantitative, accurate, and comprehensive transcriptomes of the CNS and other systems. The discovery of novel genes, diverse alternative splicing events, and noncoding RNAs has remarkably expanded the complexity of gene expression profiles and will help us to understand intricate neural circuits. Here, we discuss the procedures and advantages of RNA-Seq technology in mammalian CNS transcriptome construction, and review the approaches of sample collection as well as recent progress in building RNA-Seq-based transcriptomes from tissue samples and specific cell types. PMID:26463470

  1. Chaining sequence/structure seeds for computing RNA similarity.

    PubMed

    Bourgeade, Laetitia; Chauve, Cédric; Allali, Julien

    2015-03-01

    We describe a new method to compare a query RNA with a static set of target RNAs. Our method is based on (i) a static indexing of the sequence/structure seeds of the target RNAs; (ii) searching the target RNAs by detecting seeds of the query present in the target, chaining these seeds in promising candidate homologs; and then (iii) completing the alignment using an anchor-based exact alignment algorithm. We apply our method on the benchmark Bralibase2.1 and compare its accuracy and efficiency with the exact method LocARNA and its recent seeds-based speed-up ExpLoc-P. Our pipeline RNA-unchained greatly improves computation time of LocARNA and is comparable to the one of ExpLoc-P, while improving the overall accuracy of the final alignments.

  2. Optimizing RNA structures by sequence extensions using RNAcop

    PubMed Central

    Hecker, Nikolai; Christensen-Dalsgaard, Mikkel; Seemann, Stefan E.; Havgaard, Jakob H.; Stadler, Peter F.; Hofacker, Ivo L.; Nielsen, Henrik; Gorodkin, Jan

    2015-01-01

    A key aspect of RNA secondary structure prediction is the identification of novel functional elements. This is a challenging task because these elements typically are embedded in longer transcripts where the borders between the element and flanking regions have to be defined. The flanking sequences impact the folding of the functional elements both at the level of computational analyses and when the element is extracted as a transcript for experimental analysis. Here, we analyze how different flanking region lengths impact folding into a constrained structure by computing probabilities of folding for different sizes of flanking regions. Our method, RNAcop (RNA context optimization by probability), is tested on known and de novo predicted structures. In vitro experiments support the computational analysis and suggest that for a number of structures, choosing proper lengths of flanking regions is critical. RNAcop is available as web server and stand-alone software via http://rth.dk/resources/rnacop. PMID:26283181

  3. Aptamer-based nanobiosensors.

    PubMed

    Kim, Yeon Seok; Raston, Nurul Hanun Ahmad; Gu, Man Bock

    2016-02-15

    It has been more than two decades since aptamer and the systematic evolution of ligands by exponential enrichment (SELEX) method were discovered by Larry Gold and Andrew Ellington in 1990, respectively. Based on the various advantages of aptamers, they have become a potent rival of antibodies in therapeutics and bio-analysis. Especially, the recent advances in aptamer biosensor application are remarkable due to its intrinsic properties of aptamers as nucleic acids and target induced conformational changes, in addition to the introduction of graphene oxide-based easy and simple immobilization-free screening method even for dual aptamers. In addition, the incorporation of various nanomaterials such as metallic nanoparticles, carbon materials, and functional nanospheres in aptasensors has facilitated the improvement of analytical performance and commercial application of aptasensors. In this review, recent prominent reports on aptasensors utilizing nanomaterials were introduced to understand the principle of aptamer-based biosensors and provide an insight for new strategies of aptasensors and the application of various nanomaterials. The perspective on aptamer-based biosensors and diagnostics was also discussed in view of technology and market.

  4. Prediction of uridine modifications in tRNA sequences.

    PubMed

    Panwar, Bharat; Raghava, Gajendra P S

    2014-10-02

    In past number of methods have been developed for predicting post-translational modifications in proteins. In contrast, limited attempt has been made to understand post-transcriptional modifications. Recently it has been shown that tRNA modifications play direct role in the genome structure and codon usage. This study is an attempt to understand kingdom-wise tRNA modifications particularly uridine modifications (UMs), as majority of modifications are uridine-derived. A three-steps strategy has been applied to develop an efficient method for the prediction of UMs. In the first step, we developed a common prediction model for all the kingdoms using a dataset from MODOMICS-2008. Support Vector Machine (SVM) based prediction models were developed and evaluated by five-fold cross-validation technique. Different approaches were applied and found that a hybrid approach of binary and structural information achieved highest Area under the curve (AUC) of 0.936. In the second step, we used newly added tRNA sequences (as independent dataset) of MODOMICS-2012 for the kingdom-wise prediction performance evaluation of previously developed (in the first step) common model and achieved performances between the AUC of 0.910 to 0.949. In the third and last step, we used different datasets from MODOMICS-2012 for the kingdom-wise individual prediction models development and achieved performances between the AUC of 0.915 to 0.987. The hybrid approach is efficient not only to predict kingdom-wise modifications but also to classify them into two most prominent UMs: Pseudouridine (Y) and Dihydrouridine (D). A webserver called tRNAmod (http://crdd.osdd.net/raghava/trnamod/) has been developed, which predicts UMs from both tRNA sequences and whole genome.

  5. Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

    DOE PAGES

    Ilgu, Muslum; Ray, Judhajeet; Bendickson, Lee; ...

    2015-12-17

    The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated twomore » light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. As a result, high background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.« less

  6. Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

    SciTech Connect

    Ilgu, Muslum; Ray, Judhajeet; Bendickson, Lee; Wang, Tianjiao; Geraskin, Ivan M.; Kraus, George A.; Nilsen-Hamilton, Marit

    2015-12-17

    The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. As a result, high background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

  7. Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

    PubMed Central

    Ilgu, Muslum; Ray, Judhajeet; Bendickson, Lee; Wang, Tianjiao; Geraskin, Ivan; Kraus, George A; Nilsen-Hamilton, Marit

    2016-01-01

    The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitability as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription. PMID:26707205

  8. Legume genomics: understanding biology through DNA and RNA sequencing

    PubMed Central

    O'Rourke, Jamie A.; Bolon, Yung-Tsi; Bucciarelli, Bruna; Vance, Carroll P.

    2014-01-01

    Background The legume family (Leguminosae) consists of approx. 17 000 species. A few of these species, including, but not limited to, Phaseolus vulgaris, Cicer arietinum and Cajanus cajan, are important dietary components, providing protein for approx. 300 million people worldwide. Additional species, including soybean (Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly in animal feed. In addition, legumes are important contributors to biological nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and providing up to 30 % of available nitrogen for the next season of crops. The application of high-throughput genomic technologies including genome sequencing projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) by the legume research community has provided major insights into genome evolution, genomic architecture and domestication. Scope and Conclusions This review presents an overview of the current state of legume genomics and explores the role that next-generation sequencing technologies play in advancing legume genomics. The adoption of next-generation sequencing and implementation of associated bioinformatic tools has allowed researchers to turn each species of interest into their own model organism. To illustrate the power of next-generation sequencing, an in-depth overview of the transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The soybean transcriptome focuses on analysing seed development in two near-isogenic lines, examining the role of transporters, oil biosynthesis and nitrogen utilization. The white lupin transcriptome analysis examines how phosphate deficiency alters gene expression patterns, inducing the formation of cluster roots. Such studies illustrate the power of next-generation sequencing and bioinformatic analyses in elucidating the gene networks underlying biological processes. PMID:24769535

  9. Screening and development of DNA aptamers specific to several oral pathogens.

    PubMed

    Park, Jung-Pyo; Shin, Hye Joo; Park, Suk-Gyun; Oh, Hee-Kyun; Choi, Choong-Ho; Park, Hong-Ju; Kook, Min-Suk; Ohk, Seung-Ho

    2015-03-01

    Aptamers are composed of single-stranded oilgonucleotides that can selectively bind desired molecules. It has been reported that RNA or DNA could act as not only a genetic messenger but also a catalyst in metabolic pathways. RNA aptamers (average sizes 40-50 bp) are smaller than antibodies and have strong binding capacities to target molecules, similar to antigen-antibody interactions. Once an aptamer was selected, it can be readily produced in large quantities at low cost. The objectives of this study are to screen and develop aptamers specific to oral pathogens such as Porphyromonas gingivalis, Treponema denticola, and Streptococcus mutans. The bacterial cell pellet was fixed with formaldehyde as a target molecule for the screening of aptamers. The SELEX method was used for the screening of aptamers and a modified western blot analysis was used to verify their specificities. Through SELEX, 40 kinds of aptamers were selected and the specificity of the aptamers to the bacterial cells was confirmed by modified western blot analysis. Through the SELEX method, 40 aptamers that specifically bind to oral pathogens were screened and isolated. The aptamers showed possibility as effective candidates for the detection agents of oral infections.

  10. Determining breast cancer histological grade from RNA-sequencing data.

    PubMed

    Wang, Mei; Klevebring, Daniel; Lindberg, Johan; Czene, Kamila; Grönberg, Henrik; Rantalainen, Mattias

    2016-05-10

    The histologic grade (HG) of breast cancer is an established prognostic factor. The grade is usually reported on a scale ranging from 1 to 3, where grade 3 tumours are the most aggressive. However, grade 2 is associated with an intermediate risk of recurrence, and carries limited information for clinical decision-making. Patients classified as grade 2 are at risk of both under- and over-treatment. RNA-sequencing analysis was conducted in a cohort of 275 women diagnosed with invasive breast cancer. Multivariate prediction models were developed to classify tumours into high and low transcriptomic grade (TG) based on gene- and isoform-level expression data from RNA-sequencing. HG2 tumours were reclassified according to the prediction model and a recurrence-free survival analysis was performed by the multivariate Cox proportional hazards regression model to assess to what extent the TG model could be used to stratify patients. The prediction model was validated in N=487 breast cancer cases from the The Cancer Genome Atlas (TCGA) data set. Differentially expressed genes and isoforms associated with HGs were analysed using linear models. The classification of grade 1 and grade 3 tumours based on RNA-sequencing data achieved high accuracy (area under the receiver operating characteristic curve = 0.97). The association between recurrence-free survival rate and HGs was confirmed in the study population (hazard ratio of grade 3 versus 1 was 2.62 with 95 % confidence interval = 1.04-6.61). The TG model enabled us to reclassify grade 2 tumours as high TG and low TG gene or isoform grade. The risk of recurrence in the high TG group of grade 2 tumours was higher than in low TG group (hazard ratio = 2.43, 95 % confidence interval = 1.13-5.20). We found 8200 genes and 13,809 isoforms that were differentially expressed between HG1 and HG3 breast cancer tumours. Gene- and isoform-level expression data from RNA-sequencing could be utilised to differentiate HG1 and HG3 tumours with

  11. Approaches to sequence analysis of 125I-labeled RNA.

    PubMed Central

    Dickson, E; Pape, L K; Robertson, H D

    1979-01-01

    A method is described for the initial steps of sequence analysis of RNase T1-and pancreatic RN-ase-resistant oligonucleotides of RNA containing cytidylate residues labeled in vitro with 125I. In many cases an oligonucleotide sequence can be deduced from a consideration of (i) its relative position in the two-dimensional fingerprint (with DEAE thin layer homochromatographic second dimension), (ii) its electrophoretic mobility on DEAE paper at pH 1.9, and (iii) identification of its products of further enzymatic digestion by comparison with a set of marker oligonucleotides. Additional methods including analysis of oligonucleotides following chemical blocking of uridylate residues with CMCT and analysis of products of incomplete enzymatic digestion are also discussed. Images PMID:106369

  12. Tetramerization of an RNA oligonucleotide containing a GGGG sequence.

    PubMed

    Kim, J; Cheong, C; Moore, P B

    1991-05-23

    Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5'-GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G-quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature.

  13. Gene regulation: ancient microRNA target sequences in plants.

    PubMed

    Floyd, Sandra K; Bowman, John L

    2004-04-01

    MicroRNAs are an abundant class of small RNAs that are thought to regulate the expression of protein-coding genes in plants and animals. Here we show that the target sequence of two microRNAs, known to regulate genes in the class-III homeodomain-leucine zipper (HD-Zip) gene family of the flowering plant Arabidopsis, is conserved in homologous sequences from all lineages of land plants, including bryophytes, lycopods, ferns and seed plants. We also find that the messenger RNAs from these genes are cleaved within the same microRNA-binding site in representatives of each land-plant group, as they are in Arabidopsis. Our results indicate not only that microRNAs mediate gene regulation in non-flowering as well as flowering plants, but also that the regulation of this class of plant genes dates back more than 400 million years.

  14. Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation

    PubMed Central

    Roy, Christian K; Olson, Sara; Graveley, Brenton R; Zamore, Phillip D; Moore, Melissa J

    2015-01-01

    Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA–DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1. DOI: http://dx.doi.org/10.7554/eLife.03700.001 PMID:25866926

  15. Aptamer for imaging and therapeutic targeting of brain tumor glioblastoma.

    PubMed

    Delač, Mateja; Motaln, Helena; Ulrich, Henning; Lah, Tamara T

    2015-09-01

    Aptamers are short single-stranded nucleic acids (RNA or ssDNA), identified by an in vitro selection process, denominated SELEX, from a partially random oligonucleotide library. They bind to a molecular target, a protein or other complex macromolecular structures of interest with high affinity and specificity, comparable to those of antibodies. Recently, aptamer selection protocols were developed for targeting living cells, including tumors. Chemical modifications of the aptamers and modalities of their detection and delivery systems are already available with high selectivity and targeting ability for the desired cancer cell type, making them promising for diagnosis and therapy. Glioblastoma multiformae represents the most malignant and fatal stage of glioma, and is also the most frequent brain tumor. Glioblastoma-specific aptamers were developed by either targeting the whole cell surface or known glioma biomarkers. These aptamers may gain importance for imaging, tumor cell isolation from biopsies and drug delivery. In biomedical imaging techniques, aptamers coupled with radionuclide or fluorescent labels, bioconjugates and nanoparticles offer an advanced, noninvasive manner for defining the glioblastoma tissue border. Though single modality aptamer imaging probes have some limitations, these are overcome by the use of multimodal probes. Due to selectivity and chemical characteristics, aptamers can be coupled to functionalized nanoparticles and loaded with a drug, appeared promising for in vivo targeting of glioblastoma. Finally, aptamers are effective mediators for gene silencing when coupled to small interfering RNA and a viral vector, thus providing a novel tool with enhanced targeting capability in drug delivery, designed for tailored treatment of glioblastoma patients.

  16. Aptamer-based electrochemical sensors with aptamer-complementary DNA oligonucleotides as probe.

    PubMed

    Lu, Ying; Li, Xianchan; Zhang, Limin; Yu, Ping; Su, Lei; Mao, Lanqun

    2008-03-15

    This study describes a facile and general strategy for the development of aptamer-based electrochemical sensors with a high specificity toward the targets and a ready regeneration feature. Very different from the existing strategies for the development of electrochemical aptasensors with the aptamers as the probes, the strategy proposed here is essentially based on the utilization of the aptamer-complementary DNA (cDNA) oligonucleotides as the probes for electrochemical sensing. In this context, the sequences at both ends of the cDNA are tailor-made to be complementary and both the redox moiety (i.e., ferrocene in this study) and thiol group are labeled onto the cDNA. The labeled cDNA are hybridized with their respective aptamers (i.e., ATP- and thrombin-binding aptamers in this study) to form double-stranded DNA (ds-DNA) and the electrochemical aptasensors are prepared by self-assembling the labeled ds-DNA onto Au electrodes. Upon target binding, the aptamers confined onto electrode surface dissociate from their respective cDNA oligonucleotides into the solution and the single-stranded cDNA could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such a conformational change of the cDNA resulting from the target binding-induced dissociation of the aptamers essentially leads to the change in the voltammetric signal of the redox moiety labeled onto the cDNA and thus constitutes the mechanism for the electrochemical aptasensors for specific target sensing. The aptasensors demonstrated here with the cDNA as the probe are readily regenerated and show good responses toward the targets. This study may offer a new and relatively general approach to electrochemical aptasensors with good analytical properties and potential applications.

  17. Long Non-Coding RNA and Alternative Splicing Modulations in Parkinson's Leukocytes Identified by RNA Sequencing

    PubMed Central

    Soreq, Lilach; Guffanti, Alessandro; Salomonis, Nathan; Simchovitz, Alon; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

    2014-01-01

    The continuously prolonged human lifespan is accompanied by increase in neurodegenerative diseases incidence, calling for the development of inexpensive blood-based diagnostics. Analyzing blood cell transcripts by RNA-Seq is a robust means to identify novel biomarkers that rapidly becomes a commonplace. However, there is lack of tools to discover novel exons, junctions and splicing events and to precisely and sensitively assess differential splicing through RNA-Seq data analysis and across RNA-Seq platforms. Here, we present a new and comprehensive computational workflow for whole-transcriptome RNA-Seq analysis, using an updated version of the software AltAnalyze, to identify both known and novel high-confidence alternative splicing events, and to integrate them with both protein-domains and microRNA binding annotations. We applied the novel workflow on RNA-Seq data from Parkinson's disease (PD) patients' leukocytes pre- and post- Deep Brain Stimulation (DBS) treatment and compared to healthy controls. Disease-mediated changes included decreased usage of alternative promoters and N-termini, 5′-end variations and mutually-exclusive exons. The PD regulated FUS and HNRNP A/B included prion-like domains regulated regions. We also present here a workflow to identify and analyze long non-coding RNAs (lncRNAs) via RNA-Seq data. We identified reduced lncRNA expression and selective PD-induced changes in 13 of over 6,000 detected leukocyte lncRNAs, four of which were inversely altered post-DBS. These included the U1 spliceosomal lncRNA and RP11-462G22.1, each entailing sequence complementarity to numerous microRNAs. Analysis of RNA-Seq from PD and unaffected controls brains revealed over 7,000 brain-expressed lncRNAs, of which 3,495 were co-expressed in the leukocytes including U1, which showed both leukocyte and brain increases. Furthermore, qRT-PCR validations confirmed these co-increases in PD leukocytes and two brain regions, the amygdala and substantia

  18. SEXCMD: Development and validation of sex marker sequences for whole-exome/genome and RNA sequencing.

    PubMed

    Jeong, Seongmun; Kim, Jiwoong; Park, Won; Jeon, Hongmin; Kim, Namshin

    2017-01-01

    Over the last decade, a large number of nucleotide sequences have been generated by next-generation sequencing technologies and deposited to public databases. However, most of these datasets do not specify the sex of individuals sampled because researchers typically ignore or hide this information. Male and female genomes in many species have distinctive sex chromosomes, XX/XY and ZW/ZZ, and expression levels of many sex-related genes differ between the sexes. Herein, we describe how to develop sex marker sequences from syntenic regions of sex chromosomes and use them to quickly identify the sex of individuals being analyzed. Array-based technologies routinely use either known sex markers or the B-allele frequency of X or Z chromosomes to deduce the sex of an individual. The same strategy has been used with whole-exome/genome sequence data; however, all reads must be aligned onto a reference genome to determine the B-allele frequency of the X or Z chromosomes. SEXCMD is a pipeline that can extract sex marker sequences from reference sex chromosomes and rapidly identify the sex of individuals from whole-exome/genome and RNA sequencing after training with a known dataset through a simple machine learning approach. The pipeline counts total numbers of hits from sex-specific marker sequences and identifies the sex of the individuals sampled based on the fact that XX/ZZ samples do not have Y or W chromosome hits. We have successfully validated our pipeline with mammalian (Homo sapiens; XY) and avian (Gallus gallus; ZW) genomes. Typical calculation time when applying SEXCMD to human whole-exome or RNA sequencing datasets is a few minutes, and analyzing human whole-genome datasets takes about 10 minutes. Another important application of SEXCMD is as a quality control measure to avoid mixing samples before bioinformatics analysis. SEXCMD comprises simple Python and R scripts and is freely available at https://github.com/lovemun/SEXCMD.

  19. Nascent RNA sequencing reveals distinct features in plant transcription

    PubMed Central

    Hetzel, Jonathan; Duttke, Sascha H.; Benner, Christopher; Chory, Joanne

    2016-01-01

    Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5′GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved “TGT” core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole. PMID:27729530

  20. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences

    PubMed Central

    Laslett, Dean; Canback, Bjorn

    2004-01-01

    A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the ‘green’ algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags. PMID:14704338

  1. Aptamers against pathogenic microorganisms

    PubMed Central

    Davydova, Anna; Vorobjeva, Maria; Pyshnyi, Dmitrii; Altman, Sidney; Vlassov, Valentin; Venyaminova, Alya

    2016-01-01

    Abstract An important current issue of modern molecular medicine and biotechnology is the search for new approaches to early diagnostic assays and adequate therapy of infectious diseases. One of the promising solutions to this problem might be a development of nucleic acid aptamers capable of interacting specifically with bacteria, protozoa, and viruses. Such aptamers can be used for the specific recognition of infectious agents as well as for blocking of their functions. The present review summarizes various modern SELEX techniques used in this field, and of several currently identified aptamers against viral particles and unicellular organisms, and their applications. The prospects of applying nucleic acid aptamers for the development of novel detection systems and antibacterial and antiviral drugs are discussed. PMID:26258445

  2. Analysis of sequencing data for probing RNA secondary structures and protein-RNA binding in studying posttranscriptional regulations.

    PubMed

    Hu, Xihao; Wu, Yang; Lu, Zhi John; Yip, Kevin Y

    2016-11-01

    High-throughput sequencing has been used to study posttranscriptional regulations, where the identification of protein-RNA binding is a major and fast-developing sub-area, which is in turn benefited by the sequencing methods for whole-transcriptome probing of RNA secondary structures. In the study of RNA secondary structures using high-throughput sequencing, bases are modified or cleaved according to their structural features, which alter the resulting composition of sequencing reads. In the study of protein-RNA binding, methods have been proposed to immuno-precipitate (IP) protein-bound RNA transcripts in vitro or in vivo By sequencing these transcripts, the protein-RNA interactions and the binding locations can be identified. For both types of data, read counts are affected by a combination of confounding factors, including expression levels of transcripts, sequence biases, mapping errors and the probing or IP efficiency of the experimental protocols. Careful processing of the sequencing data and proper extraction of important features are fundamentally important to a successful analysis. Here we review and compare different experimental methods for probing RNA secondary structures and binding sites of RNA-binding proteins (RBPs), and the computational methods proposed for analyzing the corresponding sequencing data. We suggest how these two types of data should be integrated to study the structural properties of RBP binding sites as a systematic way to better understand posttranscriptional regulations.

  3. Use of Unamplified RNA/cDNA–Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses

    PubMed Central

    Kilianski, Andy; Roth, Pierce A.; Liem, Alvin T.; Hill, Jessica M.; Willis, Kristen L.; Rossmaier, Rebecca D.; Marinich, Andrew V.; Maughan, Michele N.; Karavis, Mark A.; Kuhn, Jens H.; Honko, Anna N.

    2016-01-01

    Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. PMID:27191483

  4. Assessing the 5S ribosomal RNA heterogeneity in Arabidopsis thaliana using short RNA next generation sequencing data.

    PubMed

    Szymanski, Maciej; Karlowski, Wojciech M

    2016-01-01

    In eukaryotes, ribosomal 5S rRNAs are products of multigene families organized within clusters of tandemly repeated units. Accumulation of genomic data obtained from a variety of organisms demonstrated that the potential 5S rRNA coding sequences show a large number of variants, often incompatible with folding into a correct secondary structure. Here, we present results of an analysis of a large set of short RNA sequences generated by the next generation sequencing techniques, to address the problem of heterogeneity of the 5S rRNA transcripts in Arabidopsis and identification of potentially functional rRNA-derived fragments.

  5. Programmable release of multiple protein drugs from aptamer-functionalized hydrogels via nucleic acid hybridization.

    PubMed

    Battig, Mark R; Soontornworajit, Boonchoy; Wang, Yong

    2012-08-01

    Polymeric delivery systems have been extensively studied to achieve localized and controlled release of protein drugs. However, it is still challenging to control the release of multiple protein drugs in distinct stages according to the progress of disease or treatment. This study successfully demonstrates that multiple protein drugs can be released from aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. Because both aptamer-protein interactions and aptamer-CS hybridization are sequence-specific, aptamer-functionalized hydrogels constitute a promising polymeric delivery system for the programmable release of multiple protein drugs to treat complex human diseases.

  6. Fluorescent Aptamer Sensors

    NASA Astrophysics Data System (ADS)

    Chen, Hui William; Kim, Youngmi; Meng, Ling; Mallikaratchy, Prabodhika; Martin, Jennifer; Tang, Zhiwen; Shangguan, Dihua; O'Donoghue, Meghan; Tan, Weihong

    Aptamers are single-stranded nucleic acid probes that can be evolved to have high specificity and affinity for different targets. These targets include biomar-ker proteins, small molecules, and even whole live cells that express a variety of surface proteins of interest. Aptamers offer several advantages over protein-based molecular probes such as low immunogenic activity, flexible modification, and in vitro synthesis. In addition, aptamers used as molecular probes can be made with easy signaling for binding with their corresponding targets. There are a few different fluorescence-based signal transduction mechanisms, such as direct fluorophore labeling, fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence anisotropy, and light-switching excimers. These signaling processes in combination with various labeling strategies of nucleic acid aptamers contribute to simple, rapid, sensitive, and selective biological assays. In this chapter, we discuss the optical signaling of aptamers for single proteins such as α-thrombin and platelet-derived growth factor (PDGF). We also present detailed discussion about fluorescent aptamers developed from cell-based systematic evolution of ligands by exponential enrichment (SELEX) for the recognition of different target tumor cells.

  7. DNA Aptamers to Human Immunodeficiency Virus Reverse Transcriptase Selected by a Primer-Free SELEX Method: Characterization and Comparison with Other Aptamers

    PubMed Central

    Lai, Yi-Tak

    2012-01-01

    A 30-nucleotide DNA aptamer (5′-AGGAAGGCTTTAGGTCTGAGATCTCGGAAT-3′, denoted PF1) selected for high affinity to human immunodeficiency virus reverse transcriptase (HIV RT) using a primer-free SELEX (systematic evolution of ligands by exponential enrichment) method was characterized to determine features promoting tight binding. PF1's equilibrium dissociation constant for RT was ∼80 nM, over 10-fold lower than a random 30-mer. Changing the 2 terminal diguanosine repeats (underlined above) to diadenosine or dithymidine modestly decreased binding. Any changes to the 2 central diguanosines dramatically decreased binding. Binding was highly sensitive to length, with any truncations that deleted part of the 4 diguanosine motifs resulting in a 6-fold or more decrease in affinity. Even a construct with all the diguanosine motifs but lacking the 5′ terminal A and 3 nucleotides at the 3′ end showed ∼3-fold binding decrease. Changes to the nucleotides between the diguanosines, even those that did not alter PF1's low secondary structure (free energy of folding ΔG=−0.61 kcal/mol), dramatically decreased binding, suggesting sequence specificity. Despite the diguanosine motifs, circular dichroism (CD) spectra indicated that PF1 did not form a G-quartet. PF1 inhibited HIV RT synthesis with a half-maximal inhibitory value (IC50) of ∼60 nM. Larger, more structured RT DNA aptamers based on the HIV polypurine tract and those that formed G-quartets (denoted S4 and R1T) were more potent inhibitors, with IC50 values of ∼4 and ∼1 nM, respectively. An RNA pseudoknot aptamer (denoted 1.1) showed an IC50 near 4 nM. Competition binding assays with PF1 and several previously characterized RT aptamers indicated that they all bound at or near the primer–template pocket. These other more structured and typically larger aptamers bound more tightly than PF1 to RT based on filter binding assays. Results indicate that PF1 represents a new class of RT aptamers that are

  8. Improved definition of the mouse transcriptome via targeted RNA sequencing.

    PubMed

    Bussotti, Giovanni; Leonardi, Tommaso; Clark, Michael B; Mercer, Tim R; Crawford, Joanna; Malquori, Lorenzo; Notredame, Cedric; Dinger, Marcel E; Mattick, John S; Enright, Anton J

    2016-05-01

    Targeted RNA sequencing (CaptureSeq) uses oligonucleotide probes to capture RNAs for sequencing, providing enriched read coverage, accurate measurement of gene expression, and quantitative expression data. We applied CaptureSeq to refine transcript annotations in the current murine GRCm38 assembly. More than 23,000 regions corresponding to putative or annotated long noncoding RNAs (lncRNAs) and 154,281 known splicing junction sites were selected for targeted sequencing across five mouse tissues and three brain subregions. The results illustrate that the mouse transcriptome is considerably more complex than previously thought. We assemble more complete transcript isoforms than GENCODE, expand transcript boundaries, and connect interspersed islands of mapped reads. We describe a novel filtering pipeline that identifies previously unannotated but high-quality transcript isoforms. In this set, 911 GENCODE neighboring genes are condensed into 400 expanded gene models. Additionally, 594 GENCODE lncRNAs acquire an open reading frame (ORF) when their structure is extended with CaptureSeq. Finally, we validate our observations using current FANTOM and Mouse ENCODE resources. © 2016 Bussotti et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Oligonucleotide Hybridization Combined with Competitive Antibody Binding for the Truncation of a High-Affinity Aptamer.

    PubMed

    Vu, Cong Quang; Rotkrua, Pichayanoot; Tantirungrotechai, Yuthana; Soontornworajit, Boonchoy

    2017-10-09

    Truncation can enhance the affinity of aptamers for their targets by limiting nonessential segments and therefore limiting the molecular degrees of freedom that must be overcome in the binding process. This study demonstrated a truncation protocol relying on competitive antibody binding and the hybridization of complementary oligonucleotides, using platelet derived growth factor BB (PDGF-BB) as the model target. On the basis of the immunoassay results, an initial long aptamer was truncated to a number of sequences with lengths of 36-40 nucleotides (nt). These sequences showed apparent KD values in the picomolar range, with the best case being a 36-nt truncated aptamer with a 150-fold increase in affinity over the full-length aptamer. The observed binding energies correlated well with relative energies calculated by molecular dynamics simulations. The effect of the truncated aptamer on PDGF-BB-stimulated fibroblasts was found to be equivalent to that of the full-length aptamer.

  10. Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

    PubMed Central

    Shi, Shali; Yu, Xiaoyan; Gao, Yimin; Xue, Binbin; Wu, Xinjiao; Wang, Xiaohong; Yang, Darong

    2014-01-01

    Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C. PMID:24307579

  11. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

  12. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed Central

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase. PMID:284320

  13. Deciphering Poxvirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Cao, Shuai; Martens, Craig A.; Porcella, Stephen F.; Xie, Zhi; Ma, Ming; Shen, Ben

    2015-01-01

    ABSTRACT The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. IMPORTANCE This report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological

  14. Transcriptional profiling of bovine milk using RNA sequencing

    PubMed Central

    2012-01-01

    Background Cow milk is a complex bioactive fluid consumed by humans beyond infancy. Even though the chemical and physical properties of cow milk are well characterized, very limited research has been done on characterizing the milk transcriptome. This study performs a comprehensive expression profiling of genes expressed in milk somatic cells of transition (day 15), peak (day 90) and late (day 250) lactation Holstein cows by RNA sequencing. Milk samples were collected from Holstein cows at 15, 90 and 250 days of lactation, and RNA was extracted from the pelleted milk cells. Gene expression analysis was conducted by Illumina RNA sequencing. Sequence reads were assembled and analyzed in CLC Genomics Workbench. Gene Ontology (GO) and pathway analysis were performed using the Blast2GO program and GeneGo application of MetaCore program. Results A total of 16,892 genes were expressed in transition lactation, 19,094 genes were expressed in peak lactation and 18,070 genes were expressed in late lactation. Regardless of the lactation stage approximately 9,000 genes showed ubiquitous expression. Genes encoding caseins, whey proteins and enzymes in lactose synthesis pathway showed higher expression in early lactation. The majority of genes in the fat metabolism pathway had high expression in transition and peak lactation milk. Most of the genes encoding for endogenous proteases and enzymes in ubiquitin-proteasome pathway showed higher expression along the course of lactation. Conclusions This is the first study to describe the comprehensive bovine milk transcriptome in Holstein cows. The results revealed that 69% of NCBI Btau 4.0 annotated genes are expressed in bovine milk somatic cells. Most of the genes were ubiquitously expressed in all three stages of lactation. However, a fraction of the milk transcriptome has genes devoted to specific functions unique to the lactation stage. This indicates the ability of milk somatic cells to adapt to different molecular functions

  15. Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses

    PubMed Central

    2012-01-01

    Background Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF), dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications. Results Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA) was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA) emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow chromatographic assays and a

  16. A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

    PubMed Central

    Landgraf, Pablo; Rusu, Mirabela; Sheridan, Robert; Sewer, Alain; Iovino, Nicola; Aravin, Alexei; Pfeffer, Sébastien; Rice, Amanda; Kamphorst, Alice O.; Landthaler, Markus; Lin, Carolina; Socci, Nicholas D.; Hermida, Leandro; Fulci, Valerio; Chiaretti, Sabina; Foà, Robin; Schliwka, Julia; Fuchs, Uta; Novosel, Astrid; Müller, Roman-Ulrich; Schermer, Bernhard; Bissels, Ute; Inman, Jason; Phan, Quang; Chien, Minchen; Weir, David B.; Choksi, Ruchi; De Vita, Gabriella; Frezzetti, Daniela; Trompeter, Hans-Ingo; Hornung, Veit; Teng, Grace; Hartmann, Gunther; Palkovits, Miklos; Di Lauro, Roberto; Wernet, Peter; Macino, Giuseppe; Rogler, Charles E.; Nagle, James W.; Ju, Jingyue; Papavasiliou, F. Nina; Benzing, Thomas; Lichter, Peter; Tam, Wayne; Brownstein, Michael J.; Bosio, Andreas; Borkhardt, Arndt; Russo, James J.; Sander, Chris; Zavolan, Mihaela; Tuschl, Thomas

    2007-01-01

    Summary MicroRNAs (miRNAs) are small non-coding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents, enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide novel computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the difference in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses. PMID:17604727

  17. Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications

    PubMed Central

    Kimoto, Michiko; Nakamura, Mana; Hirao, Ichiro

    2016-01-01

    A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G–C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165. Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165. PMID:27387284

  18. RNA expression profile of calcified bicuspid, tricuspid, and normal human aortic valves by RNA sequencing.

    PubMed

    Guauque-Olarte, Sandra; Droit, Arnaud; Tremblay-Marchand, Joël; Gaudreault, Nathalie; Kalavrouziotis, Dimitri; Dagenais, Francois; Seidman, Jonathan G; Body, Simon C; Pibarot, Philippe; Mathieu, Patrick; Bossé, Yohan

    2016-10-01

    The molecular mechanisms leading to premature development of aortic valve stenosis (AS) in individuals with a bicuspid aortic valve are unknown. The objective of this study was to identify genes differentially expressed between calcified bicuspid aortic valves (BAVc) and tricuspid valves with (TAVc) and without (TAVn) AS using RNA sequencing (RNA-Seq). We collected 10 human BAVc and nine TAVc from men who underwent primary aortic valve replacement. Eight TAVn were obtained from men who underwent heart transplantation. mRNA levels were measured by RNA-Seq and compared between valve groups. Two genes were upregulated, and none were downregulated in BAVc compared with TAVc, suggesting a similar gene expression response to AS in individuals with bicuspid and tricuspid valves. There were 462 genes upregulated and 282 downregulated in BAVc compared with TAVn. In TAVc compared with TAVn, 329 genes were up- and 170 were downregulated. A total of 273 upregulated and 147 downregulated genes were concordantly altered between BAVc vs. TAVn and TAVc vs. TAVn, which represent 56 and 84% of significant genes in the first and second comparisons, respectively. This indicates that extra genes and pathways were altered in BAVc. Shared pathways between calcified (BAVc and TAVc) and normal (TAVn) aortic valves were also more extensively altered in BAVc. The top pathway enriched for genes differentially expressed in calcified compared with normal valves was fibrosis, which support the remodeling process as a therapeutic target. These findings are relevant to understand the molecular basis of AS in patients with bicuspid and tricuspid valves.

  19. Comparative RNA sequencing reveals substantial genetic variation in endangered primates.

    PubMed

    Perry, George H; Melsted, Páll; Marioni, John C; Wang, Ying; Bainer, Russell; Pickrell, Joseph K; Michelini, Katelyn; Zehr, Sarah; Yoder, Anne D; Stephens, Matthew; Pritchard, Jonathan K; Gilad, Yoav

    2012-04-01

    Comparative genomic studies in primates have yielded important insights into the evolutionary forces that shape genetic diversity and revealed the likely genetic basis for certain species-specific adaptations. To date, however, these studies have focused on only a small number of species. For the majority of nonhuman primates, including some of the most critically endangered, genome-level data are not yet available. In this study, we have taken the first steps toward addressing this gap by sequencing RNA from the livers of multiple individuals from each of 16 mammalian species, including humans and 11 nonhuman primates. Of the nonhuman primate species, five are lemurs and two are lorisoids, for which little or no genomic data were previously available. To analyze these data, we developed a method for de novo assembly and alignment of orthologous gene sequences across species. We assembled an average of 5721 gene sequences per species and characterized diversity and divergence of both gene sequences and gene expression levels. We identified patterns of variation that are consistent with the action of positive or directional selection, including an 18-fold enrichment of peroxisomal genes among genes whose regulation likely evolved under directional selection in the ancestral primate lineage. Importantly, we found no relationship between genetic diversity and endangered status, with the two most endangered species in our study, the black and white ruffed lemur and the Coquerel's sifaka, having the highest genetic diversity among all primates. Our observations imply that many endangered lemur populations still harbor considerable genetic variation. Timely efforts to conserve these species alongside their habitats have, therefore, strong potential to achieve long-term success.

  20. Comparative RNA sequencing reveals substantial genetic variation in endangered primates

    PubMed Central

    Perry, George H.; Melsted, Páll; Marioni, John C.; Wang, Ying; Bainer, Russell; Pickrell, Joseph K.; Michelini, Katelyn; Zehr, Sarah; Yoder, Anne D.; Stephens, Matthew; Pritchard, Jonathan K.; Gilad, Yoav

    2012-01-01

    Comparative genomic studies in primates have yielded important insights into the evolutionary forces that shape genetic diversity and revealed the likely genetic basis for certain species-specific adaptations. To date, however, these studies have focused on only a small number of species. For the majority of nonhuman primates, including some of the most critically endangered, genome-level data are not yet available. In this study, we have taken the first steps toward addressing this gap by sequencing RNA from the livers of multiple individuals from each of 16 mammalian species, including humans and 11 nonhuman primates. Of the nonhuman primate species, five are lemurs and two are lorisoids, for which little or no genomic data were previously available. To analyze these data, we developed a method for de novo assembly and alignment of orthologous gene sequences across species. We assembled an average of 5721 gene sequences per species and characterized diversity and divergence of both gene sequences and gene expression levels. We identified patterns of variation that are consistent with the action of positive or directional selection, including an 18-fold enrichment of peroxisomal genes among genes whose regulation likely evolved under directional selection in the ancestral primate lineage. Importantly, we found no relationship between genetic diversity and endangered status, with the two most endangered species in our study, the black and white ruffed lemur and the Coquerel's sifaka, having the highest genetic diversity among all primates. Our observations imply that many endangered lemur populations still harbor considerable genetic variation. Timely efforts to conserve these species alongside their habitats have, therefore, strong potential to achieve long-term success. PMID:22207615

  1. Optimization of shRNA inhibitors by variation of the terminal loop sequence.

    PubMed

    Schopman, Nick C T; Liu, Ying Poi; Konstantinova, Pavlina; ter Brake, Olivier; Berkhout, Ben

    2010-05-01

    Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA. Most studies use the original shRNA design of the pSuper system, but no extensive study regarding optimization of the shRNA loop sequence has been performed. We tested the impact of different hairpin loop sequences, varying in size and structure, on the activity of a set of shRNAs targeting HIV-1. We were able to transform weak inhibitors into intermediate or even strong shRNA inhibitors by replacing the loop sequence. We demonstrate that the efficacy of these optimized shRNA inhibitors is improved significantly in different cell types due to increased siRNA production. These results indicate that the loop sequence is an essential part of the shRNA design. The optimized shRNA loop sequence is generally applicable for RNAi knockdown studies, and will allow us to develop a more potent gene therapy against HIV-1.

  2. A Small Aptamer with Strong and Specific Recognition of the Triphosphate of ATP

    PubMed Central

    Sazani, Peter L.; Larralde, Rosa

    2004-01-01

    We report the in vitro selection of an RNA-based ATP aptamer with the ability to discriminate between adenosine ligands based on their 5‘ phosphorylation state. Previous selection of ATP aptamers yielded molecules that do not significantly discriminate between ligands at the 5‘ position. By applying a selective pressure that demands recognition of the 5‘ triphosphate, we obtained an aptamer that binds to ATP with a Kd of approximately 5 μM, and to AMP with a Kd of approximately 5.5 mM, a difference of 1100-fold. This aptamer demonstrates the ability of small RNAs to interact with negatively charged moieties. PMID:15237981

  3. Modulations of RNA sequences by cytokinin in pumpkin cotyledons

    SciTech Connect

    Chang, C.; Ertl, J.; Chen, C.

    1987-04-01

    Polyadenylated mRNAs from excised pumpkin cotyledons treated with or without 10/sup -4/ M benzyladenine (BA) for various time periods in suspension culture were assayed by in vitro translation in the presence of (/sup 35/S) methionine. The radioactive polypeptides were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Specific sequences of mRNAs were enhanced, reduced, induced, or suppressed by the hormone within 60 min of the application of BA to the cotyledons. Four independent cDNA clones of cytokinin-modulated mRNAs have been selected and characterized. RNA blot hybridization using the four cDNA probes also indicates that the levels of specific mRNAs are modulated upward or downward by the hormone.

  4. Genome-wide analyses of Epstein-Barr virus reveal conserved RNA structures and a novel stable intronic sequence RNA

    PubMed Central

    2013-01-01

    Background Epstein-Barr virus (EBV) is a human herpesvirus implicated in cancer and autoimmune disorders. Little is known concerning the roles of RNA structure in this important human pathogen. This study provides the first comprehensive genome-wide survey of RNA and RNA structure in EBV. Results Novel EBV RNAs and RNA structures were identified by computational modeling and RNA-Seq analyses of EBV. Scans of the genomic sequences of four EBV strains (EBV-1, EBV-2, GD1, and GD2) and of the closely related Macacine herpesvirus 4 using the RNAz program discovered 265 regions with high probability of forming conserved RNA structures. Secondary structure models are proposed for these regions based on a combination of free energy minimization and comparative sequence analysis. The analysis of RNA-Seq data uncovered the first observation of a stable intronic sequence RNA (sisRNA) in EBV. The abundance of this sisRNA rivals that of the well-known and highly expressed EBV-encoded non-coding RNAs (EBERs). Conclusion This work identifies regions of the EBV genome likely to generate functional RNAs and RNA structures, provides structural models for these regions, and discusses potential functions suggested by the modeled structures. Enhanced understanding of the EBV transcriptome will guide future experimental analyses of the discovered RNAs and RNA structures. PMID:23937650

  5. Aptamers: A promising chemical antibody for cancer therapy

    PubMed Central

    Zhou, Gang; Wilson, George; Hebbard, Lionel; Duan, Wei; Liddle, Christopher; George, Jacob; Qiao, Liang

    2016-01-01

    Aptamers, also known as chemical antibodies, are single-stranded nucleic acid oligonucleotides which bind to their targets with high specificity and affinity. They are typically selected by repetitive in vitro process termed systematic evolution of ligands by exponential enrichment (SELEX). Owing to their excellent properties compared to conventional antibodies, notably their smaller physical size and lower immunogenicity and toxicity, aptamers have recently emerged as a new class of agents to deliver therapeutic drugs to cancer cells by targeting specific cancer-associated hallmarks. Aptamers can also be structurally modified to make them more flexible in order to conjugate other agents such as nano-materials and therapeutic RNA agents, thus extending their applications for cancer therapy. This review presents the current knowledge on the practical applications of aptamers in the treatment of a variety of cancers. PMID:26863567

  6. Nanomaterial-assisted aptamers for optical sensing.

    PubMed

    Wang, Guoqing; Wang, Yunqing; Chen, Lingxin; Choo, Jaebum

    2010-04-15

    Aptamers are single-strand DNA or RNA selected in vitro that bind specifically with a broad range of targets from metal ions, organic molecules, to proteins, cells and microorganisms. As an emerging class of recognition elements, aptamers offer remarkable convenience in the design and modification of their structures, which has motivated them to generate a great variety of aptamer sensors (aptasensors) that exhibit high sensitivity as well as specificity. On the other hand, the development of nanoscience and nanotechnology has generated nanomaterials with novel properties compared with their counterparts in macroscale. By integrating their strengths of both fields, recently, versatile aptamers coupling with novel nanomaterials for designing nanomaterial-assisted aptasensors (NAAs) make the combinations universal strategies for sensitive optical sensing. NAAs have been considered as an excellent sensing platform and found wide applications in analytical community. In this review, we summarize recent advances in the development of various optical NAAs, employing various detection techniques including colorimetry, fluorometry, surface-enhanced Raman scattering (SERS), magnetic resonance imaging (MRI) and surface plasmon resonance (SPR).

  7. Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

    PubMed Central

    Balmain, A; Minty, A J; Birnie, G D

    1980-01-01

    Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell. PMID:7433127

  8. A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae.

    PubMed Central

    de la Cruz, V F; Lake, J A; Simpson, A M; Simpson, L

    1985-01-01

    The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined. A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model. The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E. coli 16S structure are preserved. Within domains, however, some stems and loops have been greatly reduced or eliminated entirely. It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome. Alignment of the L. tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66. Images PMID:3856267

  9. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  10. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  11. Sequence of the 16S Ribosomal RNA from Halobacterium volcanii, an Archaebacterium.

    PubMed

    Gupta, R; Lanter, J M; Woese, C R

    1983-08-12

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  12. Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA.

    PubMed Central

    Xiao, L.; Limor, J.; Bern, C.; Lal, A. A.

    2001-01-01

    We sequenced a 173-nucleotide fragment of the small double-stranded viruslike RNA of Cryptosporidium parvum isolates from 23 calves and 38 humans. Sequence diversity was detected at 17 sites. Isolates from the same outbreak had identical double-stranded RNA sequences, suggesting that this technique may be useful for tracking Cryptosporidium infection sources. PMID:11266306

  13. High-throughput illumina strand-specific RNA sequencing library preparation

    USDA-ARS?s Scientific Manuscript database

    Conventional Illumina RNA-Seq does not have the resolution to decode the complex eukaryote transcriptome due to the lack of RNA polarity information. Strand-specific RNA sequencing (ssRNA-Seq) can overcome these limitations and as such is better suited for genome annotation, de novo transcriptome as...

  14. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14

    PubMed Central

    Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS assay, and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of AS1411-aptamers in SNU-761 HCC cells. We investigated the in vivo effects of the AS1411-aptamer using BALB/c nude mice in a subcutaneous xenograft model with SNU-761 cells. Treatment with a modified AS1411-aptamer significantly decreased in vitro (under normoxic [P = 0.035] and hypoxic [P = 0.018] conditions) and in vivo (under normoxic conditions, P = 0.041) HCC cell proliferation compared to control aptamers. AS1411- and control aptamers failed to control HCC cell proliferation. However, AS1411- and the modified AS1411-aptamer did not induce caspase activation. Decrease in cell growth by AS1411 or modified AS1411 was not prevented by caspase or necrosis inhibitors. In a microarray, AS1411 significantly enhanced galectin-14 expression. Suppression of HCC cell proliferation by the modified AS1411-aptamer was attenuated by galectin-14 siRNA transfection. Modified AS1411-aptamer suppressed HCC cell growth in vitro and in vivo by up-regulating galectin-14 expressions. Modified AS1411-aptamers may have therapeutic potential as a novel targeted therapy for HCC. PMID:27494117

  15. DNA aptamer selection in methanolic media: Adenine-aptamer as proof-of-concept.

    PubMed

    Chaou, Thinhinane; Vialet, Brune; Azéma, Laurent

    2016-03-15

    The major objective of this study is to investigate the usefulness of aptamers as in situ detection tool in organic solvents, which are often used for environmental extraction. But two problems related to the use of methanol-containing buffers have to be addressed. Firstly, the folding of nucleic acids can be impaired, because of weaker hydrogen bonding interactions. Secondly, the affinity of aptamers selected in aqueous buffers can be altered by the presence of methanol. Thus, in order to improve hydrophobicity of the DNA pool, nucleotide with hydrophobic modification 5-(octa1,7-diynyl)-2'-deoxyuridine (ODT) has been chosen instead of thymidine. As a proof of concept, an adenine aptamer operating in presence 25% of methanol has been selected. We have shown that the modified nucleotide is essential for target binding in organic media, in addition to essential structural pattern as proposed through analysing truncated sequences analysis. The strategy described in this paper offers preliminary insight on the adaptability of the implementation of aptamers as key instrument for in situ detection. It could be broaden to identify other aptamers directed against other chemical species after alcoholic extraction or for monitoring by-product traces in drugs production.

  16. FASTR: A novel data format for concomitant representation of RNA sequence and secondary structure information.

    PubMed

    Bose, Tungadri; Dutta, Anirban; Mh, Mohammed; Gandhi, Hemang; Mande, Sharmila S

    2015-09-01

    Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, 'FASTR', for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. 'FASTR' - the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant 'fastr' string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the 'FASTR' methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.

  17. Polyadenylation of RNA transcribed from mammalian SINEs by RNA polymerase III: Complex requirements for nucleotide sequences.

    PubMed

    Borodulina, Olga R; Golubchikova, Julia S; Ustyantsev, Ilia G; Kramerov, Dmitri A

    2016-02-01

    It is generally accepted that only transcripts synthesized by RNA polymerase II (e.g., mRNA) were subject to AAUAAA-dependent polyadenylation. However, we previously showed that RNA transcribed by RNA polymerase III (pol III) from mouse B2 SINE could be polyadenylated in an AAUAAA-dependent manner. Many species of mammalian SINEs end with the pol III transcriptional terminator (TTTTT) and contain hexamers AATAAA in their A-rich tail. Such SINEs were united into Class T(+), whereas SINEs lacking the terminator and AATAAA sequences were classified as T(-). Here we studied the structural features of SINE pol III transcripts that are necessary for their polyadenylation. Eight and six SINE families from classes T(+) and T(-), respectively, were analyzed. The replacement of AATAAA with AACAAA in T(+) SINEs abolished the RNA polyadenylation. Interestingly, insertion of the polyadenylation signal (AATAAA) and pol III transcription terminator in T(-) SINEs did not result in polyadenylation. The detailed analysis of three T(+) SINEs (B2, DIP, and VES) revealed areas important for the polyadenylation of their pol III transcripts: the polyadenylation signal and terminator in A-rich tail, β region positioned immediately downstream of the box B of pol III promoter, and τ region located upstream of the tail. In DIP and VES (but not in B2), the τ region is a polypyrimidine motif which is also characteristic of many other T(+) SINEs. Most likely, SINEs of different mammals acquired these structural features independently as a result of parallel evolution. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation.

    PubMed

    Daniel, Camille; Mélaïne, Feriel; Roupioz, Yoann; Livache, Thierry; Buhot, Arnaud

    2013-02-15

    Aptamers are raising an increasing interest for biosensor applications as replacements for antibodies due to their high stability and low cost. Thrombin, a key enzyme in the coagulation cascade, is an archetypical target against which two different aptamers, binding to two different exosites, have been selected. Recent studies dedicated to thrombin monitoring applications of biosensors have taken advantage of a potential sandwich-like structure between thrombin and these two aptamers for amplification purposes. However, in most cases, only end-point analysis was observed as a result of labeling requirements, thus preventing access to the kinetics of the complex formation. By using Surface Plasmon Resonance (SPR) imaging of aptamer-functionalized biosensors, we followed the binding of thrombin on the sensor and its interaction with a second reporter aptamer in real-time and in a label-free manner. Surprisingly, we showed that the injection of a second unlabeled-aptamer following the previous thrombin injection destabilized the thrombin-aptamer complex formed on the sensor surface, thus limiting any further amplification. However, the direct co-injection of thrombin, pre-complexed with a biotinylated aptamer bound to streptavidin efficiently increased the SPR signal by comparison to single thrombin detection. The various injection sequences performed may be rationalized considering a poor selectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sandwich complexation of the thrombin with its aptamers. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

    PubMed Central

    Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

    2015-01-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  20. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    PubMed

    Olson, Nathan D; Lund, Steven P; Zook, Justin M; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B

    2015-03-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.

  1. Determination of sequence and structural requirements for pathogenicity of a cucumber mosaic virus satellite RNA (Y-satRNA).

    PubMed

    Masuta, C; Takanami, Y

    1989-12-01

    We describe the use of biologically active cDNA clones to investigate genetic determinants of a satellite RNA that modulates symptoms normally induced by its helper virus, cucumber mosaic virus (CMV). For this purpose, we have investigated a CMV satellite RNA (Y-satRNA) that induces bright yellow symptoms on tobacco and necrosis on tomato. To determine the pathogenicity-modulating domain of Y-satRNA, several insertion and deletion mutants were created by using various restriction sites in the cDNA of Y-satRNA, and RNA transcripts derived from the clones were mixed with CMV and used to inoculate plants. Although the satellite RNA was able to tolerate small insertions (as much as 4 bases at present), small deletions were deleterious, indicating that the sequence requirements for viability of the satellite RNA are relatively inflexible. Biological activity assays of chimeric satellite RNAs between Y-satRNA and a non-necrogenic satellite RNA, T73-satRNA, suggested that only two (or at least one of two) specific bases (positions 318 and 325) in the 3' region direct the necrogenic property of Y-satRNA. Sequences involved in production of yellow symptoms were investigated by constructing chimeras between Y-sat cDNA and cDNA of a satellite RNA designated S19-satRNA. S19-satRNA has considerable homology to Y-satRNA but does not elicit yellow symptoms on tobacco. Chimeric clones were constructed by using a BstXI site that cuts within a stable secondary structure in the region between positions 100 and 200 (region Y). The results of infectivity tests with RNA transcripts suggest that formation of a secondary structure in region Y may be involved in induction of yellow symptoms as well as viability of Y-satRNA.

  2. Pea enation mosaic virus genoma RNA contains no polyadenylate sequences and cannot be aminoacylated.

    PubMed

    German, T L; De Zoeten, G A; Hall, T C

    1978-01-01

    An active synthetase enzyme preparation from peas (Pisum sativum L.) did not catalyze the aminoacylation of pea enation mosaic virus RNA. The viral RNA was shown not to contain polyadenylic acid sequences.

  3. Ligand Binding and Conformational Changes in the Purine-Binding Riboswitch Aptamer Domains

    NASA Astrophysics Data System (ADS)

    Noeske, Jonas; Buck, Janina; Wöhnert, Jens; Schwalbe, Harald

    Riboswitches are highly structured mRNA elements that regulate gene expression upon specific binding of small metabolite molecules. The purine-binding riboswitches bind different purine ligands by forming both canonical Watson—Crick and non-canonical intermolecular base pairs, involving a variety of hydrogen bonds between the riboswitch aptamer domain and the purine ligand. Here, we summarize work on the ligand binding modes of both purine-binding aptamer domains, their con-formational characteristics in the free and ligand-bound forms, and their ligand-induced folding. The adenine- and guanine-binding riboswitch aptamer domains display different conformations in their free forms, despite nearly identical nucleotide loop sequences that form a loop—loop interaction in the ligand-bound forms. Interestingly, the stability of helix II is crucial for the formation of the loop—loop interaction in the free form. A more stable helix II in the guanine riboswitch leads to a preformed loop—loop interaction in its free form. In contrast, a less stable helix II in the adenine riboswitch results in a lack of this loop—loop interaction in the absence of ligand and divalent cations.

  4. Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

    PubMed Central

    Martin, Dorrelyn P.; Miya, Jharna; Reeser, Julie W.; Roychowdhury, Sameek

    2017-01-01

    RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 – 100 million sequencing