Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

PubMed Central

Gao, Run-Ping; Brigstock, David R

2009-01-01

AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function. METHODS: CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-β1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry. RESULTS: In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase. CONCLUSION: Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase. PMID:19673024

A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogeneous in length with a 3' end blocked for transesterification.

PubMed Central

Grosshans, C A; Cech, T R

1991-01-01

The L-21 Scal form of the Tetrahymena ribozyme acts as a sequence-specific endonuclease. This ribozyme has a homogeneous 5' end but a somewhat heterogeneous 3' end, as is typical of RNA synthesized by transcription in vitro. To produce a more homogeneous ribozyme for both structural and enzymological studies, a hammerhead ribozyme was inserted at the 3' end of the Tetrahymena ribozyme. During transcription the hammerhead moiety self-cleaves to produce the L-21 A Tetrahymena ribozyme, which ends at A410 with a 2',3'-cyclic phosphate terminus. The new ribozyme has endoribonuclease activity equivalent to that of L-21 Scal under conditions where binding of substrate is rate-limiting, as well as under conditions where chemical cleavage by guanosine is rate-limiting. However, the L-21 A has lost activity in oligo(C) disproportionation (e.g., 2 pC5----pC4 + pC6), consistent with the previous proposal that this reaction occurs predominantly through a covalent ribozyme-substrate intermediate involving the 3'-terminal hydroxyl group of the ribozyme. Formation of such an intermediate would be prevented by the 2',3'-cyclic phosphate terminus. Thus the L-21 A ribozyme has simplified enzymatic activity, being fully active as an endonuclease but blocked for disproportionation. Images PMID:1650453

Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.

PubMed Central

Sigurdsson, S T; Tuschl, T; Eckstein, F

1995-01-01

Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure. PMID:7489517

Antibiotic interactions with the hammerhead ribozyme:tetracyclines as a new class of hammerhead inhibitor.

PubMed Central

Murray, J B; Arnold, J R

1996-01-01

A screening of a range of common laboratory antibiotics for inhibition of the hammerhead ribozyme has shown that in addition to certain aminoglycosides (most notably neomycin B) the tetracyclines are also effective inhibitors, with chlorotetracycline being more effective than tetracycline. Inhibition by chlorotetracycline is not as strong as that by neomycin B but is more complicated, with at least two binding sites apparent. As with hammerhead inhibition by neomycin B, chlorotetracycline inhibition can be overcome by raising the concentration of the Mg2+ ion cofactor. We find that around six Mg2+ ions will displace neomycin B, compared with twelve for chlorotetracycline. Inhibition observed in the presence of mixtures of neomycin B and chlorotetracycline is consistent with separate binding sites on the hammerhead for these two classes of antibiotic. Under certain conditions of the mixing order and low concentration of chlorotetracycline, enhancement of single-turnover hammerhead cleavage by up to 20% is observed, with higher concentrations of antibiotic being inhibitory. We have also found that the presence of 2.5% (v/v) DMSO causes a 30% enhancement of the single-turnover cleavage. These results thus extend the range of known inhibitors of hammerhead cleavage, and also demonstrate how the cleavage can be accelerated. PMID:8760373

Rolling Circle Transcription of Ribozymes Targeted to ras and mdr-1

DTIC Science & Technology

2001-09-01

ssDNA) to direct transcription of an tion-PCR, and recyclization were carried out to optimize active hammerhead ribozyme in E. coli cells. transcription...transcription I hammerhead ribozyme I in vitro selection and 12.5 units/ml RNase inhibitor (Promega), in a total reaction volume of 15 tk1. After a...sequence encoding a ssDNA, and splint ssDNA were ethanol-precipitated and used as hammerhead ribozyme . templates to begin the next round of in vitro

Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.

PubMed Central

Hermann, T; Auffinger, P; Scott, W G; Westhof, E

1997-01-01

In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA. PMID:9254698

Efficient trans-cleavage by the Schistosoma mansoni SMα1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus

PubMed Central

Vazquez-Tello, Alejandro; Castán, Pablo; Moreno, Renata; Smith, James M.; Berenguer, José; Cedergren, Robert

2002-01-01

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMα DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme–product dissociation step. The optimal temperature for trans-cleavage was 70°C. This result was confirmed when both the SMα1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMα1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMα1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo. PMID:11917021

Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

PubMed Central

Zhou, Jing-Min; Zhou, De-Min; Takagi, Yasuomi; Kasai, Yasuhiro; Inoue, Atsushi; Baba, Tadashi; Taira, Kazunari

2002-01-01

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses. PMID:12034824

Characterization of a native hammerhead ribozyme derived from schistosomes

PubMed Central

OSBORNE, EDITH M.; SCHAAK, JANELL E.; DEROSE, VICTORIA J.

2005-01-01

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence. PMID:15659358

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

PubMed Central

de Silva, Chamaree; Walter, Nils G.

2009-01-01

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer. PMID:19029309

The tolerance to exchanges of the Watson–Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

PubMed Central

Przybilski, Rita; Hammann, Christian

2007-01-01

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson–Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson–Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3–R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem–loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson–Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes. PMID:17666711

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277

A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

PubMed Central

Lustig, B; Lin, N H; Smith, S M; Jernigan, R L; Jeang, K T

1995-01-01

A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason. Images PMID:7567466

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

PubMed Central

Borda, Emily J.; Markley, John C.; Sigurdsson, Snorri Th.

2003-01-01

We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes. PMID:12736309

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

PubMed Central

McDowell, S. Elizabeth; Jun, Jesse M.; Walter, Nils G.

2010-01-01

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ∼10-fold higher than physiologic concentrations of Mg2+ ions. Extended versions containing native loop–loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg2+ concentrations, for reasons that are still ill-understood. Here, we use Mg2+ titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop–loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop–loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop–loop interactions in extended hammerhead ribozymes—and Mg2+ ions that bind to minimal ribozymes—may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state. PMID:20921269

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

PubMed Central

Bassi, G S; Murchie, A I; Lilley, D M

1996-01-01

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation. PMID:8752086

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

PubMed Central

Roychowdhury-Saha, Manami; Burke, Donald H.

2007-01-01

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized “RzB” hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na+ alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg2+ and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min−1 in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg2+, unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg2+, low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg2+ alone. These observations suggest that Mg2+ and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role. PMID:17456566

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

PubMed Central

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L.

2016-01-01

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction. PMID:26398724

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

PubMed

Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L

2015-10-20

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Folding of the natural hammerhead ribozyme is enhanced by interaction of auxiliary elements

PubMed Central

PENEDO, J. CARLOS; WILSON, TIMOTHY J.; JAYASENA, SUMEDHA D.; KHVOROVA, ANASTASIA; LILLEY, DAVID M.J.

2004-01-01

It has been shown that the activity of the hammerhead ribozyme at μM magnesium ion concentrations is markedly increased by the inclusion of loops in helices I and II. We have studied the effect of such loops on the magnesium ion-induced folding of the ribozyme, using fluorescence resonance energy transfer. We find that with the loops in place, folding into the active conformation occurs in a single step, in the μM range of magnesium ion concentration. Disruption of the loop–loop interaction leads to a reversion to two-step folding, with the second stage requiring mM concentrations of magnesium ion. Sodium ions also promote the folding of the natural form of the ribozyme at high concentrations, but the folding occurs as a two-stage process. The loops clearly act as important auxiliary elements in the function of the ribozyme, permitting folding to occur efficiently under physiological conditions. PMID:15100442

Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis.

PubMed

Bellon, L; Maloney, L; Zinnen, S P; Sandberg, J A; Johnson, K E

2000-08-01

Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine. Copyright 2000 Academic Press.

Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

PubMed Central

Menger, Marcus; Eckstein, Fritz; Porschke, Dietmar

2000-01-01

The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to ~200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A→G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (τ ≈200 ms). Addition of Mg2+ ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures

NASA Astrophysics Data System (ADS)

El-Murr, Nizar; Maurel, Marie-Christine; Rihova, Martina; Vergne, Jacques; Hervé, Guy; Kato, Mikio; Kawamura, Kunio

2012-09-01

The "RNA world" hypothesis proposes that—early in the evolution of life—RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg2+, which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg2+ on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na+ or K+), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

Hammerhead Ribozyme against γ‐Glutamylcysteine Synthetase Attenuates Resistance to Ionizing Radiation and Cisplatin in Human T98G Glioblastoma Cells

PubMed Central

Tani, Masaharu; Goto, Shinji; Kamada, Kensaku; Mori, Katsuharu; Urata, Yoshishige; Ihara, Yoshito; Kijima, Hiroshi; Ueyama, Yoshito; Shibata, Shobu

2002-01-01

Glioblastoma cells are highly malignant and show resistance to ionizing radiation, as well as anti‐cancer drugs. This resistance to cancer therapy is often associated with a high concentration of glutathione (GSH). In this study, the effect of continuous down‐regulation of γ‐glutamylcysteine synthetase (γ‐GCS) expression, a rate‐limiting enzyme for GSH synthesis, on resistance to ionizing radiation and cisplatin (CDDP) was studied in T98G human glioblastoma cells. We constructed a hammerhead ribozyme against a γ‐GCS heavy subunit (γ‐GCSh) mRNA and transfected it into T98G cells. (1) The transfection of the ribozyme decreased the concentration of GSH and resulted in G1 cell cycle arrest of T98G cells. (2) The transfection of the ribozyme increased the cytotoxicity of ionizing radiation and CDDP in T98G cells. Thus, hammerhead ribozyme against γ‐GCS is suggested to have potential as a cancer gene therapy to reduce the resistance of malignant cells to ionizing radiation and anti‐cancer drugs. PMID:12079521

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.

PubMed Central

Grasby, J A; Jonathan, P; Butler, G; Gait, M J

1993-01-01

The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage. PMID:8233777

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

PubMed Central

Kore, A R; Vaish, N K; Kutzke, U; Eckstein, F

1998-01-01

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH. PMID:9722629

Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

PubMed Central

Nakano, Shu-ichi; Kitagawa, Yuichi; Miyoshi, Daisuke; Sugimoto, Naoki

2014-01-01

Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol), small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds. PMID:25161873

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

PubMed Central

Schultz, Eric P.; Vasquez, Ernesto E.; Scott, William G.

2014-01-01

The hammerhead ribozyme catalyzes RNA cleavage via acid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK a of the substituted purine; in both cases inosine, which is similar to G in pK a and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

PubMed

Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

2014-09-01

The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

PubMed Central

Inoue, Atsushi; Takagi, Yasuomi; Taira, Kazunari

2004-01-01

Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway—a cooperative pathway—in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst. PMID:15302920

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

PubMed

Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

2017-03-17

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells.

PubMed Central

Homann, M; Tabler, M; Tzortzakaki, S; Sczakiel, G

1994-01-01

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo. Images PMID:7524030

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

PubMed Central

Shields, T P; Mollova, E; Ste Marie, L; Hansen, M R; Pardi, A

1999-01-01

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product. PMID:10496226

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

PubMed Central

Hammann, Christian; Norman, David G.; Lilley, David M. J.

2001-01-01

We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range. PMID:11331743

Coupling of Fast and Slow Modes in the Reaction Pathway of the Minimal Hammerhead Ribozyme Cleavage

PubMed Central

Radhakrishnan, Ravi

2007-01-01

By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg2+) ions, proceeds via an in-line-attack by CYT 17 O2′ on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack. PMID:17545240

Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.

PubMed Central

Williams, D M; Pieken, W A; Eckstein, F

1992-01-01

The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed. Images PMID:1736306

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

PubMed Central

Conaty, J; Hendry, P; Lockett, T

1999-01-01

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system. PMID:10325431

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

PubMed Central

Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

2014-01-01

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17.

PubMed Central

Ludwig, J; Blaschke, M; Sproat, B S

1998-01-01

In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17to N16.2C16.1H17(H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15. 1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2'- O -alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2. PMID:9580675

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

Ubiquitous presence of the hammerhead ribozyme motif along the tree of life

PubMed Central

de la Peña, Marcos; García-Robles, Inmaculada

2010-01-01

Examples of small self-cleaving RNAs embedded in noncoding regions already have been found to be involved in the control of gene expression, although their origin remains uncertain. In this work, we show the widespread occurrence of the hammerhead ribozyme (HHR) motif among genomes from the Bacteria, Chromalveolata, Plantae, and Metazoa kingdoms. Intergenic HHRs were detected in three different bacterial genomes, whereas metagenomic data from Galapagos Islands showed the occurrence of similar ribozymes that could be regarded as direct relics from the RNA world. Among eukaryotes, HHRs were detected in the genomes of three water molds as well as 20 plant species, ranging from unicellular algae to vascular plants. These HHRs were very similar to those previously described in small RNA plant pathogens and, in some cases, appeared as close tandem repetitions. A parallel situation of tandemly repeated HHR motifs was also detected in the genomes of lower metazoans from cnidarians to invertebrates, with special emphasis among hematophagous and parasitic organisms. Altogether, these findings unveil the HHR as a widespread motif in DNA genomes, which would be involved in new forms of retrotransposable elements. PMID:20705646

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3′ end can be exported efficiently and specifically to the cytoplasm in mammalian cells

PubMed Central

Kuwabara, Tomoko; Warashina, Masaki; Koseki, Shiori; Sano, Masayuki; Ohkawa, Jun; Nakayama, Kazuhisa; Taira, Kazunari

2001-01-01

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNAVal-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3′ end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5′ and 3′ ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells. PMID:11433023

Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

PubMed Central

KISSELEVA, NATALIA; KHVOROVA, ANASTASIA; WESTHOF, ERIC; SCHIEMANN, OLAV

2005-01-01

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding. PMID:15611296

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

Mechanistic studies of copper(II)-aminoglycoside mediated DNA damage and magnesium catalyzed nuclease activity of hammerhead ribozyme

NASA Astrophysics Data System (ADS)

Patwardhan, Anjali A.

The antibacterial activity of aminoglycosides stems from their high affinity binding to the 16S rRNA in bacteria resulting in inhibition of protein synthesis. Used to treat acute bacterial infections these antibiotics have limited applications due to their high dosage requirements and the emergence of resistant strains. We have synthesized and characterized Cu(II) derivatives of the aminoglycosides, kanamycin A, tobramycin, neamine, kanamycin B, neomycin B, and paromomycin. The first three exhibit preferential and tight binding to Cu(II) as against neomycin B and kanamycin B and paromomycin. EPR of frozen solutions and UV-visible spectroscopy suggest a change in geometry around the Cu(II) but the stabilities of the complexes in water differ. These copper derivatives efficiently cleave plasmid DNA at micromolar concentrations (hydrolytic) and at nanomolar concentrations in the presence co-reactants like hydrogen peroxide or ascorbic acid. Hydrolysis is multi turnover and exhibits Michelis-Menten kinetics with enzyme-like behavior whereas oxidative cleavage is highly specific with C-4' H abstraction resulting in characteristic base propenal and nucleotide base products. Hydroxyl radicals generated are copper based and are generated in close proximity of the substrate. Hammerhead ribozymes are selectively hydrolyzed in the presence of divalent ions with Mg2+ being the metal ion of choice in vivo . Our studies with complex ions like cobalt hexaammine and fac-triamminetriaquochromium(III) establish outer sphere interactions of Mg2+ with the hammerhead in the catalytic site. There are two sets of sites, one structural and one catalytic. Complex ions in the catalytic site and divalent ions in the structural site result in a slow but active hammerhead ribozyme suggesting that the complex ions are not inhibitory, contrary to what was suggested previously.

Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

PubMed Central

Jankowsky, E; Strunk, G; Schwenzer, B

1997-01-01

Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes. PMID:9207013

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water

NASA Technical Reports Server (NTRS)

Sarma, R. H.; Sarma, M. H.; Rein, R.; Shibata, M.; Setlik, R. S.; Ornstein, R. L.; Kazim, A. L.; Cairo, A.; Tomasi, T. B.

1995-01-01

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Development of RNAi Libraries for Target Validation and Therapeutics

DTIC Science & Technology

2006-03-01

met using a hammerhead ribozyme transgene reduces in vitro invasion and migration in prostate cancer cells. Prostate, 60: 317-324, 2004. 49...Watkins, G., Mason, M.D., Jiang, W.G. (2004) Targeting the HGF/SF receptor c-met using a hammerhead ribozyme transgene reduces in vitro invasion...reduction of tumor growth (27). In addition, when c-met was knocked-down using ribozymes , cell invasion and metastasis were inhibited both in vitro

Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes.

PubMed

Tanaka, T; Inui, O; Dohi, N; Okada, N; Okada, H; Kikuchi, Y

2001-07-01

Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

Characterization of Ribozymes Targeting a Congenital Night Blindness Mutation in Rhodopsin Mutation.

PubMed

Conley, Shannon M; Whalen, Patrick; Lewin, Alfred S; Naash, Muna I

2016-01-01

The G90D mutation in the rhodopsin gene leads to autosomal dominant congenital stationary night blindness (CSNB) in patients. This occurs because the G90D mutant protein cannot efficiently bind chromophore and is constitutively active. To combat this mutation, we designed and characterized two different hammerhead ribozymes to cleave G90D transcript. In vitro testing showed that the G90D1 ribozyme efficiently and specifically cleaved the mutant transcript while G90D2 cleaved both WT and mutant transcript. AAV-mediated delivery of G90D1 under the control of the mouse opsin promoter (MOP500) to G90D transgenic eyes showed that the ribozyme partially retarded the functional degeneration (as measured by electroretinography [ERG]) associated with this mutation. These results suggest that with additional optimization, ribozymes may be a useful part of the gene therapy knockdown strategy for dominant retinal disease.

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

PubMed Central

Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

2004-01-01

Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.

PubMed Central

Holm, P. S.; Scanlon, K. J.; Dietel, M.

1994-01-01

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line. Images Figure 1 Figure 3 Figure 2 PMID:7914421

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation

PubMed Central

Mikulecky, Peter J.; Feig, Andrew L.

2004-01-01

In proteins, empirical correlations have shown that changes in heat capacity (ΔCP) scale linearly with the hydrophobic surface area buried upon folding. The influence of ΔCP on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to ΔCPs of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a ΔCP of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated ΔCP by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the ΔCP. We weigh the strengths and limitations of this method for determining ΔCP values. Finally, we discuss the data in light of the physical origins of the ΔCPs for RNA folding and consider their impact on biological function. PMID:15282329

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

PubMed Central

Giannini, C D; Roth, W K; Piiper, A; Zeuzem, S

1999-01-01

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene. PMID:10373591

HER-2 as a Progression Factor and Therapeutic Target in Breast Cancer.

DTIC Science & Technology

1999-06-01

used gene specific targeting of HER-2 with hammerhead - ribozyme expression constructs, a technology which we have applied successfully in the...2 in MCF-7 cells by ribozyme -targeting estradiol lost its ability to induce anchorage- independent colony formation in soft agar of the tumor cells...between estrogen and HER-2 signal transduction is ongoing. 14. SUBJECT TERMS Breast Cancer HER-2, estradiol, ribozymes , apoptosis, cell cycle, cDNA

Monitoring Retroviral RNA Dimerization In Vivo via Hammerhead Ribozyme Cleavage

PubMed Central

Pal, Bijay K.; Scherer, Lisa; Zelby, Laurie; Bertrand, Edouard; Rossi, John J.

1998-01-01

We have used a strategy for colocalization of Psi (Ψ)-tethered ribozymes and targets to demonstrate that Ψ sequences are capable of specific interaction in the cytoplasm of both packaging and nonpackaging cells. These results indicate that current in vitro dimerization models may have in vivo counterparts. The methodology used may be applied to further genetic analyses on Ψ domain interactions in vivo. PMID:9733882

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

PubMed Central

Düvel, Katrin; Valerius, Oliver; Mangus, David A; Jacobson, Allan; Braus, Gerhard H

2002-01-01

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis. PMID:12003493

Study the Pathogenic Role of ErbB-3, ErbB-4 and their Ligand Heregulin in Human Breast Cancer Cell

DTIC Science & Technology

1999-07-01

implicated in human breast cancers. To delineate the biological function of ErbB-4 receptors in breast cancer, we employed a hammerhead ribozyme strategy to...receptors for neuregulin (23, 24). Activation cancer, we generated three specific hammerhead ribozymes targeted to of ErbB-2 by NRGI-a is thought to...generated three specific hammerhead ribozymes targeted to specific sites within ErbB-4 mRNA. These ErbB-4 ribozymes (Rz6, INTRODUCTION Rz2I, and Rz29

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

PubMed Central

Lott, William B.; Pontius, Brian W.; von Hippel, Peter H.

1998-01-01

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 μM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and >50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg2+–La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction. PMID:9435228

A Novel Molecular Targeting of a Tumor-Specific Oncogenic Mutant Receptor in Human Prostate Cancer

DTIC Science & Technology

2005-02-01

in cells and can generate dominant negative mutant (15). Hammerhead ribozymes are self-cleaving RNAs whose catalytic activity has been mapped to a...specific ribozyme targeted at the fusion junction of EGFRvIII. This specific EGFRvIII ribozyme is able to effectively cleave EGFRvIII mRNA under...physiological conditions in a cell-free system. While expressing this EGFRvIII- ribozyme in 32D/EGFRvIII cell, EGFRvIII- ribozyme is capable of down-regulating

Study the Pathogenic Role of ErbB-3, ErbB-4 and their Ligand Heregulin in Human Breast Cancer Cells.

DTIC Science & Technology

1997-07-01

characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes , Rz6...proposal we plan to utilize hammerhead ribozymes which target HRG and its receptors (ErbB-3, ErbB-4) to interrupt their signaling. We will then study...cancer. Results: To assist in characterizing the role of ErbB-4 in breast cancer, we have generated three specific hammerhead ribozymes (Rz6, Rz2 1

Role of mp 170 Seprase in Breast Cancer.

DTIC Science & Technology

1999-07-01

endogenous Seprase/FAPa in MDA-436 cells. Previously, three hammerhead ribozyme constructs targeting Seprase/FAPa at nucleotide residues 703, 892...breast cancer cells using the ribozyme approach. In contrast, I also constructed two hammerhead ribozymes targeting FGF-BP2 at nucleotide residues 223 and...molecule for further characterization, however, with little success. Ribozyme constructs targeting Seprase/FAPa were made, and despite their in vitro

Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

PubMed

Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

2015-05-15

Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches.

Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue.

PubMed

Mir, Aamir; Golden, Barbara L

2016-02-02

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.

An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

PubMed Central

Chartrand, P; Harvey, S C; Ferbeyre, G; Usman, N; Cedergren, R

1995-01-01

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer. Images PMID:7479070

Two active site divalent ions in the crystal structure of the hammerhead ribozyme bound to a transition state analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mir, Aamir; Golden, Barbara L.

2015-11-09

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the p K A of G12. Finally, on the basis ofmore » this crystal structure as well as a wealth of biochemical studies, in this paper we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.« less

A triplex ribozyme expression system based on a single hairpin ribozyme.

PubMed

Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

2008-09-01

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

Design of the hairpin ribozyme for targeting specific RNA sequences.

PubMed

Hampel, A; DeYoung, M B; Galasinski, S; Siwkowski, A

1997-01-01

The following steps should be taken when designing the hairpin ribozyme to cleave a specific target sequence: 1. Select a target sequence containing BN*GUC where B is C, G, or U. 2. Select the target sequence in areas least likely to have extensive interfering structure. 3. Design the conventional hairpin ribozyme as shown in Fig. 1, such that it can form a 4 bp helix 2 and helix 1 lengths up to 10 bp. 4. Synthesize this ribozyme from single-stranded DNA templates with a double-stranded T7 promoter. 5. Prepare a series of short substrates capable of forming a range of helix 1 lengths of 5-10 bp. 6. Identify these by direct RNA sequencing. 7. Assay the extent of cleavage of each substrate to identify the optimal length of helix 1. 8. Prepare the hairpin tetraloop ribozyme to determine if catalytic efficiency can be improved.

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

PubMed Central

Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M

1993-01-01

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489

Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

PubMed Central

Lee, Tai-Sung; Wong, Kin-Yiu; Giambasu, George M.; York, Darrin M.

2016-01-01

Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg2+ ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg2+ ion or multiple Na+ ions, one of which is also in a bridging coordination pattern. In the case of a single Mg2+ ion bound in the active site, the Mg2+ ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2′). As the reaction proceeds, the Mg2+ ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2′. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis. PMID:24156941

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

PubMed Central

Zillmann, M; Limauro, S E; Goodchild, J

1997-01-01

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core. PMID:9214657

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

PubMed Central

Fu, D J; McLaughlin, L W

1992-01-01

Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A11, G13, or A14. Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in Km and a decrease in kcat. These results suggest that the exocyclic amino group at-G10 and the hydroxyls at G10 and G13 are important both for ribozyme-substrate binding and for the Mg(2+)-catalyzed cleavage reaction. PMID:1570323

Development of Lead Hammerhead Ribozyme Candidates against Human Rod Opsin mRNA for Retinal Degeneration Therapy

PubMed Central

Abdelmaksoud, Heba E.; Yau, Edwin H.; Zuker, Michael; Sullivan, Jack M.

2011-01-01

To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p < 0.05). Successful lead candidates (hhRz CUC↓ 266, hhRz CUC↓ 1411, hhRz AUA↓ 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48 hr paradigm of testing. These results validate a rigorous computational

Inducible Transgenic Models of BRCA1 Function

DTIC Science & Technology

1999-10-01

inducible expression vectors were created to conditionally express four different hammerhead ribozymes designed to specifically cleave the Brca...transcript. Hammerhead ribozymes are catalytic RNAs that efficiently cleave RNA and thereby down- regulate gene expression. Hammerhead ribozymes can cleave...any RNA containing its 5’-UH-3’ consensus sequence where U can be replaced by a C, and H=C, U or A. Hammerhead ribozymes effectively and selectively

Inducible Transgenic Models of BRCA1 Function

DTIC Science & Technology

2000-10-01

four different hammerhead ribozymes designed to specifically cleave the Brcal transcript. Hammerhead ribozymes are catalytic RNAs that efficiently...cleave RNA and thereby down- regulate gene expression. Hammerhead ribozymes can cleave any RNA containing a 5’-UH-3’ consensus sequence where U can be...replaced by C, and H=C, U or A. Hammerhead ribozymes have been shown to effectively and selectively inhibit gene expression in bacteria, plants, cell

Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme

NASA Technical Reports Server (NTRS)

Setlik, R. F.; Shibata, M.; Sarma, R. H.; Sarma, M. H.; Kazim, A. L.; Ornstein, R. L.; Tomasi, T. B.; Rein, R.

1995-01-01

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

Pleiotrophin as a Growth Factor and Therapeutic Target in Breast Cancer.

DTIC Science & Technology

1998-10-01

Hs578T cells by stable transfection with hammerhead ribozymes as done for a number of different genes by my laboratory (for a review see reference [28...angiogenesis and metastasis. 14. SUBJECT TERMS Breast Cancer growth factor, pleiotrophin, hormones, retrovirus, ribozymes 15. NUMBER OF PAGES...of their endogenous PTN with specific ribozymes [10-12]. We were the first laboratory to purify PTN from human cancer cells (MDA-MB 231 breast

Effect of Hammerhead Ribozyme against Human Thymidylate Synthase on the Cytotoxicity of Thymidylate Synthase Inhibitors

PubMed Central

Takemura, Yuzuru; Miyachi, Hayato; Skelton, Lorraine; Jackman, Ann L.

1995-01-01

One of the resistance mechanisms to folate‐based thymidylate synthase (TS) inhibitors is the increase in TS activity in tumor cells. Human B lymphoblastoid cell line (W1L2) was made resistant to a lipophilic non‐polyglutamatable TS inhibitor (ZM249148), and the subline (W1L2:R179) showed a 20‐fold increase in TS enzyme activity with concomitant overexpression of TS mRNA. To overcome the resistance, we designed a ribozyme that can cleave the CUC sequences in a triple tandemly repeated sequence of TS mRNA. Expression of this ribozyme in W1L2:R179 cells transfected with Epstein Barr virus‐based expression vector resulted in sensitization to TS inhibitors concomitantly with a decrease of TS expression. The ribozyme expressed in transfectants was shown to be functional in cleaving artificial TS RNA in vitro. PMID:8567390

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal

PubMed Central

Cho, Suhyung; Kim, Ji-Eun; Lee, Bo-Rahm; Kim, June-Hyung; Kim, Byung-Gee

2005-01-01

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal. PMID:16314308

A Cellular High-Throughput Screening Approach for Therapeutic trans-Cleaving Ribozymes and RNAi against Arbitrary mRNA Disease Targets

PubMed Central

Yau, Edwin H.; Butler, Mark C.; Sullivan, Jack M.

2016-01-01

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a

Transdominant Rev and Protease Mutant Proteins of HIV-SIV as Potential Antiviral Agents in Vitro and in Vivo (AIDS)

DTIC Science & Technology

1993-10-30

hammerhead ribozymes (7-9) and a hairpin ribozyme (10) directed against HIV-l RNA has been shown to confer significant resistance to HIV-I infection...antisense oligodeoxynucleotides (ODN) directed to the Rev Response Element (RRE) and ribozymes that target viral mRNAs. The ribozyme approach, in...particular, has yielded extremely encouraging positive data. We showed that a hairpin ribozyme designed to cleave HIV-1 RNA in the 5’ leader sequence

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

PubMed Central

Sripathi, Kamali N.; Tay, Wendy W.; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G.

2014-01-01

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2′-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. PMID:24854621

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape.

PubMed

Sripathi, Kamali N; Tay, Wendy W; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G

2014-07-01

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2'-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. © 2014 Sripathi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Entropy-Driven Folding of an RNA Helical Junction: An Isothermal Titration Calorimetric Analysis of the Hammerhead Ribozyme†

PubMed Central

Mikulecky, Peter J.; Takach, Jennifer C.; Feig, Andrew L.

2008-01-01

Helical junctions are extremely common motifs in naturally occurring RNAs, but little is known about the thermodynamics that drive their folding. Studies of junction folding face several challenges: non-two-state folding behavior, superposition of secondary and tertiary structural energetics, and drastically opposing enthalpic and entropic contributions to folding. Here we describe a thermodynamic dissection of the folding of the hammerhead ribozyme, a three-way RNA helical junction, by using isothermal titration calorimetry of bimolecular RNA constructs. By using this method, we show that tertiary folding of the hammerhead core occurs with a highly unfavorable enthalpy change, and is therefore entropically driven. Furthermore, the enthalpies and heat capacities of core folding are the same whether supported by monovalent or divalent ions. These properties appear to be general to the core sequence of bimolecular hammerhead constructs. We present a model for the ion-induced folding of the hammerhead core that is similar to those advanced for the folding of much larger RNAs, involving ion-induced collapse to a structured, non-native state accompanied by rearrangement of core residues to produce the native fold. In agreement with previous enzymological and structural studies, our thermodynamic data suggest that the hammerhead structure is stabilized in vitro predominantly by diffusely bound ions. Our approach addresses several significant challenges that accompany the study of junction folding, and should prove useful in defining the thermodynamic determinants of stability in these important RNA motifs. PMID:15134461

Cripto-1 in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1999-06-01

4). T.O. 2 We have designed and tested a hammerhead ribozyme [22, 23] that recognizes nucleotides 12-28 of the murine CR-I mRNA and cuts after the GUC...III C AGCCGG U A G GA U FIG. 1. CR-i-specific hammerhead ribozyme . (A) Diagram of the processed CR-1 mRNA, showing the ribozyme recognition sequence...diagram of the CR-1 message and ribozyme is shown RT-PCR reactions were electrophoresed through 1.5% agarose-TAE in Figure 1A, and the folded hammerhead

Selenium is a Chemotherapeutic Agent for the Treatment of Prostate Cancer

DTIC Science & Technology

2006-02-01

receptor activity by a hammerhead ribozyme . Mol Endrocrinol 1998;12:1558-1566. 10. Ko YJ, Devi GR, London CA, et al. Androgen receptor down-regulation...Strategic targeting of the AR with ribozymes , antisense oligomers, and small interfering RNAs has been shown to significantly inhibit prostate cancer

Alteration of hairpin ribozyme specificity utilizing PCR.

PubMed

DeGrandis, P; Hampel, A; Galasinski, S; Borneman, J; Siwkowski, A; Altschuler, M

1994-12-01

We have developed a method by which a researcher can quickly alter the specificity of a trans hairpin ribozyme. Utilizing this PCR method, two oligonucleotides, and any target vector, new ribozyme template sequences can be generated without the synthesis of longer oligonucleotides. We have produced templates with altered specificity for both standard and modified (larger) ribozymes. After transcription, these ribozymes show specific cleavage activity with the new substrate beta-glucuronidase (GUS), and no activity against the original substrate (HIV-1, 5' leader sequence). Utilizing this technique, it is also possible to produce an inactive ribozyme that can be used as an antisense control. Applications of this procedure would provide a rapid and economical system for the assessment of trans ribozyme activity.

Cripto-1 in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

1998-06-01

and tested a hammerhead ribozyme [20, 37] that recognizes nucleotides 12-28 of the murine CR-I mRNA and cuts after the GUC triplet at nucleotides 18-20...UACACGG- 3’ "A UACICGG31 AAGGCC AUGUGCC-5’ IIII C AGCCGG U A G GA U FIG. 1. CR-l-specific hammerhead ribozyme . (A) Diagram of the processed CR-i mRNA...the CR-1 message and ribozyme is shown RT-PCR reactions were electrophoresed through 1.5% agarose-TAE in Figure 1A, and the folded hammerhead , bound

Selective Androgen Receptor Downregulators (SARDs): A New Prostate Cancer Therapy

DTIC Science & Technology

2006-10-01

of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol, 12: 1558...cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol...used to down-regulate the AR include antisense oligonucleotides (9, 10), ribozyme treatments (11, 12), AR dominant negatives (13) and small

Ribozyme-mediated signal augmentation on a mass-sensitive biosensor.

PubMed

Knudsen, Scott M; Lee, Joonhyung; Ellington, Andrew D; Savran, Cagri A

2006-12-20

Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.

Intracellular metabolism of a 2'-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis.

PubMed Central

Prasmickaite, L; Hogset, A; Maelandsmo, G; Berg, K; Goodchild, J; Perkins, T; Fodstad, O; Hovig, E

1998-01-01

The uptake and cellular metabolism of a fluorescein-labelled synthetic ribozyme stabilized by 2'- O -methyl modification and a 3' inverted thymidine have been studied, employing capillary gel electrophoresis as a novel and efficient analytical method. After internalization by DOTAP transfection, electrophoretic peaks of intact ribozyme and different degradation products were easily resolved and the amount of intracellular intact ribozyme was quantified to >10(7) molecules/cell at the peak value after 4 h transfection. On further incubation the amount of intracellular intact ribozyme decreased due to both degradation and efflux from the cell. However, even after 48 h incubation there were still >10(6) intact ribozyme molecules/cell. Clear differences both in uptake and in metabolism were seen when comparing DOTAP transfection with the uptake of free ribozyme. Fluorescence microscopy studies indicated that the ribozyme was mainly localized in intracellular granules, probably not accessible to target mRNA. This implies that agents able to release the intact ribozyme from intracellular vesicles into the cytosol should have a considerable potential for increasing the biological effects of synthetic ribozymes. PMID:9722645

Enhanced Product Stability in the Hammerhead Ribozyme†

PubMed Central

Shepotinovskaya, Irina; Uhlenbeck, Olke C.

2010-01-01

The rate of dissociation of P1, the 5′ product of hammerhead cleavage, is 100–300-fold slower in full-length hammerheads than in hammerheads that either lack or have disrupting mutations in the loop-loop tertiary interaction. The added stability requires the presence of residue 17 at the 3′ terminus of P1 but not the 2′, 3′ terminal phosphate. Since residue 17 is buried within the catalytic core of the hammerhead in the x-ray structure, we propose that the enhanced P1 stability is the result of the cooperative folding of the hammerhead around this residue. However, since the P1 is fully stabilized above 2.5 mM MgCl2 while hammerhead activity continues to increase with increasing MgCl2, it is clear that the hammerhead structure in the transition state must differ from that of the product complex. The product stabilization assay is used to test our earlier proposal that different tertiary interactions modulate the cleavage rate by differentially stabilizing the core. PMID:20423112

Studying Hammerheads in Hawaii

ERIC Educational Resources Information Center

Handler, Alex; Duncan, Kanesa

2006-01-01

In this article, the author discusses the High School Scalloped Hammerhead Shark Tagging Program in Hawaii which is an example of a successful partnership research collaboration. High school students and teachers worked with biologists from the University of Hawaii-Manoa (UHM) to conduct research on the life history of scalloped hammerhead sharks…

Inhibitory Ah Receptor-Androgen Receptor Crosstalk in Prostate Cancer

DTIC Science & Technology

2006-02-01

Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate...therapeutic implications, N. Engl. J. Med. 285 (1971) 1182–1186. [14] T. Dorai, C.A. Olsson, A.E. Katz, R. Buttyan, Development of a hammerhead ... ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer, Prostate 32 (1997) 246

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

PubMed Central

Campbell, T B; Cech, T R

1995-01-01

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519

Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.

PubMed Central

L'Huillier, P J; Davis, S R; Bellamy, A R

1992-01-01

Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell. Images PMID:1425576

Substrate specificity and reaction kinetics of an X-motif ribozyme

PubMed Central

LAZAREV, DENIS; PUSKARZ, IZABELA; BREAKER, RONALD R.

2003-01-01

The X-motif is an in vitro-selected ribozyme that catalyzes RNA cleavage by an internal phosphoester transfer reaction. This ribozyme class is distinguished by the fact that it emerged as the dominant clone among at least 12 different classes of ribozymes when in vitro selection was conducted to favor the isolation of high-speed catalysts. We have examined the structural and kinetic properties of the X-motif in order to provide a framework for its application as an RNA-cleaving agent and to explore how this ribozyme catalyzes phosphoester transfer with a predicted rate constant that is similar to those exhibited by the four natural self-cleaving ribozymes. The secondary structure of the X-motif includes four stem elements that form a central unpaired junction. In a bimolecular format, two of these base-paired arms define the substrate specificity of the ribozyme and can be changed to target different RNAs for cleavage. The requirements for nucleotide identity at the cleavage site are GD, where D = G, A, or U and cleavage occurs between the two nucleotides. The ribozyme has an absolute requirement for a divalent cation cofactor and exhibits kinetic behavior that is consistent with the obligate binding of at least two metal ions. PMID:12756327

Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae.

PubMed

Klauser, Benedikt; Rehm, Charlotte; Summerer, Daniel; Hartig, Jörg S

2015-01-01

Synthetic RNA-based switches are a growing class of genetic controllers applied in synthetic biology to engineer cellular functions. In this chapter, we detail a protocol for the selection of posttranscriptional controllers of gene expression in yeast using the Schistosoma mansoni hammerhead ribozyme as a central catalytic unit. Incorporation of a small molecule-sensing aptamer domain into the ribozyme renders its activity ligand-dependent. Aptazymes display numerous advantages over conventional protein-based transcriptional controllers, namely, the use of little genomic space for encryption, their modular architecture allowing for easy reprogramming to new inputs, the physical linkage to the message to be controlled, and the ability to function without protein cofactors. Herein, we describe the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. We also highlight how to adapt the protocol to screen for switches responsive to other ligands. Reprogramming of the sensor unit and incorporation into any RNA of interest enables the fulfillment of a variety of regulatory functions. However, proper functioning of the aptazyme is largely dependent on optimal connection between the aptamer and the catalytic core. We obtained functional switches from a pool of variants carrying randomized connection sequences by an in vivo selection in MaV203 yeast cells that allows screening of a large sequence space of up to 1×10(9) variants. The protocol given explains how to construct aptazyme libraries, carry out the in vivo selection and characterize novel ON- and OFF-switches. © 2015 Elsevier Inc. All rights reserved.

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

PubMed Central

Zhou, De-Min; Zhang, Li-He; Taira, Kazunari

1997-01-01

In a previous examination using natural all-RNA substrates that contained either a 5′-oxy or 5′-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2′-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5′-thio group (R11S) and (ii) the departure of the 5′ leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5′-oxygen, because a switch in metal ion specificity was not observed with the 5′-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis. PMID:9405614

Insight into the role of Mg2+ in hammerhead ribozyme catalysis from X-ray crystallography and molecular dynamics simulation

PubMed Central

Lee, Tai-Sung; López, Carlos Silva; Martick, Monika; Scott, William G.; York, Darrin M.

2008-01-01

Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg2+ ion), early and late transition state mimics are presented based on a recently reported crystal structure of a full-length hammerhead RNA. The simulation results support a catalytically active conformation with a Mg2+ ion bridging the A9 and scissile phosphates. In the reactant state, the Mg2+ spends significant time closely associated with the 2′OH of G8, but remains fairly distant from the leaving group O5′ position. In the early TS mimic simulation, where the nucleophilic O2′ and leaving group O5′ are equidistant from the phosphorus, the Mg2+ ion remains tightly coordinated to the 2′OH of G8, but is positioned closer to the O5′ leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg2+ ion undergoes a transition whereby the coordination with the 2′OH of G8 is replace by the leaving group O5′ that has developed significant charge. At the same time, the 2′OH of G8 forms a hydrogen bond with the leaving group O5′ and is positioned to act as a general acid catalyst. This work represents the first reported simulations of the full-length hammerhead structure and TS mimics, and provides direct evidence for the possible role of a bridging Mg2+ ion in catalysis that is consistent with both crystallographic and biochemical data. PMID:19079784

Kinetics of hairpin ribozyme cleavage in yeast.

PubMed Central

Donahue, C P; Fedor, M J

1997-01-01

Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

Toxic Neuronal Death by Glyeraldehyde-3-Phosphate Dehydrogenase and Mitochondria

DTIC Science & Technology

2003-08-01

Neuroreport, 10(5), 1149-1153. Sioud, M., & Jespersen, L. (1996). Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase...1996) Enhancemen t of hammerhead r ibozyme cata lysis by glycera ldehyde-3- phospha te dehydrogenase. J Mol Biol 257:775–789. Sirover MA (1997) Role of

Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis.

PubMed

Lee, Raymond Teck Ho; Ng, Ashley Shu Mei; Ingham, Philip W

2016-01-01

CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.

Self-Incorporation of Coenzymes by Ribozymes

NASA Technical Reports Server (NTRS)

Breaker, Ronald R.; Joyce, Gerald F.

1995-01-01

RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNAcatalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

Georgetown Institute for Cognitive and Computational Sciences

DTIC Science & Technology

2000-03-01

in neuronal apoptosis. Cerebellar granules cells (CGCs) were co-transfected with a green fluorescent protein reporter and one of several hammerhead ... ribozymes constructed to cleave caspase-3 RNA. Use of such ribozymes is highly selective. In separate experiments we co-transfected with a gene that...expressing a ribozyme against rat caspase-3. Apoptosis was assessed after 24, 36, or 48 h of serum/K+ deprivation. In negative control cells expressing ß

Selective Androgen Receptor Down-Regulators (SARDs): A New Prostate Cancer Therapy

DTIC Science & Technology

2007-10-01

PCa (9). Thus far, the techniques that have been used to down-regulate the AR include antisense oligonucleotides (10, 11), ribozyme treatments (12...Our findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or...Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol

Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta.

PubMed

Millington-Ward, Sophia; Allers, Carolina; Tuohy, Gearóid; Conget, Paulette; Allen, Danny; McMahon, Helena P; Kenna, Paul F; Humphries, Peter; Farrar, G Jane

2002-09-15

Over 100 dominant-negative mutations within the COL1A1 gene have been identified in osteogenesis imperfecta (OI). In terms of human therapeutics, targeting each of these mutations independently is unlikely to be feasible. Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1. Downregulation was confirmed at the protein level. Transducing stem cells with Rzpol1A1 ex vivo followed by autologous transplantation could provide a gene therapy for a large proportion of OI patients with gain-of-function mutations using a single therapeutic.

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

PubMed Central

Trang, P; Hsu, A W; Liu, F

1999-01-01

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications. PMID:10556315

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

NASA Technical Reports Server (NTRS)

Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.;

Repairing RNA Transcripts that Mediate Breast Cancer Susceptibility

DTIC Science & Technology

2005-08-01

is actually the yield of TES product plus the yield of cryptic is in contrast to hammerhead and hairpin ribozymes , which products. This increases the...therapeutics. To this end, we have developed a novel biomolecule (a ribozyme ) that can specifically excise regions from RNA transcripts. In this work, we...designed a ribozyme that excises an insertion mutation that is linked to breast cancer predisposition from a short mimic of the p53 transcript in a

Cripto-1 in Mammary Gland Development and Carcinogenesis

DTIC Science & Technology

2000-12-01

Task 4). T.O. 2 We have designed and tested a hammerhead ribozyme [21, 22] that recognizes nucleotides 12-28 of the murine CR-I mRNA and cuts after the...Growth Factors, Ribozymes 23 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF...Underexpression of CR-1 will decrease tumorigenicity of highly or moderately tumorigenic cell lines. (T.O. 2) 4. Efficient delivery of a ribozyme or

Puberty, Differentiation and Breast Cancer Risk

DTIC Science & Technology

1999-09-01

tetracycline or doxocycline to culture medium. We designed hammerhead ribozymes which recognize four different regions of mBrca2, 6 Proprietary Data DAMD17...containing a ribozyme directed against the 5’ end of mBrca2 appear to show dox-dependent regulation of Brca2 levels (as measured by Western blot...for possible modest regulation), we have designed ribozymes which recognize human and mouse BRCA2 sequence, and we intend to create tet-inducible

Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

DTIC Science & Technology

2009-10-01

CT-cells have endog- enous CT stably knocked down to undetectable levels using anti-CT hammerhead ribozymes [25]. Salmon CT (BAChem, Torrance, CA) was...and cells called CTR-, derived from PC-3M cells after anti-CT receptor ribozyme knock- down of CTR[18]. CTR-cells have very low levels of CD44v

Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

DTIC Science & Technology

2008-04-01

hammerhead ribozymes .25 Salmon CT (BAChem, Torrance, CA) was used at physiologic 50 nM dose14,16, which effectively alters CD44,6 or at 250 nM. To detect...receptor14), and cells called CTR-, derived from PC-3M cells after anti-CT receptor ribozyme knockdown of CTR.18 CTR- cells have very low levels of

A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

NASA Technical Reports Server (NTRS)

Jaeger, L.; Wright, M. C.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

1999-01-01

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

Delta ribozyme has the ability to cleave in transan mRNA.

PubMed Central

Roy, G; Ananvoranich, S; Perreault, J P

1999-01-01

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724

Signal Propagation and Detection via Catalytically Immolative Biopolymer-Programmed Nanomaterials

DTIC Science & Technology

2015-02-09

hammerhead   ribozymes   as   therapeutic   tools   to   control  disease  genes.  Curr.  Gene  Ther.  5,  11-­‐24  (2005...Incorporation  of  Coenzymes  by   Ribozymes .  Journal   of  Molecular  Evolution  40,  551-­‐558  (1995).   36   Liou,  H.-­‐C

Microtubule-Targeting Therapy for Prostate Cancer

DTIC Science & Technology

2007-02-01

that were done to achieve the above specific goals. 1. Biological effects of ribozyme -carrying adenoviruses that target stathmin mRNA in human...prostate cancer cells: A ribozyme is a small RNA molecule that acts stoichiometrically to cleave multiple target RNA molecules [1]. This unique ability...of a ribozyme to degrade multiple target RNA molecules is a more efficient approach for down regulating genes that are expressed at very high levels

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

PubMed

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

2017-06-13

Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

NASA Technical Reports Server (NTRS)

Hager, A. J.; Szostak, J. W.

1997-01-01

BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

NASA Technical Reports Server (NTRS)

Tsang, Joyce; Joyce, Gerald F.

1996-01-01

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

An anti-VEGF ribozyme embedded within the adenoviral VAI sequence inhibits glioblastoma cell angiogenic potential in vitro.

PubMed

Ciafrè, Silvia Anna; Niola, Francesco; Wannenes, Francesca; Farace, Maria Giulia

2004-01-01

Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis, where it functions as one of the major angiogenic factors sustaining growth and draining catabolites. In this study, we developed an anti-VEGF ribozyme targeted to the 5' part of human VEGF mRNA. We endowed this ribozyme with an additional feature expected to improve its activity in vivo, by cloning it into a VAI transcriptional cassette. VAI is originally part of the adenovirus genome, and is characterized by high transcription rates, good stability due to its strong secondary structure and cytoplasmic localization. Transfection of U87 human glioblastoma cells with plasmid vectors encoding for this ribozyme resulted in a strong (-56%) reduction of VEGF secreted in the extracellular medium, indicating a good biological activity of the ribozyme. Moreover, this reduction in VEGF secretion had the important functional consequence of drastically diminishing the formation of tube-like structures of human umbilical vascular endothelial cells in a Matrigel in vitro angiogenesis assay. In conclusion, our VAI-embedded anti-VEGF ribozyme is a good inhibitor of angiogenesis in vitro, in a glioblastoma cell context. Thus, it may represent a useful tool for future applications in vivo, for antiangiogenic gene therapy of glioblastoma and of highly vascularized tumors. Copyright 2004 S. Karger AG, Basel

Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

PubMed

Chowrira, B M; Burke, J M

1991-09-03

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

In vitro evolution of a ribozyme that contains 5-bromouridine

NASA Technical Reports Server (NTRS)

Dai, X.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

2000-01-01

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure

PubMed Central

Tobé, Sylvia; Heams, Thomas; Vergne, Jacques; Hervé, Guy; Maurel, Marie-Christine

2005-01-01

The discovery of ribozymes strengthened the RNA world hypothesis, which assumes that these precursors of modern life both stored information and acted as catalysts. For the first time among extensive studies on ribozymes, we have investigated the influence of hydrostatic pressure on the hairpin ribozyme catalytic activity. High pressures are of interest when studying life under extreme conditions and may help to understand the behavior of macromolecules at the origins of life. Kinetic studies of the hairpin ribozyme self-cleavage were performed under high hydrostatic pressure. The activation volume of the reaction (34 ± 5 ml/mol) calculated from these experiments is of the same order of magnitude as those of common protein enzymes, and reflects an important compaction of the RNA molecule during catalysis, associated to a water release. Kinetic studies were also carried out under osmotic pressure and confirmed this interpretation and the involvement of water movements (78 ± 4 water molecules per RNA molecule). Taken together, these results are consistent with structural studies indicating that loops A and B of the ribozyme come into close contact during the formation of the transition state. While validating baro-biochemistry as an efficient tool for investigating dynamics at work during RNA catalysis, these results provide a complementary view of ribozyme catalytic mechanisms. PMID:15870387

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

PubMed Central

Gleitsman, Kristin R.

2014-01-01

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. PMID:25246656

Engineering integrated digital circuits with allosteric ribozymes for scaling up molecular computation and diagnostics.

PubMed

Penchovsky, Robert

2012-10-19

Here we describe molecular implementations of integrated digital circuits, including a three-input AND logic gate, a two-input multiplexer, and 1-to-2 decoder using allosteric ribozymes. Furthermore, we demonstrate a multiplexer-decoder circuit. The ribozymes are designed to seek-and-destroy specific RNAs with a certain length by a fully computerized procedure. The algorithm can accurately predict one base substitution that alters the ribozyme's logic function. The ability to sense the length of RNA molecules enables single ribozymes to be used as platforms for multiple interactions. These ribozymes can work as integrated circuits with the functionality of up to five logic gates. The ribozyme design is universal since the allosteric and substrate domains can be altered to sense different RNAs. In addition, the ribozymes can specifically cleave RNA molecules with triplet-repeat expansions observed in genetic disorders such as oculopharyngeal muscular dystrophy. Therefore, the designer ribozymes can be employed for scaling up computing and diagnostic networks in the fields of molecular computing and diagnostics and RNA synthetic biology.

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

DOE PAGES

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; ...

2014-12-05

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

PubMed Central

Yao, Chengguo; Müller, Ulrich F.

2011-01-01

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3′-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world. PMID:21622900

Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach

PubMed Central

Beger, Carmela; Pierce, Leigh N.; Krüger, Martin; Marcusson, Eric G.; Robbins, Joan M.; Welcsh, Piri; Welch, Peter J.; Welte, Karl; King, Mary-Claire; Barber, Jack R.; Wong-Staal, Flossie

2001-01-01

Expression of the breast and ovarian cancer susceptibility gene BRCA1 is down-regulated in sporadic breast and ovarian cancer cases. Therefore, the identification of genes involved in the regulation of BRCA1 expression might lead to new insights into the pathogenesis and treatment of these tumors. In the present study, an “inverse genomics” approach based on a randomized ribozyme gene library was applied to identify cellular genes regulating BRCA1 expression. A ribozyme gene library with randomized target recognition sequences was introduced into human ovarian cancer-derived cells stably expressing a selectable marker [enhanced green fluorescence protein (EGFP)] under the control of the BRCA1 promoter. Cells in which BRCA1 expression was upregulated by particular ribozymes were selected through their concomitant increase in EGFP expression. The cellular target gene of one ribozyme was identified to be the dominant negative transcriptional regulator Id4. Modulation of Id4 expression resulted in inversely regulated expression of BRCA1. In addition, increase in Id4 expression was associated with the ability of cells to exhibit anchorage-independent growth, demonstrating the biological relevance of this gene. Our data suggest that Id4 is a crucial gene regulating BRCA1 expression and might therefore be important for the BRCA1 regulatory pathway involved in the pathogenesis of sporadic breast and ovarian cancer. PMID:11136250

Crystal structure of Pistol, a class of self-cleaving ribozyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Laura A.; Wang, Jimin; Steitz, Thomas A.

2017-01-17

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile bymore » deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.« less

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

PubMed Central

Daròs, J A; Marcos, J F; Hernández, C; Flores, R

1994-01-01

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7809126

Pressure modulates the self-cleavage step of the hairpin ribozyme

NASA Astrophysics Data System (ADS)

Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

2017-03-01

The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

Evaluation of a Petition Requesting National Marine Fisheries Service (NMFS) to List the Smooth Hammerhead Shark (Sphryna zygaena) as a Threatened or Endangered Species Under the Endangered Species Act (ESA)

NASA Astrophysics Data System (ADS)

Sturm, A. B.

2016-12-01

The wildlife conservation organization, Defenders of Wildlife, petitioned NMFS to list the smooth hammerhead shark, Sphryna zygaena, as endangered or threatened throughout its range under the ESA. The petition was critically evaluated to determine if the petitioners presented substantial scientific or commercial information indicating that the smooth hammerhead shark may warrant listing under the ESA. The petition and the cited scientific literature (as well as scientific literature readily available in NMFS files) were evaluated to determine if the smooth hammerhead shark may be threatened or endangered because of any one or a combination of the following five ESA section 4(a)(1) factors: (1) present or threatened destruction, modification, or curtailment of its habitat or range; (2) over utilization for commercial, recreational, scientific, or educational purposes; (3) disease or predation; (4) inadequacy of existing regulatory mechanisms; (5) or other natural or manmade factors affecting its continued existence. The available scientific literature indicates that the smooth hammerhead shark populations have declined in multiple regions. Smooth hammerhead sharks may warrant listing due to ongoing threats of over utilization for commercial purposes by global fisheries that target and retain incidental catch of these species to obtain their high-value fins, possible inadequacies in global regulatory mechanisms to control this level of exploitation, and natural factors (such as inherent biological vulnerabilities) that may be exacerbating these threats. Based on these findings, the smooth hammerhead shark may warrant listing as a threatened or endangered species under the ESA and a status review of the species is currently being conducted.

Evaluation of a Petition Requesting National Marine Fisheries Service (NMFS) to List the Smooth Hammerhead Shark (Sphryna zygaena) as a Threatened or Endangered Species Under the Endangered Species Act (ESA)

NASA Astrophysics Data System (ADS)

Sturm, A. B.

2016-02-01

The wildlife conservation organization, Defenders of Wildlife, petitioned NMFS to list the smooth hammerhead shark, Sphryna zygaena, as endangered or threatened throughout its range under the ESA. The petition was critically evaluated to determine if the petitioners presented substantial scientific or commercial information indicating that the smooth hammerhead shark may warrant listing under the ESA. The petition and the cited scientific literature (as well as scientific literature readily available in NMFS files) were evaluated to determine if the smooth hammerhead shark may be threatened or endangered because of any one or a combination of the following five ESA section 4(a)(1) factors: (1) present or threatened destruction, modification, or curtailment of its habitat or range; (2) over utilization for commercial, recreational, scientific, or educational purposes; (3) disease or predation; (4) inadequacy of existing regulatory mechanisms; (5) or other natural or manmade factors affecting its continued existence. The available scientific literature indicates that the smooth hammerhead shark populations have declined in multiple regions. Smooth hammerhead sharks may warrant listing due to ongoing threats of over utilization for commercial purposes by global fisheries that target and retain incidental catch of these species to obtain their high-value fins, possible inadequacies in global regulatory mechanisms to control this level of exploitation, and natural factors (such as inherent biological vulnerabilities) that may be exacerbating these threats. Based on these findings, the smooth hammerhead shark may warrant listing as a threatened or endangered species under the ESA and a status review of the species is currently being conducted.

Ribozyme-catalysed RNA synthesis using triplet building blocks.

PubMed

Attwater, James; Raguram, Aditya; Morgunov, Alexey S; Gianni, Edoardo; Holliger, Philipp

2018-05-15

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit '+' and '-' strands in segments and assemble them into a new active ribozyme. © 2018, Attwater et al.

Applicability of PM3 to transphosphorylation reaction path: Toward designing a minimal ribozyme

NASA Technical Reports Server (NTRS)

Manchester, John I.; Shibata, Masayuki; Setlik, Robert F.; Ornstein, Rick L.; Rein, Robert

1993-01-01

A growing body of evidence shows that RNA can catalyze many of the reactions necessary both for replication of genetic material and the possible transition into the modern protein-based world. However, contemporary ribozymes are too large to have self-assembled from a prebiotic oligonucleotide pool. Still, it is likely that the major features of the earliest ribozymes have been preserved as molecular fossils in the catalytic RNA of today. Therefore, the search for a minimal ribozyme has been aimed at finding the necessary structural features of a modern ribozyme (Beaudry and Joyce, 1990). Both a three-dimensional model and quantum chemical calculations are required to quantitatively determine the effects of structural features of the ribozyme on the reaction it catalyzes. Using this model, quantum chemical calculations must be performed to determine quantitatively the effects of structural features on catalysis. Previous studies of the reaction path have been conducted at the ab initio level, but these methods are limited to small models due to enormous computational requirements. Semiempirical methods have been applied to large systems in the past; however, the accuracy of these methods depends largely on a simple model of the ribozyme-catalyzed reaction, or hydrolysis of phosphoric acid. We find that the results are qualitatively similar to ab initio results using large basis sets. Therefore, PM3 is suitable for studying the reaction path of the ribozyme-catalyzed reaction.

Selection of ribozymes that catalyse multiple-turnover Diels–Alder cycloadditions by using in vitro compartmentalization

PubMed Central

Agresti, Jeremy J.; Kelly, Bernard T.; Jäschke, Andres; Griffiths, Andrew D.

2005-01-01

In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 1011 ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By coencapsulating the gene, RNA, and the substrates/products of the catalyzed reaction, ribozymes can be selected for all enzymatic properties: substrate recognition, product formation, rate acceleration, and turnover. Here we exploit the complementarity of IVC with systematic evolution of ligands by exponential enrichment (SELEX), which allows selection of larger libraries (≥1015) and for very small rate accelerations (kcat/kuncat) but only selects for intramolecular single-turnover reactions. We selected ≈1014 random RNAs for Diels–Alderase activity with five rounds of SELEX, then six to nine rounds with IVC. All selected ribozymes catalyzed the Diels–Alder reaction in a truly bimolecular fashion and with multiple turnover. Nearly all ribozymes selected by using eleven rounds of SELEX alone contain a common catalytic motif. Selecting with SELEX then IVC gave ribozymes with significant sequence variations in this catalytic motif and ribozymes with completely novel motifs. Interestingly, the catalytic properties of all of the selected ribozymes were quite similar. The ribozymes are strongly product inhibited, consistent with the Diels–Alder transition state closely resembling the product. More efficient Diels–Alderases may need to catalyze a second reaction that transforms the product and prevents product inhibition. PMID:16260754

The Hydrodynamics and Odorant Transport Phenomena of Olfaction in the Hammerhead Shark

NASA Astrophysics Data System (ADS)

Rygg, Alex; Craven, Brent

2013-11-01

The hammerhead shark possesses a unique head morphology that is thought to facilitate enhanced olfactory performance. The olfactory organs, located at the distal ends of the cephalofoil, contain numerous lamellae that increase the surface area for olfaction. Functionally, for the shark to detect chemical stimuli, water-borne odors must reach the olfactory sensory epithelium that lines these lamellae. Thus, odorant transport from the aquatic environment to the sensory epithelium is the first critical step in olfaction. Here we investigate the hydrodynamics and odorant transport phenomena of olfaction in the hammerhead shark based on an anatomically-accurate reconstruction of the head and olfactory chamber from high-resolution micro-CT and MRI scans of a cadaver specimen. Computational fluid dynamics (CFD) simulations of water flow in the reconstructed model reveal the external and internal hydrodynamics of olfaction during swimming. Odorant transport in the olfactory organ is investigated using a multi-scale approach, whereby molecular dynamics (MD) simulations are used to calculate odorant partition coefficients that are subsequently utilized in macro-scale CFD simulations of odorant deposition. The hydrodynamic and odorant transport results are used to elucidate several important features of olfactory function in the hammerhead shark.

Nucleotide synthetase ribozymes may have emerged first in the RNA world

PubMed Central

Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

2007-01-01

Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells. PMID:17878321

Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures.

PubMed Central

Hernández, C; Flores, R

1992-01-01

Peach latent mosaic viroid (PLMVd), the causal agent of peach latent mosaic disease, has been sequenced and found to be a circular RNA molecule of 337 nucleotide residues, which adopts a branched conformation when it is folded in the model of lowest free energy. PLMVd exhibits limited homologies with other viroids and some satellite RNAs, but it does not have any of the central conserved sequences characteristic of the subgroups of typical viroids. However, a segment of approximately one-third of the PLMVd sequence has the elements required to form in the RNAs of both polarities the hammerhead structures proposed to act in the in vitro self-cleavage of avocado sunblotch viroid (ASBVd) and some satellite RNAs. Plus and minus partial- and full-length RNA transcripts of PLMVd containing the hammerhead structures displayed self-cleavage during transcription and after purification as predicted by these structures. These data are consistent with the high stability of the PLMVd hammerhead structures, more similar to the corresponding structures of some satellite RNAs than to those of ASBVd, and indicate that the self-cleavage reactions of PLMVd are most probably mediated by single hammerhead structures. Our results support the inclusion of PLMVd in a viroid subgroup represented by ASBVd, whose members are characterized by their ability to self-cleave in vitro, and probably in vivo, through hammerhead structures. A consensus phylogenetic tree has been obtained suggesting that PLMVd, together with ASBVd, may represent an evolutionary link between viroids and viroid-like satellite RNAs. Images PMID:1373888

Inventing and improving ribozyme function: rational design versus iterative selection methods

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

Two major strategies for generating novel biological catalysts exist. One relies on our knowledge of biopolymer structure and function to aid in the 'rational design' of new enzymes. The other, often called 'irrational design', aims to generate new catalysts, in the absence of detailed physicochemical knowledge, by using selection methods to search a library of molecules for functional variants. Both strategies have been applied, with considerable success, to the remodeling of existing ribozymes and the development of ribozymes with novel catalytic function. The two strategies are by no means mutually exclusive, and are best applied in a complementary fashion to obtain ribozymes with the desired catalytic properties.

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Evolution in vitro: analysis of a lineage of ribozymes

NASA Technical Reports Server (NTRS)

Lehman, N.; Joyce, G. F.

1993-01-01

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

The glmS Ribozyme Cofactor is a General Acid-Base Catalyst

PubMed Central

Viladoms, Julia; Fedor, Martha J.

2012-01-01

The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The D-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst. PMID:23113700

The glmS ribozyme cofactor is a general acid-base catalyst.

PubMed

Viladoms, Júlia; Fedor, Martha J

2012-11-21

The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.

Generation and development of RNA ligase ribozymes with modular architecture through "design and selection".

PubMed

Fujita, Yuki; Ishikawa, Junya; Furuta, Hiroyuki; Ikawa, Yoshiya

2010-08-26

In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed "design and selection," new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection.

A thiamin-utilizing ribozyme decarboxylates a pyruvate-like substrate

NASA Astrophysics Data System (ADS)

Cernak, Paul; Sen, Dipankar

2013-11-01

Vitamins are hypothesized to be relics of an RNA world, and were probably participants in RNA-mediated primordial metabolism. If catalytic RNAs, or ribozymes, could harness vitamin cofactors to aid their function in a manner similar to protein enzymes, it would enable them to catalyse a much larger set of chemical reactions. The cofactor thiamin diphosphate, a derivative of vitamin B1 (thiamin), is used by enzymes to catalyse difficult metabolic reactions, including decarboxylation of stable α-keto acids such as pyruvate. Here, we report a ribozyme that uses free thiamin to decarboxylate a pyruvate-based suicide substrate (LnkPB). Thiamin conjugated to biotin was used to isolate catalytic individuals from a pool of random-sequence RNAs attached to LnkPB. Analysis of a stable guanosine adduct obtained via digestion of an RNA sequence (clone dc4) showed the expected decarboxylation product. The discovery of a prototypic thiamin-utilizing ribozyme has implications for the role of RNA in orchestrating early metabolic cycles.

Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

PubMed Central

Ben Aissa, Manel; April, Marie-Claude; Bergeron, Lucien-Junior; Perreault, Jean-Pierre; Levesque, Georges

2012-01-01

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ 40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD. PMID:22482079

Molecular crowding favors reactivity of a human ribozyme under physiological ionic conditions.

PubMed

Strulson, Christopher A; Yennawar, Neela H; Rambo, Robert P; Bevilacqua, Philip C

2013-11-19

In an effort to relate RNA folding to function under cellular-like conditions, we monitored the self-cleavage reaction of the human hepatitis delta virus-like CPEB3 ribozyme in the background of physiological ionic concentrations and various crowding and cosolute agents. We found that at physiological free Mg(2+) concentrations (∼0.1-0.5 mM), both crowders and cosolutes stimulate the rate of self-cleavage, up to ∼6-fold, but that in 10 mM Mg(2+) (conditions widely used for in vitro ribozyme studies) these same additives have virtually no effect on the self-cleavage rate. We further observe a dependence of the self-cleavage rate on crowder size, wherein the level of rate stimulation is diminished for crowders larger than the size of the unfolded RNA. Monitoring effects of crowding and cosolute agents on rates in biological amounts of urea revealed additive-promoted increases at both low and high Mg(2+) concentrations, with a maximal stimulation of more than 10-fold and a rescue of the rate to its urea-free values. Small-angle X-ray scattering experiments reveal a structural basis for this stimulation in that higher-molecular weight crowding agents favor a more compact form of the ribozyme in 0.5 mM Mg(2+) that is essentially equivalent to the form under standard ribozyme conditions of 10 mM Mg(2+) without a crowder. This finding suggests that at least a portion of the rate enhancement arises from favoring the native RNA tertiary structure. We conclude that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions.

Marfan syndrome, magnesium status and medical prevention of cardiovascular complications by hemodynamic treatments and antisense gene therapy.

PubMed

Igondjo-Tchen, S; Pagès, N; Bac, P; Godeau, G; Durlach, J

2003-03-01

The medical management of Marfan Syndrome (MFS) mainly relies on early prevention of the aortic complications. Hemodynamic treatments try to diminish the forcefulness of cardiac contractions and to reduce blood pressure: for example long term administration of propranolol may significantly reduce the rate of increase in aortic ratio (aortic diameter/expected aortic diameter). Retardation of aortic dilatation may be most often observed by early treatment started when the baseline end-diastolic aortic root diameter is < 40 mm. It seems better to use beta-blockers without intrinsic sympathomimetic activity. Successful acceptance of beta-blockers may be limited by side-effects, but the efficiency of alternative hypotensive agents (calcium channel inhibitors, ACE inhibitors) is not yet validated. Gene therapy might constitute an etiologic specific treatment of MFS. FBN1-RZ1 hammerhead antisense ribozyme is able to suppress expression of the mutant FBN1 allele. The use of ribozymes as systemic therapeutic agents will depend on efficient delivery to its target, but the various proposed vectors raise yet unsolved problems. A hydrogel angioplasty balloon might be a possible vector for delivering an antisense ribozyme in the aortic wall specifically. Ribozymes--as deoxyribonucleotides--may be taken up by tissue upon local application. Further research should study ex vivo local application of antisense ribozyme on human aortic wall, before assessing in vivo efficiency and tolerance of this aortic local vectorisation. It is always necessary to maintain a balanced magnesium intake in patients with MFS. Firstly to prevent the multiple noxious effects of magnesium deficiency on cardiovascular targets. Secondly to ensure the best efficiency and the least toxicity of the hemodynamic drugs used as long term prophylactic treatment for cardiovascular complications and of the etiologic antisense magnesium-dependent gene therapy, in the future.

Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

PubMed Central

Walter, N G; Burke, J M

1997-01-01

Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems. PMID:9085846

Role of the Active Site Guanine in the glmS Ribozyme Self-Cleavage Mechanism: Quantum Mechanical/Molecular Mechanical Free Energy Simulations

PubMed Central

2015-01-01

The glmS ribozyme catalyzes a self-cleavage reaction at the phosphodiester bond between residues A-1 and G1. This reaction is thought to occur by an acid–base mechanism involving the glucosamine-6-phosphate cofactor and G40 residue. Herein quantum mechanical/molecular mechanical free energy simulations and pKa calculations, as well as experimental measurements of the rate constant for self-cleavage, are utilized to elucidate the mechanism, particularly the role of G40. Our calculations suggest that an external base deprotonates either G40(N1) or possibly A-1(O2′), which would be followed by proton transfer from G40(N1) to A-1(O2′). After this initial deprotonation, A-1(O2′) starts attacking the phosphate as a hydroxyl group, which is hydrogen-bonded to deprotonated G40, concurrent with G40(N1) moving closer to the hydroxyl group and directing the in-line attack. Proton transfer from A-1(O2′) to G40 is concomitant with attack of the scissile phosphate, followed by the remainder of the cleavage reaction. A mechanism in which an external base does not participate, but rather the proton transfers from A-1(O2′) to a nonbridging oxygen during nucleophilic attack, was also considered but deemed to be less likely due to its higher effective free energy barrier. The calculated rate constant for the favored mechanism is in agreement with the experimental rate constant measured at biological Mg2+ ion concentration. According to these calculations, catalysis is optimal when G40 has an elevated pKa rather than a pKa shifted toward neutrality, although a balance among the pKa’s of A-1, G40, and the nonbridging oxygen is essential. These results have general implications, as the hammerhead, hairpin, and twister ribozymes have guanines at a similar position as G40. PMID:25526516

3'-End labeling of nucleic acids by a polymerase ribozyme.

PubMed

Samanta, Biswajit; Horning, David P; Joyce, Gerald F

2018-06-13

A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.

Continuous In Vitro Evolution of a Ribozyme that Catalyzes Three Successive Nucleotidyl Addition Reactions

NASA Technical Reports Server (NTRS)

McGinness, Kathleen E.; Wright, Martin C.; Joyce, Gerald F.

2002-01-01

Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(exp 406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5' yieldig 3' or a 3' yielding 5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

PubMed

Kolev, Nikolay G; Hartland, Emilia I; Huber, Paul W

2008-10-01

The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

Restoration of chemosensitivity in cancer cells with MDR phenotype by deoxyribozyme, compared with ribozyme.

PubMed

Xing, Ai-Yan; Shi, Duan-bo; Liu, Wei; Chen, Xu; Sun, Yan-Lin; Wang, Xiao; Zhang, Jian-ping; Gao, Peng

2013-06-01

One of the main mechanisms for multidrug resistance (MDR) involves multidrug resistance gene 1 (MDR1) which encodes P-glycoprotein (Pgp). Pgp acts as a drug efflux pump and exports chemotherapeutic agents from cancer cells. Specific inhibition of Pgp expression by gene therapy is considered a well-respective strategy having less innate toxicities. At present, the investigation of DRz in reversal MDR is scarce. In the study, phosphorothioate DRz that targets to the translation initiation codon AUG was synthesized and transfected into breast cancer cells and leukemia cells with MDR phenotype. ASODN (antisense oligonucleotide) and ribozyme targets to the same region were also synthesized for comparison analysis. Alterations in MDR1 mRNA and Pgp were determined by RT-PCR, Northern blot, flow cytometry and Rh123 retention tests. Chemosensitivity of the treated cells was determined by MTT assay. The results showed that DRz could significantly suppress expression of MDR1 mRNA and inhibit synthesis of Pgp. The efflux activity of Pgp was inhibited accordingly. Chemosensitivity assay showed that a 21-fold reduction in drug resistance for Adriamycin and a 45-fold reduction in drug resistance for Vinblastine were found in the treated cells 36h after transfection. These data suggest that DRz targeted to the translation initiation codon AUG can reverse MDR phenotype in cancer cells and restore their chemosensitivity. Moreover, the reversal efficiency of DRz is better than that of ribozyme and ASODN targets to the same region of MDR1 mRNA. Copyright © 2013 Elsevier Inc. All rights reserved.

Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

PubMed Central

Downs, W D; Cech, T R

1996-01-01

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures. PMID:8756414

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

PubMed Central

Serganov, Alexander; Keiper, Sonja; Malinina, Lucy; Tereshko, Valentina; Skripkin, Eugene; Höbartner, Claudia; Polonskaia, Anna; Phan, Anh Tuân; Wombacher, Richard; Micura, Ronald; Dauter, Zbigniew; Jäschke, Andres; Patel, Dinshaw J

2015-01-01

The majority of structural efforts addressing RNA’s catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions. PMID:15723077

Crossing lines: a multidisciplinary framework for assessing connectivity of hammerhead sharks across jurisdictional boundaries

NASA Astrophysics Data System (ADS)

Chin, A.; Simpfendorfer, C. A.; White, W. T.; Johnson, G. J.; McAuley, R. B.; Heupel, M. R.

2017-04-01

Conservation and management of migratory species can be complex and challenging. International agreements such as the Convention on Migratory Species (CMS) provide policy frameworks, but assessments and management can be hampered by lack of data and tractable mechanisms to integrate disparate datasets. An assessment of scalloped (Sphyrna lewini) and great (Sphyrna mokarran) hammerhead population structure and connectivity across northern Australia, Indonesia and Papua New Guinea (PNG) was conducted to inform management responses to CMS and Convention on International Trade in Endangered Species listings of these species. An Integrated Assessment Framework (IAF) was devised to systematically incorporate data across jurisdictions and create a regional synopsis, and amalgamated a suite of data from the Australasian region. Scalloped hammerhead populations are segregated by sex and size, with Australian populations dominated by juveniles and small adult males, while Indonesian and PNG populations included large adult females. The IAF process introduced genetic and tagging data to produce conceptual models of stock structure and movement. Several hypotheses were produced to explain stock structure and movement patterns, but more data are needed to identify the most likely hypothesis. This study demonstrates a process for assessing migratory species connectivity and highlights priority areas for hammerhead management and research.

Crossing lines: a multidisciplinary framework for assessing connectivity of hammerhead sharks across jurisdictional boundaries.

PubMed

Chin, A; Simpfendorfer, C A; White, W T; Johnson, G J; McAuley, R B; Heupel, M R

2017-04-21

Conservation and management of migratory species can be complex and challenging. International agreements such as the Convention on Migratory Species (CMS) provide policy frameworks, but assessments and management can be hampered by lack of data and tractable mechanisms to integrate disparate datasets. An assessment of scalloped (Sphyrna lewini) and great (Sphyrna mokarran) hammerhead population structure and connectivity across northern Australia, Indonesia and Papua New Guinea (PNG) was conducted to inform management responses to CMS and Convention on International Trade in Endangered Species listings of these species. An Integrated Assessment Framework (IAF) was devised to systematically incorporate data across jurisdictions and create a regional synopsis, and amalgamated a suite of data from the Australasian region. Scalloped hammerhead populations are segregated by sex and size, with Australian populations dominated by juveniles and small adult males, while Indonesian and PNG populations included large adult females. The IAF process introduced genetic and tagging data to produce conceptual models of stock structure and movement. Several hypotheses were produced to explain stock structure and movement patterns, but more data are needed to identify the most likely hypothesis. This study demonstrates a process for assessing migratory species connectivity and highlights priority areas for hammerhead management and research.

Crossing lines: a multidisciplinary framework for assessing connectivity of hammerhead sharks across jurisdictional boundaries

PubMed Central

Chin, A.; Simpfendorfer, C. A.; White, W. T.; Johnson, G. J.; McAuley, R. B.; Heupel, M. R.

2017-01-01

Conservation and management of migratory species can be complex and challenging. International agreements such as the Convention on Migratory Species (CMS) provide policy frameworks, but assessments and management can be hampered by lack of data and tractable mechanisms to integrate disparate datasets. An assessment of scalloped (Sphyrna lewini) and great (Sphyrna mokarran) hammerhead population structure and connectivity across northern Australia, Indonesia and Papua New Guinea (PNG) was conducted to inform management responses to CMS and Convention on International Trade in Endangered Species listings of these species. An Integrated Assessment Framework (IAF) was devised to systematically incorporate data across jurisdictions and create a regional synopsis, and amalgamated a suite of data from the Australasian region. Scalloped hammerhead populations are segregated by sex and size, with Australian populations dominated by juveniles and small adult males, while Indonesian and PNG populations included large adult females. The IAF process introduced genetic and tagging data to produce conceptual models of stock structure and movement. Several hypotheses were produced to explain stock structure and movement patterns, but more data are needed to identify the most likely hypothesis. This study demonstrates a process for assessing migratory species connectivity and highlights priority areas for hammerhead management and research. PMID:28429742

Local neutral networks help maintain inaccurately replicating ribozymes.

PubMed

Szilágyi, András; Kun, Ádám; Szathmáry, Eörs

2014-01-01

The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world.

A novel strategy for selection of allosteric ribozymes yields RiboReporter™ sensors for caffeine and aspartame

PubMed Central

Ferguson, Alicia; Boomer, Ryan M.; Kurz, Markus; Keene, Sara C.; Diener, John L.; Keefe, Anthony D.; Wilson, Charles; Cload, Sharon T.

2004-01-01

We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter™ sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids. PMID:15026535

Kinetic and Binding Analysis of the Catalytic Involvement of Ribose Moieties of a trans-Acting δ Ribozyme*

PubMed Central

Fiola, Karine; Perreault, Jean-Pierre

2010-01-01

We have identified ribose 2′-hydroxyl groups (2′-OHs) that are critical for the activity of a trans-cleaving δ ribozyme derived from the antigenomic strand of the hepatitis δ virus. Initially, an RNA-DNA mixed ribozyme composed of 26 deoxyribo- (specifically the nucleotides forming the P2 stem and the P4 stem-loop) and 31 ribonucleotides (those forming the catalytic center) was engineered. This mixed ribozyme catalyzed the cleavage of a small substrate with kinetic parameters virtually identical to those of the all-RNA ribozyme. The further substitution of deoxyribose for ribose residues permitted us to investigate the contribution of all 2′-OHs to catalysis. Determination of the kinetic parameters for the cleavage reaction of the resulting ribozymes revealed (i) 10 2′-OH groups appear to be important in supporting the formation of several hydrogen bonds within the catalytic core, (ii) none of the important 2′-OHs seem to coordinate a magnesium cation, and (iii) 1 of the tested RNA-DNA mixed polymers appeared to stabilize the ribozyme-substrate transition-state complex, resulting in an improvement over the all-RNA counterpart. The contribution of the 2′-OHs to the catalytic mechanism is discussed, and differences with the crystal structure of a genomic δ self-cleaved product are explained. Clearly, the 2′-OHs are essential components of the network of interactions involved in the formation of the catalytic center of the δ ribozyme. PMID:12015324

Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Dagenais, Pierre; Legault, Pascale

2016-08-19

The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics

PubMed Central

Bingaman, Jamie L.; Messina, Kyle J.; Bevilacqua, Philip C.

2017-01-01

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand-mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions. PMID:28315484

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

PubMed

Lacroix-Labonté, Julie; Girard, Nicolas; Lemieux, Sébastien; Legault, Pascale

2012-03-01

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

General acid-base catalysis mediated by nucleobases in the hairpin ribozyme

PubMed Central

Kath-Schorr, Stephanie; Wilson, Timothy J.; Li, Nan-Sheng; Lu, Jun; Piccirilli, Joseph A.; Lilley, David M. J.

2012-01-01

The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data. PMID:22958171

RNA Synthesis by in Vitro Selected Ribozymes for Recreating an RNA World

PubMed Central

Martin, Lyssa L.; Unrau, Peter J.; Müller, Ulrich F.

2015-01-01

The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme’s possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory. PMID:25610978

Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

PubMed

Bingaman, Jamie L; Messina, Kyle J; Bevilacqua, Philip C

2017-05-01

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change.

PubMed

Tanaka, Yoichiro; Tagaya, Mitsuhiro; Hori, Tamaki; Sakamoto, Taiichi; Kurihara, Yasuyuki; Katahira, Masato; Uesugi, Seiichi

2002-06-01

Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans-acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site. We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3' cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5'-terminal cytidine residue prevents this conformational change and its absence stabilizes the product-ribozyme complex in the presence of Mg2+. The structure of the Mg2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.

Evidence That Nucleophile Deprotonation Exceeds Bond Formation in the HDV Ribozyme Transition State.

PubMed

Lu, Jun; Koo, Selene C; Weissman, Benjamin P; Harris, Michael E; Li, Nan-Sheng; Piccirilli, Joseph A

2018-06-26

Steric constraints imposed by the active sites of protein and RNA enzymes pose major challenges to the investigation of structure-function relationships within these systems. As a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized phosphoramidites from propanediol derivatives and incorporated them at the 5'-termini of RNA and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution reduces the maximal rate and the kinetic p K a , consistent with the expected electron-withdrawing effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo transphosphorylation relatively efficiently, suggesting that the conformational constraints imposed by a ribofuranose ring are not strictly required for ribozyme catalysis. In contrast to the nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction rate, consistent with a mechanism in which (1) the 2'-hydroxyl nucleophile exists predominantly in its neutral, protonated form in the ground state and (2) the 2'-hydroxyl bears some negative charge in the rate-determining step, consistent with a transition state in which the extent of 2'-OH deprotonation exceeds the extent of P-O bond formation.

Hammerhead Shark Research Immersion Program: Experiential Learning Leads to Lasting Educational Benefits

ERIC Educational Resources Information Center

Handler, Alex; Duncan, Kanesa

2006-01-01

High school students (n = 45) participated in a 5-day research immersion study on juvenile scalloped hammerhead sharks in Kaneohe Bay, Oahu, Hawaii. Self-surveys were used to evaluate scientific concepts and skills taught during the program. There was a significant shift in students' perceived level of understanding for all categories of concepts…

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

PubMed Central

Scanlon, K J; Jiao, L; Funato, T; Wang, W; Tone, T; Rossi, J J; Kashani-Sabet, M

1991-01-01

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression. Images PMID:1660142

Trans-activation of the Tetrahymena group I intron ribozyme via a non-native RNA-RNA interaction.

PubMed Central

Ikawa, Y; Shiraishi, H; Inoue, T

1999-01-01

The peripheral P2.1 domain of the Tetrahymena group I intron ribozyme has been shown to be non-essential for splicing. We found, however, that separately prepared P2.1 RNA efficiently accelerates the 3' splice-site-specific hydrolysis reaction of a mutant ribozyme lacking both P2.1 and its upstream region in trans. We report here the unusual properties of this trans-activation. Compensatory mutational analysis revealed that non-native long-range base-pairings between the loop region of P2.1 RNA and L5c region of the mutant ribozyme are needed for the activation in spite of the fact that P2.1 forms base-pairings with P9.1 in the Tetrahymena ribozyme. The trans -activation depends on the non-native RNA-RNA interaction together with the higher order structure of P2.1 RNA. This activation is unique among the known trans-activations that utilize native tertiary interactions or RNA chaperons. PMID:10075996

Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing

PubMed Central

Kisseleva, Natalia; Kraut, Stefanie; Jäschke, Andres; Schiemann, Olav

2007-01-01

In ribozyme catalysis, metal ions are generally known to make structural and∕or mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance (EPR) spectroscopy. Manganese ion titrations revealed five high-affinity Mn2+ binding sites with an upper Kd of 0.6±0.2 μM. In order to characterize each binding site individually, EPR-silent Cd2+ ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn2+ binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn2+ dimer. Based on simulations, the Mn2+-Mn2+ distance within the dimer was found to be ∼6 Å, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions. PMID:19404418

Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme.

PubMed

White, Neil A; Hoogstraten, Charles G

2017-09-01

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2'-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure. Copyright © 2017 Elsevier B.V. All rights reserved.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts.

PubMed

Li, Li-Hua; Guo, Zi-Jian; Yan, Ling-Ling; Yang, Ji-Cheng; Xie, Yu-Feng; Sheng, Wei-Hua; Huang, Zhao-Hui; Wang, Xue-Hao

2007-12-21

To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7,721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed. VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7,721 cells and xenografted tumor. Compared to the control group, the transfected cells grew slower in cell cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

Computed Energetics of Nucleotides in Spatial Ribozyme Structures: An Accurate Identification of Functional Regions from Structure

PubMed Central

Torshin, Ivan Y.

2004-01-01

Ribozymes are functionally diverse RNA molecules with intrinsic catalytic activity. Multiple structural and biochemical studies are required to establish which nucleotide bases are involved in the catalysis. The relative energetic properties of the nucleotide bases have been analyzed in a set of the known ribozyme structures. It was found that many of the known catalytic nucleotides can be identified using only the structure without any additional biochemical data. The results of the calculations compare well with the available biochemical data on RNA stability. Extensive in silico mutagenesis suggests that most of the nucleotides in ribozymes stabilize the RNA. The calculations show that relative contribution of the catalytic bases to RNA stability observably differs from contributions of the noncatalytic bases. Distinction between the concepts of “relative stability” and “mutational stability” is suggested. As results of prediction for several models of ribozymes appear to be in agreement with the published data on the potential active site regions, the method can potentially be used for prediction of functional nucleotides from nucleic sequence. PMID:15105962

Targeting of the Nuclear Receptor Coactivator Isoform DELTA3AIB1 in Breast Cancer

DTIC Science & Technology

2007-03-01

lab showed that the downregulation of overall levels of AIB1 plus ∆3AIB1, using a regulatable AIB1 directed ribozyme , resulted in reduced tumor...overall levels of AIB1 plus ∆3AIB1, using a regulatable AIB1 directed ribozyme , resulted in reduced tumor growth in vivo. Overall, these data indicate a...Reiter R, Powers C, Wellstein A, Riegel AT. Ribozyme targeting shows that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

PubMed Central

Ananvoranich, S; Lafontaine, D A; Perreault, J P

1999-01-01

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway. PMID:10037808

A speculated ribozyme site in the herpes simplex virus type 1 latency-associated transcript gene is not essential for a wild-type reactivation phenotype

PubMed Central

Carpenter, Dale; Singh, Sukhpreet; Osorio, Nelson; Hsiang, Chinhui; Jiang, Xianzhi; Jin, Ling; Jones, Clinton; Wechsler, Steven L

2010-01-01

During herpes simplex virus-1 (HSV-1) latency in sensory neurons, LAT (latency-associated transcript) is the only abundantly expressed viral gene. LAT plays an important role in the HSV-1 latency-reactivation cycle, because LAT deletion mutants have a significantly decreased reactivation phenotype. Based solely on sequence analysis, it was speculated that LAT encodes a ribozyme that plays an important role in how LAT enhances the virus’ reactivation phenotype. Because LAT ribozyme activity has never been reported, we decided to test the converse hypothesis, namely, that this region of LAT does not encode a ribozyme function important for LAT’s ability to enhance the reactivation phenotype. We constructed a viral mutant (LAT-Rz) in which the speculated ribozyme consensus sequence was altered such that no ribozyme was encoded. We report here that LAT-Rz had a wild-type reactivation phenotype in mice, confirming the hypothesis that the speculated LAT ribozyme is not a dominant factor in stimulating the latency-reactivation cycle in mice. PMID:18982533

Wobble pairs of the HDV ribozyme play specific roles in stabilization of active site dynamics.

PubMed

Sripathi, Kamali N; Banáš, Pavel; Réblová, Kamila; Šponer, Jiří; Otyepka, Michal; Walter, Nils G

2015-02-28

The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5') hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5') general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5') hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs.

Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

PubMed Central

Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

2015-01-01

The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

PubMed

Aroonsri, Aiyada; Akinola, Olugbenga; Posayapisit, Navaporn; Songsungthong, Warangkhana; Uthaipibull, Chairat; Kamchonwongpaisan, Sumalee; Gbotosho, Grace O; Yuthavong, Yongyuth; Shaw, Philip J

2016-07-01

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Natural and unnatural ribozymes: back to the primordial RNA world.

PubMed

Talini, Giulia; Gallori, Enzo; Maurel, Marie-Christine

2009-09-01

We review natural and in vitro selected ribozymes, for which combined studies could provide us with both insight into the functions performed by ancient RNA molecules in a primitive RNA world and a hypothesis about evolutionary steps that led to the contemporary world.

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

PubMed Central

Mustoe, Anthony M.; Al-Hashimi, Hashim M.; Brooks, Charles L.

2016-01-01

A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding. PMID:26481360

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

PubMed Central

Harris, M E; Kazantsev, A V; Chen, J L; Pace, N R

1997-01-01

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme. PMID:9174092

Bcl2-low-expressing MCF7 cells undergo necrosis rather than apoptosis upon staurosporine treatment.

PubMed Central

Poliseno, Laura; Bianchi, Laura; Citti, Lorenzo; Liberatori, Sabrina; Mariani, Laura; Salvetti, Alessandra; Evangelista, Monica; Bini, Luca; Pallini, Vitaliano; Rainaldi, Giuseppe

2004-01-01

We present a ribozyme-based strategy for studying the effects of Bcl2 down-regulation. The anti-bcl2 hammerhead ribozyme Rz-bcl2 was stably transfected into MCF7 cancer cells and the cleavage of Bcl2 mRNA was demonstrated using a new assay for cleavage product detection, while Western blot analysis showed a concomitant depletion of Bcl2 protein. Rz-bcl2-expressing cells were more sensitive to staurosporine than control cells. Moreover, both molecular and cellular read-outs indicated that staurosporine-induced cell death was necrosis rather than apoptosis in these cells. The study of the effects of Bcl2 down-regulation was extended to the global MCF7 protein expression profile, exploiting a proteomic approach. Two reference electro-pherograms of Rz-bcl2-transfected cells, one with the ribozyme in a catalytically active form and the other with the ribozyme in a catalytically inactive form, were obtained. When comparing the two-dimensional maps, 53 differentially expressed spots were found, four of which were identified by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS as calreticulin, nucleophosmin, phosphoglycerate kinase and pyruvate kinase. How the up-regulation of these proteins might help to explain the modification of Bcl2 activity is discussed. PMID:14748742

Architecture of a Diels-Alderase ribozyme with a preformed catalytic pocket.

PubMed

Keiper, Sonja; Bebenroth, Dirk; Seelig, Burckhard; Westhof, Eric; Jäschke, Andres

2004-09-01

Artificial ribozymes catalyze a variety of chemical reactions. Their structures and reaction mechanisms are largely unknown. We have analyzed a ribozyme catalyzing Diels-Alder cycloaddition reactions by comprehensive mutation analysis and a variety of probing techniques. New tertiary interactions involving base pairs between nucleotides of the 5' terminus and a large internal loop forming a pseudoknot fold were identified. The probing data indicate a preformed tertiary structure that shows no major changes on substrate or product binding. Based on these observations, a molecular architecture featuring a Y-shaped arrangement is proposed. The tertiary structure is formed in a rather unusual way; that is, the opposite sides of the asymmetric internal loop are clamped by the four 5'-terminal nucleotides, forming two adjacent two base-pair helices. It is proposed that the catalytic pocket is formed by a wedge within one of these helices.

Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme

PubMed Central

Lévesque, Dominique; Reymond, Cédric; Perreault, Jean-Pierre

2012-01-01

The HDV ribozyme’s folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U23 and G28 nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U23 and G28 can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction. PMID:22768274

The effects of anti-Fas ribozyme on T lymphocyte apoptosis in mice model with chronic obstructive pulmonary disease.

PubMed

Zhuo, Song-Ming; Li, Si-Cong; Lin, Yong-Qun; Yu, Hai-Bin; Li, Na

2017-10-01

In this study, we aimed to investigate the effects of anti-Fas ribozyme on the apoptosis of T lymphocytes (T cells) in mice model with chronic obstructive pulmonary disease (COPD). Male 6-week-old C57BL/6 mice were used to establish the COPD model by exposure to cigarette smoke. The COPD mice were sacrificed for spleen dissection and T cell isolation. T cells were randomly divided into four groups (n=10 per group). Group A was used as the control. B, C, and D groups were transfected with empty lentivirus, anti-Fas ribozyme, and an anti-Fas ribozyme mutant, respectively. The expression of Fas mRNA and protein in the T cells were evaluated using qPCR and Western blot, respectively. Flow cytometry was used to evaluate the apoptosis of CD 4+ T cells and calculate the ratio of CD 4+ to CD 8+ T cells (CD 4+ /CD 8+ ). Anti-Fas ribozyme significantly inhibited the expression of Fas in the T cells of COPD mice. In addition, the number of apoptotic CD 4+ T cells and CD 4+ /CD 8+ of the C and D groups were significantly lower and higher than those of group A, respectively ( P <0.05). The apoptotic CD 4+ T cells and CD 4+ CD 8+ of the C group were significantly lower and higher than those of group D, respectively ( P <0.05). Anti-Fas ribozyme significantly inhibited the expression of Fas, increased CD 4+ /CD 8+ , and inhibited the apoptosis of T cells in COPD mice.

Complexity in pH-Dependent Ribozyme Kinetics: Dark pKa Shifts and Wavy Rate-pH Profiles.

PubMed

Frankel, Erica A; Bevilacqua, Philip C

2018-02-06

Charged bases occur in RNA enzymes, or ribozymes, where they play key roles in catalysis. Cationic bases donate protons and perform electrostatic catalysis, while anionic bases accept protons. We previously published simulations of rate-pH profiles for ribozymes in terms of species plots for the general acid and general base that have been useful for understanding how ribozymes respond to pH. In that study, we did not consider interaction between the general acid and general base or interaction with other species on the RNA. Since that report, diverse small ribozyme classes have been discovered, many of which have charged nucleobases or metal ions in the active site that can either directly interact and participate in catalysis or indirectly interact as "influencers". Herein, we simulate experimental rate-pH profiles in terms of species plots in which reverse protonated charged nucleobases interact. These analyses uncover two surprising features of pH-dependent enzyme kinetics. (1) Cooperativity between the general acid and general base enhances population of the functional forms of a ribozyme and manifests itself as hidden or "dark" pK a shifts, real pK a shifts that accelerate the reaction but are not readily observed by standard experimental approaches, and (2) influencers favorably shift the pK a s of proton-transferring nucleobases and manifest themselves as "wavy" rate-pH profiles. We identify parallels with the protein enzyme literature, including reverse protonation and wavelike behavior, while pointing out that RNA is more prone to reverse protonation. The complexities uncovered, which arise from simple pairwise interactions, should aid deconvolution of complex rate-pH profiles for RNA and protein enzymes and suggest veiled catalytic devices for promoting catalysis that can be tested by experiment and calculation.

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

PubMed Central

Kraut, Stefanie; Bebenroth, Dirk; Nierth, Alexander; Kobitski, Andrei Y.; Nienhaus, G. Ulrich; Jäschke, Andres

2012-01-01

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function. PMID:21976731

The electrosensorial pore system of the cephalofoil in the four most common species of hammerhead shark (Elasmobranchii: Sphyrnidae) from the Southwestern Atlantic.

PubMed

Mello, Waldiney

2009-04-01

The laterally expanded head is the principal character distinguishing hammerhead sharks, and its morphology is important for interpreting their ontogeny and species diversity. Because their head shape changes during its ontogeny, it is vital to evaluate it in order to establish other taxonomical characteristics to correctly identify Sphyrna species. This study examines the distribution of electrosensorial pore regions on the ventral surface of the cephalofoil (VSC) in Sphyrna lewini, S. tiburo, S. tudes and S. zygaena from the Southwestern Atlantic Ocean. The pore distribution patterns in the VSC can distinguish these species. Use of those patterns, with the head shape, confirms the identification of the four most common species of hammerhead sharks in the Southwestern Atlantic.

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PubMed

Merdan, Thomas; Kunath, Klaus; Fischer, Dagmar; Kopecek, Jindrich; Kissel, Thomas

2002-02-01

Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

76 FR 72891 - Endangered and Threatened Wildlife; 90-Day Finding on a Petition To List the Scalloped Hammerhead...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-28

... hammerhead shark as threatened or endangered under the Endangered Species Act (ESA), and to designate... NatureServe for listing under the U.S. Endangered Species Act'' because NatureServe assessments ``have... Headquarters Office (see ADDRESSES). Authority The authority for this action is the Endangered Species Act of...

Chemical synthesis of oligoribonucleotides containing 2-aminopurine: substrates for the investigation of ribozyme function

NASA Technical Reports Server (NTRS)

Doudna, J. A.; Szostak, J. W.; Rich, A.; Usman, N.

1990-01-01

The chemical synthesis of a fully protected ribonucleoside phosphoramidite, containing 2-aminopurine as the base component, and its incorporation into short oligoribonucleotides as substrates for an engineered ribozyme from Tetrahymena is described.

An Adaptive Defect Weighted Sampling Algorithm to Design Pseudoknotted RNA Secondary Structures

PubMed Central

Zandi, Kasra; Butler, Gregory; Kharma, Nawwaf

2016-01-01

Computational design of RNA sequences that fold into targeted secondary structures has many applications in biomedicine, nanotechnology and synthetic biology. An RNA molecule is made of different types of secondary structure elements and an important RNA element named pseudoknot plays a key role in stabilizing the functional form of the molecule. However, due to the computational complexities associated with characterizing pseudoknotted RNA structures, most of the existing RNA sequence designer algorithms generally ignore this important structural element and therefore limit their applications. In this paper we present a new algorithm to design RNA sequences for pseudoknotted secondary structures. We use NUPACK as the folding algorithm to compute the equilibrium characteristics of the pseudoknotted RNAs, and describe a new adaptive defect weighted sampling algorithm named Enzymer to design low ensemble defect RNA sequences for targeted secondary structures including pseudoknots. We used a biological data set of 201 pseudoknotted structures from the Pseudobase library to benchmark the performance of our algorithm. We compared the quality characteristics of the RNA sequences we designed by Enzymer with the results obtained from the state of the art MODENA and antaRNA. Our results show our method succeeds more frequently than MODENA and antaRNA do, and generates sequences that have lower ensemble defect, lower probability defect and higher thermostability. Finally by using Enzymer and by constraining the design to a naturally occurring and highly conserved Hammerhead motif, we designed 8 sequences for a pseudoknotted cis-acting Hammerhead ribozyme. Enzymer is available for download at https://bitbucket.org/casraz/enzymer. PMID:27499762

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

PubMed

Ledbetter, Michael P; Hwang, Tony W; Stovall, Gwendolyn M; Ellington, Andrew D

2013-01-01

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule. Copyright © 2013 Wiley-Liss, Inc.

The role of an active site Mg(2+) in HDV ribozyme self-cleavage: insights from QM/MM calculations.

PubMed

Mlýnský, Vojtěch; Walter, Nils G; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

2015-01-07

The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H(+) in the active site, which acts as the general acid, and a partially hydrated Mg(2+) ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with a distinct position and coordination of the catalytically important active site Mg(2+) ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal mol(-1), indicating that the specific position of the Mg(2+) ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2'-OH) nucleophile and the nucleophilic attack of the resulting U-1(2'-O(-)) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimate the pKa of the U-1(2'-OH) group to range from 8.8 to 11.2, suggesting that it is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg(2+) ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg(2+) ion, facilitate deprotonation and activation of the 2'-OH nucleophile.

Recovery of infectious type Asia1 foot-and-mouth disease virus from suckling mice directly inoculated with an RNA polymerase I/II-driven unidirectional transcription plasmid.

PubMed

Lian, Kaiqi; Yang, Fan; Zhu, Zixiang; Cao, Weijun; Jin, Ye; Li, Dan; Zhang, Keshan; Guo, Jianhong; Zheng, Haixue; Liu, Xiangtao

2015-10-02

We developed an RNA polymerase (pol) I- and II-driven plasmid-based reverse genetics system to rescue infectious foot-and-mouth disease virus (FMDV) from cloned cDNA. In this plasmid-based transfection, the full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences, which were arranged downstream of the two promoters (cytomegalovirus (CMV) and pol I promoter) and upstream of the terminators and polyadenylation signal, respectively. The utility of this method was demonstrated by the recovery of FMDV Asia1 HN/CHA/06 in BHK-21 cells transfected with cDNA plasmids. Furthermore, infectious FMDV Asia1 HN/CHA/06 could be rescued from suckling mice directly inoculated with cDNA plasmids. Thus, this reverse genetics system can be applied to fundamental research and vaccine studies, most notably to rescue those viruses for which there is currently an absence of a suitable cell culture system. Copyright © 2015 Elsevier B.V. All rights reserved.

Competition Between Co(NH3)63+ and Inner Sphere Mg2+ Ions in the HDV Ribozyme

PubMed Central

Gong, Bo; Chen, Jui-Hui; Bevilacqua, Philip C.; Golden, Barbara L.; Carey, Paul R.

2009-01-01

Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg2+ are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH3)63+, an analog of Mg(H2O)62+. Here, the binding of Mg2+ and Co(NH3)63+ to the HDV ribozyme are studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg2+ alone is introduced to the ribozyme, inner sphere coordination of Mg(H2O)x2+ (x≤5) to non-bridging PO2− oxygen, and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg2+ induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH3)63+ alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg2+ binding, Co(NH3)63+ binding does not perturb PO2− group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH3)63+ ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO2− groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH3)63+ ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg2+-PO2− binding sites: sites that Co(NH3)63+ can displace, and others it cannot. PMID:19888753

Molecular Dynamics Study of Twister Ribozyme: Role of Mg(2+) Ions and the Hydrogen-Bonding Network in the Active Site.

PubMed

Ucisik, Melek N; Bevilacqua, Philip C; Hammes-Schiffer, Sharon

2016-07-12

The recently discovered twister ribozyme is thought to utilize general acid-base catalysis in its self-cleavage mechanism, but the roles of nucleobases and metal ions in the mechanism are unclear. Herein, molecular dynamics simulations of the env22 twister ribozyme are performed to elucidate the structural and equilibrium dynamical properties, as well as to examine the role of Mg(2+) ions and possible candidates for the general base and acid in the self-cleavage mechanism. The active site region and the ends of the pseudoknots were found to be less mobile than other regions of the ribozyme, most likely providing structural stability and possibly facilitating catalysis. A purported catalytic Mg(2+) ion and the closest neighboring Mg(2+) ion remained chelated and relatively immobile throughout the microsecond trajectories, although removal of these Mg(2+) ions did not lead to any significant changes in the structure or equilibrium motions of the ribozyme on the microsecond time scale. In addition, a third metal ion, a Na(+) ion remained close to A1(O5'), the leaving group atom, during the majority of the microsecond trajectories, suggesting that it might stabilize the negative charge on A1(O5') during self-cleavage. The locations of these cations and their interactions with key nucleotides in the active site suggest that they may be catalytically relevant. The P1 stem is partially melted at its top and bottom in the crystal structure and further unwinds in the trajectories. The simulations also revealed an interconnected network comprised of hydrogen-bonding and π-stacking interactions that create a relatively rigid network around the self-cleavage site. The nucleotides involved in this network are among the highly conserved nucleotides in twister ribozymes, suggesting that this interaction network may be important to structure and function.

On Improving CRISPR for Editing Plant Genes: Ribozyme-Mediated Guide RNA Production and Fluorescence-Based Technology for Isolating Transgene-Free Mutants Generated by CRISPR.

PubMed

He, Yubing; Wang, Rongchen; Dai, Xinhua; Zhao, Yunde

2017-01-01

CRISPR/Cas9-mediated genome editing technology has been used to successfully edit numerous genes in various organisms including plants. There are still two major challenges in using CRISPR/Cas9 technology for gene editing in plants. First, there are very limited choices of promoters that are suitable for in vivo production of single-guide RNAs (sgRNAs), which is complementary to the target sequence and which guides Cas9 to generate double-strand breaks at the target site. It is especially difficult to produce sgRNA molecules with temporal and spatial precision. Second, there is a lack of efficient methods for identifying plants that (1) contain heritable and stable mutations generated by CRISPR/Cas9, and (2) no longer harbor the CRISPR/Cas9 construct and other transgenes. In this chapter, we describe the development of a ribozyme-based strategy that enables the production of sgRNA molecules from any chosen promoter. More importantly, the ribozyme-based technology makes it feasible to produce sgRNAs with temporal and spatial precision, greatly expanding the scope and applications of CRISPR/Cas9 technology. We also developed a fluorescence-based technology that allows us to efficiently and reliably isolate Cas9-free stable Arabidopsis mutants. Thus, we provide effective protocols to overcome two important obstacles in using CRISPR/Cas9 for editing genes in plants. © 2017 Elsevier Inc. All rights reserved.

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

PubMed Central

Harris, M E; Pace, N R

1995-01-01

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs. PMID:7585250

ICTV Virus Taxonomy Profile: Avsunviroidae.

PubMed

Di Serio, Francesco; Li, Shi-Fang; Matoušek, Jaroslav; Owens, Robert A; Pallás, Vicente; Randles, John W; Sano, Teruo; Verhoeven, Jacobus Th J; Vidalakis, Georgios; Flores, Ricardo; Ictv Report Consortium

2018-05-01

Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at http://www.ictv.global/report/avsunviroidae.

Optimal self-cleavage activity of the hepatitis delta virus RNA is dependent on a homopurine base pair in the ribozyme core.

PubMed Central

Been, M D; Perrotta, A T

1995-01-01

A non-Watson-Crick G.G interaction within the core region of the hepatitis delta virus (HDV) antigenomic ribozyme is required for optimal rates of self-cleavage activity. Base substitutions for either one or both G's revealed that full activity was obtained only when both G's were replaced with A's. At those positions, substitutions that generate potential Watson-Crick, G.U, heteropurine, or homopyrimidine combinations resulted in dramatically lower cleavage activity. A homopurine symmetric base pair, of the same type identified in the high-affinity binding site of the HIV RRE, is most consistent with this data. Additional features shared between the antigenomic ribozyme and the Rev binding site in the vicinity of the homopurine pairs suggest some structural similarity for this region of the two RNAs and a possible motif associated with this homopurine interaction. Evidence for a homopurine pair at the equivalent position in a modified form of the HDV genomic ribozyme was also found. With the postulated symmetric pairing scheme, large distortions in the nucleotide conformation, the sugar-phosphate backbone, or both would be necessary to accommodate this interaction at the end of a helix; we hypothesize that this distortion is critical to the structure of the active site of the ribozyme and it is stabilized by the homopurine base pair. PMID:8595561

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme

PubMed Central

Xie, Zheng; Srividya, Narayanan; Sosnick, Tobin R.; Pan, Tao; Scherer, Norbert F.

2004-01-01

The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-Ieq-to-N, and focused on the Ieq-to-N transition. The present study focuses on the U-to-Ieq transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5′ end and Cy3 at the 3′ end show that modifications required for labeling do not interfere with folding and help to define the Mg2+ concentration range for the U-to-Ieq transition. Histogram analysis of the Mg2+-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg2+ concentrations on the time scale of seconds. The trajectories at intermediate Mg2+ concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of “nonsudden jump” FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many “local” conformational switches. A free energy contour is constructed to illustrate the complex folding surface. PMID:14704266

Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions.

PubMed Central

Earnshaw, D J; Gait, M J

1998-01-01

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells. PMID:9837982

The GlcN6P cofactor serves multiple catalytic roles in the glmS ribozyme

PubMed Central

Bingaman, Jamie L.; Zhang, Sixue; Stevens, David R.; Yennawar, Neela H.; Hammes-Schiffer, Sharon; Bevilacqua, Philip C.

2017-01-01

RNA enzymes have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors. The naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies implicated GlcN6P as the general acid. Here we describe new catalytic roles for GlcN6P through experiments and calculations. Large stereospecific normal thio effects and lack of metal ion rescue in the holoribozyme show that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal ion rescue in the aporibozyme reveals this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, allowing RNA to compensate for its limited functional diversity. PMID:28192411

RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme

PubMed Central

Biesiada, Marcin; Boniecki, Michał J.; Chou, Fang-Chieh; Ferré-D'Amaré, Adrian R.; Das, Rhiju; Dunin-Horkawicz, Stanisław; Geniesse, Caleb; Kappel, Kalli; Kladwang, Wipapat; Krokhotin, Andrey; Łach, Grzegorz E.; Major, François; Mann, Thomas H.; Pachulska-Wieczorek, Katarzyna; Patel, Dinshaw J.; Piccirilli, Joseph A.; Popenda, Mariusz; Purzycka, Katarzyna J.; Ren, Aiming; Rice, Greggory M.; Santalucia, John; Tandon, Arpit; Trausch, Jeremiah J.; Wang, Jian; Weeks, Kevin M.; Williams, Benfeard; Xiao, Yi; Zhang, Dong; Zok, Tomasz

2017-01-01

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5′-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson–Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/. PMID:28138060

A catalytic metal ion interacts with the cleavage Site G.U wobble in the HDV ribozyme.

PubMed

Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C; Carey, Paul R; Golden, Barbara L

2009-02-24

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.

Montmorillonite protection of an UV-irradiated hairpin ribozyme: evolution of the RNA world in a mineral environment

PubMed Central

Biondi, Elisa; Branciamore, Sergio; Maurel, Marie-Christine; Gallori, Enzo

2007-01-01

Background The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis. Results The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay. Conclusion These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization. PMID:17767730

Cleavage of an amide bond by a ribozyme

NASA Technical Reports Server (NTRS)

Dai, X.; De Mesmaeker, A.; Joyce, G. F.; Miller, S. L. (Principal Investigator)

1995-01-01

A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

PubMed Central

Lynch, S R; Tinoco, I

1998-01-01

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented. PMID:9461457

Unravelling RNA-substrate interactions in a ribozyme-catalysed reaction using fluorescent turn-on probes.

PubMed

Gaffarogullari, Ece Cazibe; Greulich, Peter; Kobitski, Andrei Yu; Nierth, Alexander; Nienhaus, G Ulrich; Jäschke, Andres

2015-04-07

The Diels-Alder reaction is one of the most important C-C bond-forming reactions in organic chemistry, and much effort has been devoted to controlling its enantio- and diastereoselectivity. The Diels-Alderase ribozyme (DAse) catalyses the reaction between anthracene dienes and maleimide dienophiles with multiple-turnover, stereoselectivity, and up to 1100-fold rate acceleration. Here, a new generation of anthracene-BODIPY-based fluorescent probes was developed to monitor catalysis by the DAse. The brightness of these probes increases up to 93-fold upon reaction with N-pentylmaleimide (NPM), making these useful tools for investigating the stereochemistry of the ribozyme-catalysed reaction. With these probes, we observed that the DAse catalyses the reaction with >91% de and >99% ee. The stereochemistry of the major product was determined unambiguously by rotating-frame nuclear Overhauser NMR spectroscopy (ROESY-NMR) and is in agreement with crystallographic structure information. The pronounced fluorescence change of the probes furthermore allowed a complete kinetic analysis, which revealed an ordered bi uni type reaction mechanism, with the dienophile binding first. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting Micrornas With Small Molecules: A Novel Approach to Treating Breast Cancer

DTIC Science & Technology

2010-10-01

ribozymes and the DNAzymes, small interfering RNAs and short hairpin RNAs, and anti-miRNA agents such as antisense oligo- nucleotides, locked nucleic...of the antagomir Preclinical studies Ribozymes or DNAzymes A ribozyme , or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. A

Great hammerhead sharks swim on their side to reduce transport costs

PubMed Central

Payne, Nicholas L.; Iosilevskii, Gil; Barnett, Adam; Fischer, Chris; Graham, Rachel T.; Gleiss, Adrian C.; Watanabe, Yuuki Y.

2016-01-01

Animals exhibit various physiological and behavioural strategies for minimizing travel costs. Fins of aquatic animals play key roles in efficient travel and, for sharks, the functions of dorsal and pectoral fins are considered well divided: the former assists propulsion and generates lateral hydrodynamic forces during turns and the latter generates vertical forces that offset sharks' negative buoyancy. Here we show that great hammerhead sharks drastically reconfigure the function of these structures, using an exaggerated dorsal fin to generate lift by swimming rolled on their side. Tagged wild sharks spend up to 90% of time swimming at roll angles between 50° and 75°, and hydrodynamic modelling shows that doing so reduces drag—and in turn, the cost of transport—by around 10% compared with traditional upright swimming. Employment of such a strongly selected feature for such a unique purpose raises interesting questions about evolutionary pathways to hydrodynamic adaptations, and our perception of form and function. PMID:27457414

Comparative study of polyethylene polyamines as activator molecules for a structurally unstable group I ribozyme.

PubMed

Gulshan, Mst Ara; Matsumura, Shigeyoshi; Higuchi, Tsunehiko; Umezawa, Naoki; Ikawa, Yoshiya

2018-04-26

Polyamines are a promising class of molecules that can modulate RNA enzyme activities. To analyze the effects of the number of amine moieties systematically, we employed four polyamines sharing dimethylene units to connect amine moieties. As a model RNA enzyme, we used a structurally unstable group I ribozyme, which was activated most and least efficiently by tetraethylenepentamine and diethylenetriamine respectively.

In vivo Knock-down of the HSV-1 Latency-Associated Transcript Reduces Reactivation from Latency.

PubMed

Watson, Zachary L; Washington, Shannan D; Phelan, Dane M; Lewin, Alfred S; Tuli, Sonal S; Schultz, Gregory S; Neumann, Donna M; Bloom, David C

2018-06-06

During Herpes Simplex Virus (HSV) latency, most viral genes are silenced with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long non-coding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilize an Adeno-associated Virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness. Importance Herpes Simplex Virus (HSV) establishes a life long infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This

Targeting MicroRNAs with Small Molecules a Novel Approach to Treating Breast Cancer

DTIC Science & Technology

2011-10-01

pathogenesis of a disease. To date, the main RNA inhibition agents used in pre- clinical and clinical studies include antisense oligonucleotides, ribozymes ...antagomir Preclinical studies Ribozymes or DNAzymes A ribozyme , or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. A DNAzyme

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

PubMed Central

Sengupta, Raghuvir N.; Van Schie, Sabine N.S.; Giambaşu, George; Dai, Qing; Yesselman, Joseph D.; York, Darrin; Piccirilli, Joseph A.; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

PubMed

Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. © 2015 Sengupta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

In silico ribozyme evolution in a metabolically coupled RNA population.

PubMed

Könnyű, Balázs; Szilágyi, András; Czárán, Tamás

2015-05-27

The RNA World hypothesis offers a plausible bridge from no-life to life on prebiotic Earth, by assuming that RNA, the only known molecule type capable of playing genetic and catalytic roles at the same time, could have been the first evolvable entity on the evolutionary path to the first living cell. We have developed the Metabolically Coupled Replicator System (MCRS), a spatially explicit simulation modelling approach to prebiotic RNA-World evolution on mineral surfaces, in which we incorporate the most important experimental facts and theoretical considerations to comply with recent knowledge on RNA and prebiotic evolution. In this paper the MCRS model framework has been extended in order to investigate the dynamical and evolutionary consequences of adding an important physico-chemical detail, namely explicit replicator structure - nucleotide sequence and 2D folding calculated from thermodynamical criteria - and their possible mutational changes, to the assumptions of a previously less detailed toy model. For each mutable nucleotide sequence the corresponding 2D folded structure with minimum free energy is calculated, which in turn is used to determine the fitness components (degradation rate, replicability and metabolic enzyme activity) of the replicator. We show that the community of such replicators providing the monomer supply for their own replication by evolving metabolic enzyme activities features an improved propensity for stable coexistence and structural adaptation. These evolutionary advantages are due to the emergent uniformity of metabolic replicator fitnesses imposed on the community by local group selection and attained through replicator trait convergence, i.e., the tendency of replicator lengths, ribozyme activities and population sizes to become similar between the coevolving replicator species that are otherwise both structurally and functionally different. In the most general terms it is the surprisingly high extra viability of the metabolic

Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

PubMed Central

Weinberg, Marc S.; Michod, Richard E.

2017-01-01

In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3′ end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life. PMID:28989747

Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

NASA Astrophysics Data System (ADS)

Dhar, Nisha; Weinberg, Marc S.; Michod, Richard E.; Durand, Pierre M.

2017-09-01

In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3' end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life.

Global phylogeography of the scalloped hammerhead shark (Sphyrna lewini).

PubMed

Duncan, K M; Martin, A P; Bowen, B W; DE Couet, H G

2006-07-01

Large marine fishes typically have little population genetic structure. The exceptions are associated with sedentary behaviour, disjunct distributions, or reproductive philopatry. Scalloped hammerhead sharks (Sphyrna lewini) incorporate the contrasting traits of oceanic habitat (usually associated with high dispersal) and possible fidelity to nursery grounds (for reproductive females). To evaluate the expectations of these contrasting behaviours, we examined the global genetic structure of S. lewini based on collections (n = 271 individuals) from 20 nursery areas. A 548-bp fragment of mitochondrial DNA control region revealed 22 polymorphic sites, 24 haplotypes, and three lineages distinguished by 2.56-3.77% sequence divergence. Coalescence analyses based on a provisional molecular clock indicate an origin in the Indo-West Pacific with late Pleistocene radiations into the central Pacific (Hawaii) and eastern Pacific (Central America), as well as recent interchange between oceans via southern Africa. Population subdivisions are strong (overall Phi(ST) = 0.749, P < 0.0001 and among oceans Phi(ST) = 0.598, P < 0.0098). Genetic discontinuity within oceans (Phi(ST) = 0.519, P < 0.0001) is primarily associated with oceanic barriers (migration across oceans M approximately 0), with much less structure along continental margins (M > 10). We conclude that nursery populations linked by continuous coastline have high connectivity, but that oceanic dispersal by females is rare. Although we cannot rule out philopatry to natal nurseries, oceanic barriers appear to have a much stronger influence on the genetic architecture of this species and may indicate a mechanism for recent evolutionary radiations in the genus Sphyrna.

A Computational Study of the Hydrodynamics in the Nasal Region of a Hammerhead Shark (Sphyrna tudes): Implications for Olfaction

PubMed Central

Rygg, Alex D.; Cox, Jonathan P. L.; Abel, Richard; Webb, Andrew G.; Smith, Nadine B.; Craven, Brent A.

2013-01-01

The hammerhead shark possesses a unique head morphology that is thought to facilitate enhanced olfactory performance. The olfactory chambers, located at the distal ends of the cephalofoil, contain numerous lamellae that increase the surface area for olfaction. Functionally, for the shark to detect chemical stimuli, water-borne odors must reach the olfactory sensory epithelium that lines these lamellae. Thus, odorant transport from the aquatic environment to the sensory epithelium is the first critical step in olfaction. Here we investigate the hydrodynamics of olfaction in Sphyrna tudes based on an anatomically-accurate reconstruction of the head and olfactory chamber from high-resolution micro-CT and MRI scans of a cadaver specimen. Computational fluid dynamics simulations of water flow in the reconstructed model reveal the external and internal hydrodynamics of olfaction during swimming. Computed external flow patterns elucidate the occurrence of flow phenomena that result in high and low pressures at the incurrent and excurrent nostrils, respectively, which induces flow through the olfactory chamber. The major (prenarial) nasal groove along the cephalofoil is shown to facilitate sampling of a large spatial extent (i.e., an extended hydrodynamic “reach”) by directing oncoming flow towards the incurrent nostril. Further, both the major and minor nasal grooves redirect some flow away from the incurrent nostril, thereby limiting the amount of fluid that enters the olfactory chamber. Internal hydrodynamic flow patterns are also revealed, where we show that flow rates within the sensory channels between olfactory lamellae are passively regulated by the apical gap, which functions as a partial bypass for flow in the olfactory chamber. Consequently, the hammerhead shark appears to utilize external (major and minor nasal grooves) and internal (apical gap) flow regulation mechanisms to limit water flow between the olfactory lamellae, thus protecting these delicate

Model systems: how chemical biologists study RNA

PubMed Central

Rios, Andro C.; Tor, Yitzhak

2009-01-01

Ribonucleic acids are structurally and functionally sophisticated biomolecules and the use of models, frequently truncated or modified sequences representing functional domains of the natural systems, is essential to their exploration. Functional non-coding RNAs such as miRNAs, riboswitches, and, in particular, ribozymes, have changed the view of RNA’s role in biology and its catalytic potential. The well-known truncated hammerhead model has recently been refined and new data provide a clearer molecular picture of the elements responsible for its catalytic power. A model for the spliceosome, a massive and highly intricate ribonucleoprotein, is also emerging, although its true utility is yet to be cemented. Such catalytic model systems could also serve as “chemo-paleontological” tools, further refining the RNA world hypothesis and its relevance to the origin and evolution of life. PMID:19879179

Intelligent Therapeutics and Metabolic Programming Through Tailormade, Ligand-Controlled RNA Switches

DTIC Science & Technology

2007-02-05

lines. Three regulatory mechanisms have been examined in our laboratory: antisense inhibition, ribozyme cleavage, and RNA interference (RNAi...cell lines. However, the latter two regulatory mechanisms, ribozyme -based inactivation and RNAi-mediated silencing, demonstrated significant activity...in these cell lines as is briefly described below. Microswitches responsive to the small molecule theophylline and targeting GFP based on a ribozyme

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.

PubMed

Xu, Li; Zhao, Lixia; Gao, Yandi; Xu, Jing; Han, Renzhi

2017-03-17

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

How the discovery of ribozymes cast RNA in the roles of both chicken and egg in origin-of-life theories.

PubMed

Sankaran, Neeraja

2012-12-01

Scientific theories about the origin-of-life theories have historically been characterized by the chicken-and-egg problem of which essential aspect of life was the first to appear, replication or self-sustenance. By the 1950s the question was cast in molecular terms and DNA and proteins had come to represent the carriers of the two functions. Meanwhile, RNA, the other nucleic acid, had played a capricious role in origin theories. Because it contained building blocks very similar to DNA, biologists recognized early that RNA could store information in its linear sequences. With the discovery in the 1980s that RNA molecules were capable of biological catalysis, a function hitherto ascribed to proteins alone, RNA took on the role of the single entity that could act as both chicken and egg. Within a few years of the discovery of these catalytic RNAs (ribozymes) scientists had formulated an RNA World hypothesis that posited an early phase in the evolution of life where all key functions were performed by RNA molecules. This paper traces the history the role of RNA in origin-of-life theories with a focus on how the discovery of ribozymes influenced the discourse. Copyright © 2012 Elsevier Ltd. All rights reserved.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

PubMed

Kim, Yun-Hee; Kim, Kyung Tae; Lee, Sang-Jin; Hong, Seung-Hee; Moon, Ju Young; Yoon, Eun Kyung; Kim, Sukyoung; Kim, Eun Ok; Kang, Se Hun; Kim, Seok Ki; Choi, Sun Il; Goh, Sung Ho; Kim, Daehong; Lee, Seong-Wook; Ju, Mi Ha; Jeong, Jin Sook; Kim, In-Hoo

2016-01-01

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone.

PubMed

Liu, Zhong-Yu; Yu, Jiu-Yang; Huang, Xing-Yao; Fan, Hang; Li, Xiao-Feng; Deng, Yong-Qiang; Ji, Xue; Cheng, Meng-Li; Ye, Qing; Zhao, Hui; Han, Jian-Feng; An, Xiao-Ping; Jiang, Tao; Zhang, Bo; Tong, Yi-Gang; Qin, Cheng-Feng

2017-11-01

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis -acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable

Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

PubMed Central

Liu, Zhong-Yu; Yu, Jiu-Yang; Huang, Xing-Yao; Fan, Hang; Li, Xiao-Feng; Deng, Yong-Qiang; Ji, Xue; Cheng, Meng-Li; Ye, Qing; Zhao, Hui; Han, Jian-Feng; An, Xiao-Ping; Jiang, Tao; Zhang, Bo; Tong, Yi-Gang

2017-01-01

ABSTRACT Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a

Overcoming Temozolomide Resistance in Glioblastoma Multiforme with MGMT-Targeting Spherical Nucleic Acids

NASA Astrophysics Data System (ADS)

Sita, Timothy L.

Glioblastoma multiforme (GBM) is the most prevalent primary central nervous system malignancy. Due to the aggressive nature of these tumors and our inability to adequately treat them, only 3-5% of patients survive longer than 3 years post-diagnosis. The standard of care for newly diagnosed GBM is surgical resection followed by concomitant and adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. TMZ cytotoxicity is mediated primarily through methylation of the O 6 -position of guanine. In the majority of patients, this methyl group is rapidly removed by the enzyme O6 -methylguanine-DNA methyltransferase (MGMT), conferring resistance to the chemotherapy. However, in a small subset of GBM patients, the promoter region for MGMT is methylated over the course of tumor development. This epigenetic silencing of MGMT activity allows TMZ to induce apoptosis in glioblastoma cells and drastically increases survival in GBM patients. The following work seeks to recapitulate this improved survival phenotype by combining TMZ with a novel nanoconstruct capable of silencing MGMT expression. The nanoconstruct consists of gold nanoparticles densely conjugated with either an MGMT-targeting ribozyme (ribozyme-Spherical Nucleic Acids (SNAs)), or small interfering RNA (siRNA) duplexes designed against MGMT (siMGMT-SNAs), and has been found to have unique characteristics, including (1) the rapid internalization by all glioma cell types studied including glioma initiating cells (GICs), (2) the capacity to potently silence MGMT expression, (3) increased apoptotic response in GBM cells, (4) the ability to cross the blood-brain barrier (BBB), blood-tumor barrier (BTB), and accumulate in GBM xenografts, and (5) no observable acute toxicity at high doses in animal models. In summary, preliminary data suggest ribozyme-SNA and siMGMT-SNAs sensitize GBM cells in vitro and in vivo, enhancing the therapeutic response to TMZ.

Enhanced Antitumorigenic Effects in Glioblastoma on Double Targeting of Pleiotrophin and Its Receptor ALK1

PubMed Central

Grzelinski, Marius; Steinberg, Florian; Martens, Tobias; Czubayko, Frank; Lamszus, Katrin; Aigner, Achim

2009-01-01

In adults, glioblastomas are the most lethal and most frequent malignant brain tumors, and the poor prognosis despite aggressive treatment indicates the need to establish novel targets for molecular intervention. The secreted growth factor pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) shows mitogenic, chemotactic, and transforming activity. Whereas PTN expression is tightly regulated during embryogenesis and is very limited in normal adult tissues, a marked PTN up-regulation is seen in tumors including glioblastomas. Likewise, the PTN receptor anaplastic lymphoma kinase (ALK) has been shown previously to be upregulated and functionally relevant in glioblastoma. In this study, we explore the antitumorigenic effects of the simultaneous ribozyme-mediated knockdown of both receptor and ligand. Various glioblastoma cell lines are analyzed for PTN and ALK expression. Beyond the individual efficacies of several specific ribozymes against PTN or ALK, respectively, antiproliferative and proapoptotic effects of a single gene targeting approach are strongly enhanced on double knockdown of both genes in vitro. More importantly, this results in the abolishment of tumor growth in an in vivo subcutaneous tumor xenograft model. Finally, the analysis of various downstream signaling pathways by antibody arrays reveals a distinct pattern of changes in the activation of signal transduction molecules on PTN/ALK double knockdown. Beyond the already known ones, it identifies additional pathways relevant for PTN/ALK signaling. We conclude that double targeting of PTN and ALK leads to enhanced antitumorigenic effects over single knockdown approaches, which offers novel therapeutic options owing to increased efficacy also after prolonged knockdown. PMID:19177199

Mechanism of Telomerase Inhibition Using Small Inibitory RNAs and Induction of Breast Tumor Cell Sensitivity

DTIC Science & Technology

2007-03-01

RTb motif mutants hTERT Senescence Apoptosis Long lag period [20,25] Ribozymes Hairpin hTR, hTERT Apoptosis Incomplete knockdown of target [26...O-(2-Methoxyethyl) oligomers. b Reverse transcriptase motif.the growth and viability of cancer cells (Table 1). Ribozymes and short-interfering RNA...recent studies indicate that complete knockdown is not essential for efficient and rapid apoptosis in reference to siRNA against hTR and ribozymes

Targeting of the Nuclear Receptor Coativator Isoform Delta 3aib1 in Breast Cancer. Addendum

DTIC Science & Technology

2007-07-01

using a regulatable AIB1 directed ribozyme , resulted in reduced tumor growth in vivo. Overall, these data indicate a major role for AIB1 and its isoform...regulatable AIB1 directed ribozyme , resulted in reduced tumor growth in vivo. Overall, these data indicate a major role for AIB1 and its isoform ∆3AIB1 in

Knocking on FXR's door: the "hammerhead"-structure series of FXR agonists - amphiphilic isoxazoles with potent in vitro and in vivo activities.

PubMed

Gege, Christian; Kinzel, Olaf; Steeneck, Christoph; Schulz, Andreas; Kremoser, Claus

2014-01-01

The Farnesoid X Receptor (FXR) was recently validated in clinical studies using the bile acid analogue Obeticholic Acid (OCA) as an attractive drug target for liver diseases such as Primary Biliary Cirrhosis (PBC) or Non-alcoholic Steatohepatitis (NASH). OCA, however, turned out to induce cholesterol- related side effects upon prolonged treatment and it shows bile acid like pharmacokinetics. The quest for synthetic non-steroidal FXR agonists with general drug likeliness and improved pharmacokinetic and - dynamic properties has started more than a decade ago: The first non-steroidal and selective FXR agonist with decent submicromolar potency, GW4064, was patented in 1998 and published in 2000. Since then, many pharmaceutical companies have taken GW4064 as a structural template for their efforts in identifying novel patentable FXR agonists with the GW-derived trisubstituted isoxazole general structure. However, so far only one compound out of these different series has made it into the early stages of clinical development: The Px-102/Px-104 from Phenex is currently tested in a phase IIa study in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). In this review we try to summarize from the patent and scientific literature the attempts to improve the GW4064 structure into different directions. Furthermore, we suggest directions for further improvements of this special class of synthetic FXR agonists which all display the typical "hammerhead"-conformation in the FXR ligand binding pocket that provides the basis for their impressive in vitro and in vivo potencies.

Detailed analysis of stem I and its 5' and 3' neighbor regions in the trans-acting HDV ribozyme.

PubMed Central

Nishikawa, F; Roy, M; Fauzi, H; Nishikawa, S

1999-01-01

To determine the stem I structure of the human hepatitis delta virus (HDV) ribozyme, which is related to the substrate sequence in the trans -acting system, we kinetically studied stem I length and sequences. Stem I extension from 7 to 8 or 9 bp caused a loss of activity and a low amount of active complex with 9 bp in the trans -acting system. In a previous report, we presented cleavage in a 6 bp stem I. The observed reaction rates indicate that the original 7 bp stem I is in the most favorable location for catalytic reaction among the possible 6-8 bp stems. To test base specificity, we replaced the original GC-rich sequence in stem I with AU-rich sequences containing six AU or UA base pairs with the natural +1G.U wobble base pair at the cleavage site. The cis -acting AU-rich molecules demonstrated similar catalytic activity to that of the wild-type. In trans -acting molecules, due to stem I instability, reaction efficiency strongly depended on the concentration of the ribozyme-substrate complex and reaction temperature. Multiple turnover was observed at 37 degreesC, strongly suggesting that stem I has no base specificity and more efficient activity can be expected under multiple turnover conditions by substituting several UA or AU base pairs into stem I. We also studied the substrate damaging sequences linked to both ends of stem I for its development in therapeutic applications and confirmed the functions of the unique structure. PMID:9862958

VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

DTIC Science & Technology

2012-10-01

selective of all gene-targeted, oligonucleotide-based drug approaches (better than ribozymes, antisense oligonucleotides ( ASO ), or microRNAs).(4) We will...respect to a scrambled siRNA control. For the migration assay, a circular region in the middle of the well was removed using a gel removal solution...oligonucleotides, ASOs ) into rabbit corneal cells and found that technique was very effective in delivering ASOs into the stroma and even into the endothelial cell

Self-assembly Controls Self-cleavage of HHR from ASBVd (-): a Combined SANS and Modeling Study

NASA Astrophysics Data System (ADS)

Leclerc, Fabrice; Zaccai, Giuseppe; Vergne, Jacques; Řìhovà, Martina; Martel, Anne; Maurel, Marie-Christine

2016-07-01

In the Avocado Sunblotch Viroid (ASBVd: 249-nt) from the Avsunviroidae family, a symmetric rolling-circle replication operates through an autocatalytic mechanism mediated by hammerhead ribozymes (HHR) embedded in both polarity strands. The concatenated multimeric ASBVd (+) and ASBVd (-) RNAs thus generated are processed by cleavage to unit-length where ASBVd (-) self-cleaves with more efficiency. Absolute scale small angle neutron scattering (SANS) revealed a temperature-dependent dimer association in both ASBVd (-) and its derived 79-nt HHR (-). A joint thermodynamic analysis of SANS and catalytic data indicates the rate-determining step corresponds to the dimer/monomer transition. 2D and 3D models of monomeric and dimeric HHR (-) suggest that the inter-molecular contacts stabilizing the dimer (between HI and HII domains) compete with the intra-molecular ones stabilizing the active conformation of the full-length HHR required for an efficient self-cleavage. Similar competing intra- and inter-molecular contacts are proposed in ASBVd (-) though with a remoter region from an extension of the HI domain.

Robust Sub-harmonic Mixer at 340 GHz Using Intrinsic Resonances of Hammer-Head Filter and Improved Diode Model

NASA Astrophysics Data System (ADS)

Wang, Cheng; He, Yue; Lu, Bin; Jiang, Jun; Miao, Li; Deng, Xian-Jin; Xiong, Yong-zhong; Zhang, Jian

2017-11-01

This paper presents a sub-harmonic mixer at 340 GHz based on anti-parallel Schottky diodes (SBDs). Intrinsic resonances in low-pass hammer-head filter have been adopted to enhance the isolation for different harmonic components, while greatly minimizing the transmission loss. The application of new DC grounding structure, impedance matching structure, and suspended micro-strip mitigates the negative influences of fabrication errors from metal cavity, quartz substrate, and micro-assembly. An improved lumped element equivalent circuit model of SBDs guarantees the accuracy of simulation, which takes current-voltage (I/V) behavior, capacitance-voltage (C/V) behavior, carrier velocity saturation, DC series resistor, plasma resonance, skin effect, and four kinds of noise generation mechanisms into consideration thoroughly. The measurement indicates that with local oscillating signal of 2 mW, the lowest double sideband conversion loss is 5.5 dB at 339 GHz; the corresponding DSB noise temperature is 757 K. The 3 dB bandwidth of conversion loss is 50 GHz from 317 to 367 GHz.

Mechanistic characterization of the HDV genomic ribozyme: a mutant of the C41 motif provides insight into the positioning and thermodynamic linkage of metal ions and protons.

PubMed

Nakano, Shu-ichi; Bevilacqua, Philip C

2007-03-20

Binding of two Mg2+ and two H+ ions influences the self-cleavage activity of the genomic HDV ribozyme. The positioning of these four ligands and their thermodynamic linkage are not fully resolved. Protonated C41 engages in a base triple, whereas protonated C75 has been implicated as an acid-base catalyst in bond cleavage. Prior studies led to the identification of one structural inner-sphere ion and one catalytic outer-sphere ion. In the present study, the contributions of the C41 base triple to the metal ion- and pH-dependence of the reaction are examined. Experiments were conducted on a CG to UA double mutant (DM), which changes the base triple to one involving an unprotonated C41. Below pH 6, the DM has a steeper dependence on pH than the wild-type (WT), consistent with a single protonation misfolding the core; this conclusion is also supported by thermal denaturation studies. Between pH 6 and 8, the WT and DM display nearly identical catalytic metal ion and H+ binding profiles. In contrast, over the same pH range, the WT and DM have distinct structural ion binding profiles; for the WT, binding is favored at lower pH, whereas the DM shows no pH dependence. These data localize the structural ion to the vicinity of the C41 motif. An overall model is presented that accommodates binding affinity, coupling, and positioning of the two metal ions and the two protons within the ribozyme. The data suggest that a protonated base triple allows the WT ribozyme to maintain appreciable activity at acidic pH, which could play an important role in the life cycle of the virus.

Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides.

PubMed Central

Komatsu, Y; Kumagai, I; Ohtsuka, E

1999-01-01

We introduced 4-thio- ((4S)U), 2-thio- ((2S)U), 4- O -methyluridine ((4Me)U) and cytidine substitutions for U+2, which is an important base for cleavage in a substrate RNA. Oligonucleotides containing 4-thio- and 4- O -methyluridine were prepared by a new convenient post-synthetic modification method using a 4- O - p -nitrophenyl-uridine derivative. A hairpin ribozyme cleaved the substrate RNA with either C+2, (4S)U+2 or (4Me)U+2 at approximately 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a (2S)U+2 was cleaved with the same activity as the natural substrate. These results suggest that the O4 of U+2 is involved in hydrogen bonding at loop A, but the O2 of U+2 is not recognized by the active residues. Circular dichroism data of the ribozymes containing (4S)U+2 and (2S)U+2, as well as the susceptibility of the thiocarbonyl group to hydrogen peroxide, suggest that a conformational change of U+2 occurs during the domain docking in the cleavage reaction. We propose here the conformational change of U+2 from the ground state to the active molecule during the reaction. PMID:10536137

Selenium is a Chemotherapeutic Agent for the Treatment of Prostate Cancer

DTIC Science & Technology

2007-12-01

to the AR without decreasing AR levels. Strategic targeting of the AR with ribozymes , antisense oligomers, and small interfering RNAs has been shown... ribozyme . Mol Endrocrinol 1998;12:1558-1566. 20 10. Ko YJ, Devi GR, London CA, et al. Androgen receptor down-regulation in prostate cancer with

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

PubMed Central

Duan, L; Pomerantz, R J

1994-01-01

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA. Images PMID:7816635

Quantum mechanical/molecular mechanical free energy simulations of the self-cleavage reaction in the hepatitis delta virus ribozyme.

PubMed

Ganguly, Abir; Thaplyal, Pallavi; Rosta, Edina; Bevilacqua, Philip C; Hammes-Schiffer, Sharon

2014-01-29

The hepatitis delta virus (HDV) ribozyme catalyzes a self-cleavage reaction using a combination of nucleobase and metal ion catalysis. Both divalent and monovalent ions can catalyze this reaction, although the rate is slower with monovalent ions alone. Herein, we use quantum mechanical/molecular mechanical (QM/MM) free energy simulations to investigate the mechanism of this ribozyme and to elucidate the roles of the catalytic metal ion. With Mg(2+) at the catalytic site, the self-cleavage mechanism is observed to be concerted with a phosphorane-like transition state and a free energy barrier of ∼13 kcal/mol, consistent with free energy barrier values extrapolated from experimental studies. With Na(+) at the catalytic site, the mechanism is observed to be sequential, passing through a phosphorane intermediate, with free energy barriers of 2-4 kcal/mol for both steps; moreover, proton transfer from the exocyclic amine of protonated C75 to the nonbridging oxygen of the scissile phosphate occurs to stabilize the phosphorane intermediate in the sequential mechanism. To explain the slower rate observed experimentally with monovalent ions, we hypothesize that the activation of the O2' nucleophile by deprotonation and orientation is less favorable with Na(+) ions than with Mg(2+) ions. To explore this hypothesis, we experimentally measure the pKa of O2' by kinetic and NMR methods and find it to be lower in the presence of divalent ions rather than only monovalent ions. The combined theoretical and experimental results indicate that the catalytic Mg(2+) ion may play three key roles: assisting in the activation of the O2' nucleophile, acidifying the general acid C75, and stabilizing the nonbridging oxygen to prevent proton transfer to it.

Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions.

PubMed

Hayden, Eric J

2016-08-15

RNA molecules provide a realistic but tractable model of a genotype to phenotype relationship. This relationship has been extensively investigated computationally using secondary structure prediction algorithms. Enzymatic RNA molecules, or ribozymes, offer access to genotypic and phenotypic information in the laboratory. Advancements in high-throughput sequencing technologies have enabled the analysis of sequences in the lab that now rivals what can be accomplished computationally. This has motivated a resurgence of in vitro selection experiments and opened new doors for the analysis of the distribution of RNA functions in genotype space. A body of computational experiments has investigated the persistence of specific RNA structures despite changes in the primary sequence, and how this mutational robustness can promote adaptations. This article summarizes recent approaches that were designed to investigate the role of mutational robustness during the evolution of RNA molecules in the laboratory, and presents theoretical motivations, experimental methods and approaches to data analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms Down-Regulating Sprouty1, a Growth Inhibitor in Prostate Cancer

DTIC Science & Technology

2008-10-01

cancer cells all express FGF-BP. Using ribozymes to FGF-BP, Aigner et al. (2002) were able to demonstrate that decreased FGF-BP is associated with...International Journal of Cancer 92 510–517. Aigner A, Renneberg H, Bojunga J, Apel J, Nelson PS & Czubayko F 2002 Ribozyme -targeting of a secreted FGF- binding

Mechanistic characterization of the HDV genomic ribozyme: solvent isotope effects and proton inventories in the absence of divalent metal ions support C75 as the general acid.

PubMed

Cerrone-Szakal, Andrea L; Siegfried, Nathan A; Bevilacqua, Philip C

2008-11-05

The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg(2+) ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg(2+)-independent and two Mg(2+)-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg(2+)-free channel is inverted relative to the fully Mg(2+)-dependent channel, with each having a near-neutral pKa. Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg(2+). However, subsequent studies on a double mutant (DM) ribozyme suggested that the pKa observed for WT in the absence of Mg(2+) arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg(2+). Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg(2+)-free pKa observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Brønsted base appears to be water or hydroxide ion depending on pH. The observed pKa's are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg(2+) being similar to that in the presence of Mg(2+) and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Brønsted base.

Eggplant latent viroid: a friendly experimental system in the family Avsunviroidae.

PubMed

Daròs, José-Antonio

2016-10-01

Eggplant latent viroid (ELVd) is the only species of the genus Elaviroid (family Avsunviroidae). All the viroids in the family Avsunviroidae contain hammerhead ribozymes in the strands of both polarities, and are considered to replicate in the chloroplasts of infected cells. This family includes two other genera: Avsunviroid and Pelamoviroid. ELVd consists of a single-stranded, circular, non-coding RNA of 332-335 nucleotides that folds in a branched quasi-rod-like minimum free-energy conformation. RNAs of complementary polarity exist in infected cells and are considered to be replication intermediates. Plus (+) polarity is assigned arbitrarily to the strand that accumulates at a higher concentration in infected tissues. HOST: To date, ELVd has only been shown to infect eggplant (Solanum melongena L.), the species in which it was discovered. A very narrow host range seems to be a common property in members of the family Avsunviroidae. ELVd infections of eggplants are apparently symptomless. ELVd is transmitted mechanically and by seed. http://subviral.med.uottawa.ca. © 2015 BSPP and John Wiley & Sons Ltd.

Inverse Thio Effects in the Hepatitis Delta Virus Ribozyme Reveal that the Reaction Pathway Is Controlled by Metal Ion Charge Density

PubMed Central

2015-01-01

The hepatitis delta virus (HDV) ribozyme self-cleaves in the presence of a wide range of monovalent and divalent ions. Prior theoretical studies provided evidence that self-cleavage proceeds via a concerted or stepwise pathway, with the outcome dictated by the valency of the metal ion. In the present study, we measure stereospecific thio effects at the nonbridging oxygens of the scissile phosphate under a wide range of experimental conditions, including varying concentrations of diverse monovalent and divalent ions, and combine these with quantum mechanical/molecular mechanical (QM/MM) free energy simulations on the stereospecific thio substrates. The RP substrate gives large normal thio effects in the presence of all monovalent ions. The SP substrate also gives normal or no thio effects, but only for smaller monovalent and divalent cations, such as Li+, Mg2+, Ca2+, and Sr2+; in contrast, sizable inverse thio effects are found for larger monovalent and divalent cations, including Na+, K+, NH4+, and Ba2+. Proton inventories are found to be unity in the presence of the larger monovalent and divalent ions, but two in the presence of Mg2+. Additionally, rate–pH profiles are inverted for the low charge density ions, and only imidazole plus ammonium ions rescue an inactive C75Δ variant in the absence of Mg2+. Results from the thio effect experiments, rate–pH profiles, proton inventories, and ammonium/imidazole rescue experiments, combined with QM/MM free energy simulations, support a change in the mechanism of HDV ribozyme self-cleavage from concerted and metal ion-stabilized to stepwise and proton transfer-stabilized as the charge density of the metal ion decreases. PMID:25799319

Domain structure of the ribozyme from eubacterial ribonuclease P.

PubMed Central

Loria, A; Pan, T

1996-01-01

Large RNAs can be composed of discrete domains that fold independently. One such "folding domain" has been identified previously in the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA). This domain contains roughly one-third of all residues. Folding of an RNA construct consisting of the remaining two-thirds of B. subtilis P RNA was examined by Fe(II)-EDTA hydroxyl radical protection. This molecule folds into the proper higher-order structure under identical conditions as the full-length P RNA, suggesting the presence of a second folding domain in B. subtilis P RNA. Folding analysis of the Escherichia coli P RNA by hydroxyl radical protection shows that this P RNA is completely folded at 5-6 mM Mg2+. In order to analyze the structural organization of folding domains in E. coli P RNA, constructs were designed based on the domain structure of B. subtilis P RNA. Fe(II)-EDTA protection indicates that E. coli P RNA also contains two folding domains. Despite the significant differences at the secondary structure level, both P RNAs appear to converge structurally at the folding domain level. The pre-tRNA substrate, localized in previous studies, may bind across the folding domains with the acceptor stem/3'CCA contacting the domain including the active site and the T stem-loop contacting the other. Because all eubacterial P RNAs share considerable homology in secondary structure to either B. subtilis or E. coli P RNA, these results suggest that this domain structure may be applicable for most, if not all, eubacterial P RNAs. Identification of folding domains should be valuable in dissecting structure-function relationship of large RNAs. PMID:8718684

Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

PubMed

Petrie, Katherine L; Joyce, Gerald F

2014-10-01

The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

Evolution of ribozymes in the presence of a mineral surface

PubMed Central

Stephenson, James D.; Popović, Milena; Bristow, Thomas F.

2016-01-01

Mineral surfaces are often proposed as the sites of critical processes in the emergence of life. Clay minerals in particular are thought to play significant roles in the origin of life including polymerizing, concentrating, organizing, and protecting biopolymers. In these scenarios, the impact of minerals on biopolymer folding is expected to influence evolutionary processes. These processes include both the initial emergence of functional structures in the presence of the mineral and the subsequent transition away from the mineral-associated niche. The initial evolution of function depends upon the number and distribution of sequences capable of functioning in the presence of the mineral, and the transition to new environments depends upon the overlap between sequences that evolve on the mineral surface and sequences that can perform the same functions in the mineral's absence. To examine these processes, we evolved self-cleaving ribozymes in vitro in the presence or absence of Na-saturated montmorillonite clay mineral particles. Starting from a shared population of random sequences, RNA populations were evolved in parallel, along separate evolutionary trajectories. Comparative sequence analysis and activity assays show that the impact of this clay mineral on functional structure selection was minimal; it neither prevented common structures from emerging, nor did it promote the emergence of new structures. This suggests that montmorillonite does not improve RNA's ability to evolve functional structures; however, it also suggests that RNAs that do evolve in contact with montmorillonite retain the same structures in mineral-free environments, potentially facilitating an evolutionary transition away from a mineral-associated niche. PMID:27793980

Click nucleic acid ligation: applications in biology and nanotechnology.

PubMed

El-Sagheer, Afaf H; Brown, Tom

2012-08-21

Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have

Expression of death decoy receptor-3 (DcR3) in human breast cancer and its functional effects on breast cancer cells in vitro.

PubMed

Ge, Zhicheng; Sanders, Andrew J; Ye, Lin; Wang, Yu; Jiang, Wen G

2011-01-01

Death Decoy Receptor-3 (DcR3), otherwise known as tumour necrosis factor receptor superfamily member 6b, is suggested to be involved in the progression and immune evasion of malignant tumours. Its ligands include FASL and LIGHT (Tumour necrosis factor ligand superfamily member 14). DcR3 has been found to be amplified in certain solid tumours. However, its role in breast tumours remains unclear. In the present study, we examined the role played by DcR3 in MCF7 and MDA-MB-231 cell lines. The expression of DcR3 was examined in MCF7 and MDA-MB-231 cell lines using immunocytochemical staining and RT-PCR. Anti-DcR3 hammerhead ribozyme transgenes were constructed and transfected into cells to create DcR3 knock-down cell sublines. The biological impact of modifying DcR3 expression in breast cancer cells was evaluated using a variety of in vitro assays, including growth, adhesion, migration and invasion models. MCF7 and MDA-MB-231 cells, usually expressing DcR3, were transfected with the anti-DcR3 ribozyme transgene. Stable transfectants containing the DcR3 ribozyme transgene (MCF7DcR3KO, MDA-MB-231DcR3KO) displayed a reduction of DcR3 expression at mRNA and protein levels. DcR3 knockdown in MCF7 cells was found to significantly reduce invasive capacity compared to pEF6 control cell lines (30.78 +/- 6.40 vs.151.67 +/- 17.67 P < 0.001). The rate of migration in MCF7DcR3KO was significantly lower than MCF7pEF6 (P < 0.001). In contrast, no such significant differences was seen between MDA-MB-231DcR3KO and MDA-MB-231pEF6. Suppressing DcR3 expression was found to have an inhibitory effect on cellular invasion and migration in MCF7 breast cancer cells. This suggests that the invasion and migration capacity of this breast cancer cell line may, at least partly, depend on DcR3. DcR3 may be regarded as a negative regulator for aggressiveness during the development and progression of certain types of breast cancer.

Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

PubMed

Zhang, Zhixiang; Qi, Shuishui; Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

2014-12-01

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

PubMed Central

Tang, Nan; Zhang, Xinxin; Chen, Shanshan; Zhu, Pengfei; Ma, Lin; Cheng, Jinping; Xu, Yun; Lu, Meiguang; Wang, Hongqing; Ding, Shou-Wei; Li, Shifang; Wu, Qingfa

2014-01-01

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs. PMID:25503469

Demographic processes underlying subtle patterns of population structure in the scalloped hammerhead shark, Sphyrna lewini.

PubMed

Nance, Holly A; Klimley, Peter; Galván-Magaña, Felipe; Martínez-Ortíz, Jimmy; Marko, Peter B

2011-01-01

Genetic diversity (θ), effective population size (N(e)), and contemporary levels of gene flow are important parameters to estimate for species of conservation concern, such as the globally endangered scalloped hammerhead shark, Sphyrna lewini. Therefore, we have reconstructed the demographic history of S. lewini across its Eastern Pacific (EP) range by applying classical and coalescent population genetic methods to a combination of 15 microsatellite loci and mtDNA control region sequences. In addition to significant population genetic structure and isolation-by-distance among seven coastal sites between central Mexico and Ecuador, the analyses revealed that all populations have experienced a bottleneck and that all current values of θ are at least an order of magnitude smaller than ancestral θ, indicating large decreases in N(e) (θ = 4N(e)μ), where μ is the mutation rate. Application of the isolation-with-migration (IM) model showed modest but significant genetic connectivity between most sampled sites (point estimates of Nm = 0.1-16.7), with divergence times (t) among all populations significantly greater than zero. Using a conservative (i.e., slow) fossil-based taxon-specific phylogenetic calibration for mtDNA mutation rates, posterior probability distributions (PPDs) for the onset of the decline in N(e) predate modern fishing in this region. The cause of decline over the last several thousand years is unknown but is highly atypical as a post-glacial demographic history. Regardless of the cause, our data and analyses suggest that S. lewini was far more abundant throughout the EP in the past than at present.

Demographic Processes Underlying Subtle Patterns of Population Structure in the Scalloped Hammerhead Shark, Sphyrna lewini

PubMed Central

Nance, Holly A.; Klimley, Peter; Galván-Magaña, Felipe; Martínez-Ortíz, Jimmy; Marko, Peter B.

2011-01-01

Genetic diversity (θ), effective population size (Ne), and contemporary levels of gene flow are important parameters to estimate for species of conservation concern, such as the globally endangered scalloped hammerhead shark, Sphyrna lewini. Therefore, we have reconstructed the demographic history of S. lewini across its Eastern Pacific (EP) range by applying classical and coalescent population genetic methods to a combination of 15 microsatellite loci and mtDNA control region sequences. In addition to significant population genetic structure and isolation-by-distance among seven coastal sites between central Mexico and Ecuador, the analyses revealed that all populations have experienced a bottleneck and that all current values of θ are at least an order of magnitude smaller than ancestral θ, indicating large decreases in Ne (θ = 4Neμ), where μ is the mutation rate. Application of the isolation-with-migration (IM) model showed modest but significant genetic connectivity between most sampled sites (point estimates of Nm = 0.1–16.7), with divergence times (t) among all populations significantly greater than zero. Using a conservative (i.e., slow) fossil-based taxon-specific phylogenetic calibration for mtDNA mutation rates, posterior probability distributions (PPDs) for the onset of the decline in Ne predate modern fishing in this region. The cause of decline over the last several thousand years is unknown but is highly atypical as a post-glacial demographic history. Regardless of the cause, our data and analyses suggest that S. lewini was far more abundant throughout the EP in the past than at present. PMID:21789171

Development of Quenching-qPCR (Q-Q) assay for measuring absolute intracellular cleavage efficiency of ribozyme.

PubMed

Kim, Min Woo; Sun, Gwanggyu; Lee, Jung Hyuk; Kim, Byung-Gee

2018-06-01

Ribozyme (Rz) is a very attractive RNA molecule in metabolic engineering and synthetic biology fields where RNA processing is required as a control unit or ON/OFF signal for its cleavage reaction. In order to use Rz for such RNA processing, Rz must have highly active and specific catalytic activity. However, current methods for assessing the intracellular activity of Rz have limitations such as difficulty in handling and inaccuracies in the evaluation of correct cleavage activity. In this paper, we proposed a simple method to accurately measure the "intracellular cleavage efficiency" of Rz. This method deactivates unwanted activity of Rz which may consistently occur after cell lysis using DNA quenching method, and calculates the cleavage efficiency by analyzing the cleaved fraction of mRNA by Rz from the total amount of mRNA containing Rz via quantitative real-time PCR (qPCR). The proposed method was applied to measure "intracellular cleavage efficiency" of sTRSV, a representative Rz, and its mutant, and their intracellular cleavage efficiencies were calculated as 89% and 93%, respectively. Copyright © 2018 Elsevier Inc. All rights reserved.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The

Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

PubMed Central

Tan, Chee Wah; Tee, Han Kang; Lee, Michelle Hui Pheng; Sam, I-Ching; Chan, Yoke Fun

2016-01-01

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3’ ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71. PMID:27617744

Multifrequency Pulsed EPR Studies of Biologically Relevant Manganese(II) Complexes

PubMed Central

Stich, T. A.; Lahiri, S.; Yeagle, G.; Dicus, M.; Brynda, M.; Gunn, A.; Aznar, C.; DeRose, V. J.; Britt, R. D.

2011-01-01

Electron paramagnetic resonance studies at multiple frequencies (MF EPR) can provide detailed electronic structure descriptions of unpaired electrons in organic radicals, inorganic complexes, and metalloenzymes. Analysis of these properties aids in the assignment of the chemical environment surrounding the paramagnet and provides mechanistic insight into the chemical reactions in which these systems take part. Herein, we present results from pulsed EPR studies performed at three different frequencies (9, 31, and 130 GHz) on [Mn(II)(H2O)6]2+, Mn(II) adducts with the nucleotides ATP and GMP, and the Mn(II)-bound form of the hammerhead ribozyme (MnHH). Through line shape analysis and interpretation of the zero-field splitting values derived from successful simulations of the corresponding continuous-wave and field-swept echo-detected spectra, these data are used to exemplify the ability of the MF EPR approach in distinguishing the nature of the first ligand sphere. A survey of recent results from pulsed EPR, as well as pulsed electron-nuclear double resonance and electron spin echo envelope modulation spectroscopic studies applied to Mn(II)-dependent systems, is also presented. PMID:22190766

Antisense oligonucleotides effectively inhibit the co-transcriptional splicing of a Candida group I intron in vitro and in vivo: Implications for antifungal therapeutics.

PubMed

Zhang, Libin; Leibowitz, Michael J; Zhang, Yi

2009-02-18

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3-P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

PubMed Central

Ohmichi, T; Okumoto, Y; Sugimoto, N

1998-01-01

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis. PMID:9837996

78 FR 42021 - Atlantic Highly Migratory Species; Commercial Gulf of Mexico Aggregated Large Coastal Shark and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-15

... Large Coastal Shark and Gulf of Mexico Hammerhead Shark Management Groups AGENCY: National Marine... coastal sharks (LCS) and hammerhead sharks in the Gulf of Mexico region. This action is necessary because... aggregated LCS and Gulf of Mexico hammerhead shark management groups are closed effective 11:30 p.m. local...

Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme

PubMed Central

Rublack, Nico

2014-01-01

Summary Over the past 20 years, the generation of functional RNAs by in vitro selection has become a standard technique. Apart from aptamers for simple binding of defined ligands, also RNAs for catalysis of chemical reactions have been selected. In the latter case, a key step often is the conjugation of one of the two reactants to the library, requiring suitable strategies for terminal or internal RNA functionalization. With the aim of selecting a ribozyme for deamination of cytidine, we have set up a selection scheme involving the attachment of the cytidine acting as deamination substrate to the 3'-terminus of the RNAs in the library, and library immobilization. Here, we report the synthesis of a bifunctional cytidine derivative suitable for conjugation to RNA and linkage of the conjugated library to a streptavidine-coated surface. Successful conjugation of the cytidine derivative to the 3'-terminus of a model RNA is demonstrated. PMID:25246949

Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic Cofactor Analogs for the glmS Ribozyme

PubMed Central

Posakony, Jeffrey J.; Ferré-D'Amaré, Adrian R.

2013-01-01

Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactor of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogs, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz) and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogs. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection. PMID:23578404

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

PubMed Central

Piazza, Carol Lyn; Smith, Dorie

2018-01-01

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting.

PubMed

Qu, Guosheng; Piazza, Carol Lyn; Smith, Dorie; Belfort, Marlene

2018-06-15

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis , inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. © 2018, Qu et al.

Detection of innersphere interactions between magnesium hydrate and the phosphate backbone of the HDV ribozyme using Raman crystallography.

PubMed

Gong, Bo; Chen, Yuanyuan; Christian, Eric L; Chen, Jui-Hui; Chase, Elaine; Chadalavada, Durga M; Yajima, Rieko; Golden, Barbara L; Bevilacqua, Philip C; Carey, Paul R

2008-07-30

A Raman microscope and Raman difference spectroscopy are used to detect the vibrational signature of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme and to follow the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone. There is a correlation between the Raman intensity of the innersphere magnesium hydrate signature peak, near 322 cm-1, and the intensity of the PO2- symmetric stretch, near 1100 cm-1, perturbed by magnesium binding, demonstrating direct observation of -PO2-...Mg2+(H2O)x innersphere complexes. The complexes may be pentahydrates (x = 5) and tetrahydrates (x = 4). The assignment of the Raman feature near 322 cm-1 to a magnesium hydrate species is confirmed by isotope shifts observed in D2O and H218O that are semiquantitatively reproduced by calculations. The standardized intensity changes in the 1100 cm-1 PO2- feature seen upon magnesium hydrate binding indicates that there are approximately 5 innersphere Mg2+...-O2P contacts per HDV molecule when the crystal is exposed to a solution containing 20 mM magnesium.

2002 Airborne Geophysical Survey at Pueblo of Isleta Bombing Targets, New Mexico, April 10 May 6, 2002 (Rev 1)

DTIC Science & Technology

2005-12-01

helicopter geophysical survey performed by US Army Engineering Support Center, Huntsville (USAESCH) and Oak Ridge National Laboratory ( ORNL ) over areas...Array Detection System NAD North American Datum ORAGS Oak Ridge Airborne Geophysical System ORNL Oak Ridge National Laboratory RMS Root...used by ORNL in 1999 for.....................5 Figure 2.4 ORAGS-Hammerhead airborne magnetometer system used at Badlands Bombing Range in FY2000

Liver as a target for oligonucleotide therapeutics.

PubMed

Sehgal, Alfica; Vaishnaw, Akshay; Fitzgerald, Kevin

2013-12-01

Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to

The mouse Pol I terminator is more efficient than the hepatitis delta virus ribozyme in generating influenza-virus-like RNAs with precise 3' ends in a plasmid-only-based virus rescue system.

PubMed

Feng, Liqiang; Li, Feng; Zheng, Xuehua; Pan, Weiqi; Zhou, Kai; Liu, Yichu; He, Hongxuan; Chen, Ling

2009-01-01

Reverse genetics systems for generating recombinant influenza viruses are based on two different mechanisms for obtaining the 3' end of the viral RNA: one uses the self-cleaving hepatitis delta virus ribozyme (HDVR), and the other uses the murine RNA polymerase I (Pol I) terminator. In this study, we employed EGFP and Renilla luciferase reporter constructs to compare the efficiency of both methods. Our results indicate that the murine Pol I terminator was more efficient than the HDVR, which will be helpful in choosing an influenza virus rescue system, as well as in establishing other RNA virus rescue systems.

Publications - GMC 116 | Alaska Division of Geological & Geophysical

Science.gov Websites

hydrocarbon data) of cuttings from OCS Y-0849-1 (Hammerhead #1) well Authors: Unocal Corporation Publication data) of cuttings from OCS Y-0849-1 (Hammerhead #1) well: Alaska Division of Geological &

RNA-Catalyzed RNA Ligation on an External RNA Template

NASA Technical Reports Server (NTRS)

McGinness, Kathleen E.; Joyce, Gerald F.

2002-01-01

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside '5-triphosphates onto the '3 end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

Prospects for nucleic acid-based therapeutics against hepatitis C virus.

PubMed

Lee, Chang Ho; Kim, Ji Hyun; Lee, Seong-Wook

2013-12-21

In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.

A simple physical mechanism enables homeostasis in primitive cells

NASA Astrophysics Data System (ADS)

Engelhart, Aaron E.; Adamala, Katarzyna P.; Szostak, Jack W.

2016-05-01

The emergence of homeostatic mechanisms that enable maintenance of an intracellular steady state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles, and ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation. This simple physical system enables a primitive homeostatic behaviour: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest that such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.

Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

PubMed

Pang, Ka Ming; Castanotto, Daniela; Li, Haitang; Scherer, Lisa; Rossi, John J

2018-01-09

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Glutamate Receptor Aptamers and ALS

DTIC Science & Technology

2009-01-01

that the evolutionary conversion of a ribozyme (RNA) to a deoxribozyme (DNA) of the same function can be accomplished but only with some critical...2006) Conversion of a ribozyme to a deoxyribozyme through in vitro evolution. Chem Biol. 13, 329-338. 18. Schultes, E. A., and Bartel, D. P. (2000...One sequence, two ribozymes : implications for the emergence of new ribozyme folds. Science 289, 448-452. 19. Wilkinson, K. A., Merino, E. J., and

Engineering RNA for Targeted siRNA Delivery and Medical Application

PubMed Central

Guo, Peixuan; Coban, Oana; Snead, Nick; Trebley, Joe; Hoeprich, Steve; Guo, Songchuan; Shu, Yi

2010-01-01

RNA engineering for nanotechnology and medical applications is an exciting emerging research field. RNA has intrinsically defined features on the nanometer scale and is a particularly interesting candidate for such applications due to its amazing diversity, flexibility and versatility in structure and function. Specifically, the current use of siRNA to silence target genes involved in disease has generated much excitement in the scientific community. The intrinsic ability to sequence-specifically down-regulate gene expression in a temporally- and spatially-controlled fashion has led to heightened interest and rapid development of siRNA-based therapeutics. Though methods for gene silencing with high efficacy and specificity have been achieved in vitro, the effective delivery of nucleic acids to specific cells in vivo has been a hurdle for RNA therapeutics. This review covers different RNA-based approaches for diagnosis, prevention and treatment of human disease, with a focus on the latest developments of nonviral carriers of siRNA for delivery in vivo. The applications and challenges of siRNA therapy, as well as potential solutions to these problems, the approaches for using phi29 pRNA-based vectors as polyvalent vehicles for specific delivery of siRNA, ribozymes, drugs or other therapeutic agents to specific cells for therapy will also be addressed. PMID:20230868

Lateral Mixing DRI Analysis: Submesoscale Water-Mass Spectra

DTIC Science & Technology

2013-09-30

program to determine submesoscale variability in the Sargasso Sea under weak-to-moderate mesoscale conditions. Two sites were examined, a quiet site...anomalies and dye streaks. Hammerhead carries finescale Sea -Bird sensors for temperature, conductivity and pressure as well as Chelsea and WetLab...m of dye-injection target densities. They were embedded in 35-km towyo grid surveys by Craig Lee’s Triaxus and 15-km butterfly surveys by Jody

An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction.

PubMed Central

Shan, S O; Herschlag, D

2000-01-01

The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA. PMID:10864040

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

PubMed Central

Shu, Yi; Haque, Farzin; Shu, Dan; Li, Wei; Zhu, Zhenqi; Kotb, Malak; Lyubchenko, Yuri; Guo, Peixuan

2013-01-01

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field. PMID:23604636

Publications - GMC 90 | Alaska Division of Geological & Geophysical Surveys

Science.gov Websites

and Facilities Staff Seismic and Well Data Data Reports Contact Us Frequently Asked Questions Ask a -1 (Hammerhead) well Authors: Unknown Publication Date: 1988 Publisher: Alaska Division of Geological for the Union Oil Company OCS-Y-0849-1 (Hammerhead) well: Alaska Division of Geological &

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

PubMed Central

Petkovic, Sonja; Badelt, Stefan; Flamm, Christoph; Delcea, Mihaela

2015-01-01

Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and self-concatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions. PMID:25999318

Publications - GMC 118 | Alaska Division of Geological & Geophysical

Science.gov Websites

and Facilities Staff Seismic and Well Data Data Reports Contact Us Frequently Asked Questions Ask a Company OCS Y-0849-2 (Hammerhead #2) well Authors: Unknown Publication Date: 1989 Publisher: Alaska reflectance data of cuttings from the Union Oil Company OCS Y-0849-2 (Hammerhead #2) well: Alaska Division of

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

PubMed

Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

2016-04-01

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthetic RNA Controllers for Programming Mammalian Cell Fate and Function

DTIC Science & Technology

2015-11-04

concentration of β- catenin owing to induction of the Wnt signaling pathway. We also extended the ribozyme -based device platform to respond to protein...magnesium concentrations. Localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or...based regulatory components by developing a platform that combines a ligand-responsive ribozyme switch and synthetic miRNA regulators to create an

Publications - GMC 197 | Alaska Division of Geological & Geophysical

Science.gov Websites

and Facilities Staff Seismic and Well Data Data Reports Contact Us Frequently Asked Questions Ask a (9,134-9,147', and 11,129') of Northstar #1 well, from unwashed cuttings (5,280-5,400) of OCS Y-0849-1 (Hammerhead #1) well, and from unwashed cuttings (5,100-5,520') of OCS Y-0849-2 (Hammerhead #2) well Authors

VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

DTIC Science & Technology

2013-03-01

oligonucleotide-based drug approaches (better than ribozymes, antisense oligonucleotides ( ASO ), or microRNAs). (4) To accomplish these objectives, we...negative control scrambled ASO (designated NC). The combination of siRNAs T1 and R1 produced a knockdown of ~80% of TGFb1 protein in the conditioned...sequences (antisense oligonucleotides, ASOs ) into rabbit corneal cells and found that technique was very effective in delivering ASOs into the stroma and

Maximizing RNA folding rates: a balancing act.

PubMed Central

Thirumalai, D; Woodson, S A

2000-01-01

Large ribozymes typically require very long times to refold into their active conformation in vitro, because the RNA is easily trapped in metastable misfolded structures. Theoretical models show that the probability of misfolding is reduced when local and long-range interactions in the RNA are balanced. Using the folding kinetics of the Tetrahymena ribozyme as an example, we propose that folding rates are maximized when the free energies of forming independent domains are similar to each other. A prediction is that the folding pathway of the ribozyme can be reversed by inverting the relative stability of the tertiary domains. This result suggests strategies for optimizing ribozyme sequences for therapeutics and structural studies. PMID:10864039

Characterization of a Fluorescent Protein Reporter System

DTIC Science & Technology

2008-03-01

pathways are initiated with the binding of a small molecule to a catalytic ribonucleic acid molecule (RNA), called a ribozyme (Thodima et al., 2006). The... ribozyme is part of a larger RNA construct, called a riboswitch, which initiates translation of a specific genetic sequence on a plasmid (circular...protein gene. Yen et al. (2004) reported insertion of a self-cleaving ribozyme upstream of the reporter gene. In the absence of a regulator (“off

An Image Processing Approach to Computing Distances Between RNA Secondary Structures Dot Plots (PREPRINT)

DTIC Science & Technology

2007-03-01

the P5abc subdomain of the tetrahymena thermophila ribozyme that was studied by Wu and Tinoco [24]. The results for the second sequence are found in...virus ribozyme that was studied by Lazinski et al. [25], for its regulation of self-cleavage activity. The results for the third sequence are found...mention the existence of eight possible mutations that provide the desired non-linear effect in the ribozyme structure, and this may explain the

Testing Delivery Platforms for New Anticancer tRNA-Based Drugs

DTIC Science & Technology

2011-03-01

measurement of aminoacylated killer tRNA. Killer tRNA aminoacylated with lysine via a ribozyme is sufficiently stable in the culture media to enable...translational efficiency which helps the design of optimal killer tRNAs. - Determined that the flexi- ribozyme can be used to attach non-serine amino acids to...tRNAs using the flexi- ribozyme strategy. REPORTABLE OUTCOME - A dual GFP-mCherry reporter plasmid DNA useful to monitor delivery of tRNA in

Directed evolution of an RNA enzyme

NASA Technical Reports Server (NTRS)

Beaudry, Amber A.; Joyce, Gerald F.

1992-01-01

An in vitro evolution procedures was used to obtain RNA enzymes with a particular catalytic function. A population of 10 exp 13 variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce 'progeny' ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

Targeting gene therapy to cancer: a review.

PubMed

Dachs, G U; Dougherty, G J; Stratford, I J; Chaplin, D J

1997-01-01

discussed is the regulation of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia. We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail. Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue. This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target. We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors. The list of genes that have been considered for use in the treatment of cancer is extensive. It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop. Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes). This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E. (ABSTRACT TRUNCATED)

Programmable formation of catalytic RNA triangles and squares by assembling modular RNA enzymes.

PubMed

Oi, Hiroki; Fujita, Daisuke; Suzuki, Yuki; Sugiyama, Hiroshi; Endo, Masayuki; Matsumura, Shigeyoshi; Ikawa, Yoshiya

2017-05-01

RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM). © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Evolution of the Division of Labor between Genes and Enzymes in the RNA World

PubMed Central

Boza, Gergely; Szilágyi, András; Kun, Ádám; Santos, Mauro; Szathmáry, Eörs

2014-01-01

The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA. PMID:25474573

Evolution of the division of labor between genes and enzymes in the RNA world.

PubMed

Boza, Gergely; Szilágyi, András; Kun, Ádám; Santos, Mauro; Szathmáry, Eörs

2014-12-01

The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

PubMed

Juergens, Hannes; Varela, Javier A; Gorter de Vries, Arthur R; Perli, Thomas; Gast, Veronica J M; Gyurchev, Nikola Y; Rajkumar, Arun S; Mans, Robert; Pronk, Jack T; Morrissey, John P; Daran, Jean-Marc G

2018-05-01

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

A molecular description of the evolution of resistance

NASA Technical Reports Server (NTRS)

Ordoukhanian, P.; Joyce, G. F.

1999-01-01

BACKGROUND: In vitro evolution has been used to obtain nucleic acid molecules with interesting functional properties. The evolution process usually is carried out in a stepwise manner, involving successive rounds of selection, amplification and mutation. Recently, a continuous in vitro evolution system was devised for RNAs that catalyze the ligation of oligonucleotide substrates, allowing the evolution of catalytic function to be studied in real time. RESULTS: Continuous in vitro evolution of an RNA ligase ribozyme was carried out in the presence of a DNA enzyme that was capable of cleaving, and thereby inactivating, the ribozyme. The DNA concentration was increased steadily over 33.5 hours of evolution, reaching a final concentration that would have been sufficient to inactivate the starting population in one second. The evolved population of ribozymes developed resistance to the DNA enzyme, reducing their vulnerability to cleavage by 2000-fold but retaining their own catalytic function. Based on sequencing and kinetic analysis of the ribozymes, two mechanisms are proposed for this resistance. One involves three nucleotide substitutions, together with two compensatory mutations, that alter the site at which the DNA enzyme binds the ribozyme. The other involves enhancement of the ribozyme's ability to bind its own substrate in a way that protects it from cleavage by the DNA enzyme. CONCLUSIONS: The ability to direct the evolution of an enzyme's biochemical properties in response to the behavior of another macromolecule provides insight into the evolution of resistance and may be useful in developing enzymes with novel or enhanced function.

Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

PubMed

Scarborough, Robert J; Adams, Kelsey L; Daher, Aïcha; Gatignol, Anne

2015-09-01

We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

PubMed

Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

2017-09-01

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Controlled evolution of an RNA enzyme

NASA Technical Reports Server (NTRS)

Joyce, G. F.

1991-01-01

It is generally thought that prior to the origin of protein synthesis, life on earth was based on self-replicating RNA molecules. This idea has become especially popular recently due to the discovery of catalytic RNA (ribozymes). RNA has both genotypic and phenotypic properties, suggesting that it is capable of undergoing Darwinian evolution. RNA evolution is likely to have played a critical role in the early history of life on earth, and thus is important in considering the possibility of life elsewhere in the solar system. We have constructed an RNA-based evolving system in the laboratory, combining amplification and mutation of an RNA genotype with selection of a corresponding RNA phenotype. This system serves as a functional model of a primitive organism. It can also be used as a tool to explore the catalytic potential of RNA. By altering the selection constraints, we are attempting to modify the substrate specificity of an existing ribozyme in order to develop ribozymes with novel catalytic function. In this way, we hope to gain a better understanding of RNA's catalytic versatility and to assess its suitability for the role of primordial catalyst. All of the RNA enzymes that are known to exist in contemporary biology carry out cleavage/ligation reactions involving RNA substrates. The Tetrahymena ribozyme, for example, catalyzes phosphoester transfer between a guanosine containing and an oligopyrimidine containing substrate. We tested the ability of mutant forms of the Tetrahymena ribozyme to carry out a comparable reaction using DNA, rather than RNA substrate. An ensemble of structural variants of the ribozyme was prepared and tested for their ability to specifically cleave d(GGCCCTCT-A3TA3TA) at the phosphodiester bond following the sequence CCCTCT. We recovered a mutant form of the enzyme that cleaves DNA more efficiently than does the wild-type. Beginning with this selected mutant we have now scattered random mutations throughout the ribozyme and have begun

Evolution in a Test Tube: Exploring the Structure and Function of RNA Probes

DTIC Science & Technology

2008-05-02

Bartel, D.P. and Szostak, J.W. (1993) Isolation of New Ribozymes from a Large Pool of Random Sequences. Science, New Series 261, 1141-1418. 24...Szostak, J.W. (1993) Isolation of New Ribozymes from a Large Pool of Random Sequences. Science, New Series 261, 1141-1418. Chen, Ying; Carlini

Shark predation on cephalopods in the Mexican and Ecuadorian Pacific Ocean

NASA Astrophysics Data System (ADS)

Galván-Magaña, Felipe; Polo-Silva, Carlos; Berenice Hernández-Aguilar, Sandra; Sandoval-Londoño, Alejandro; Ruth Ochoa-Díaz, Maria; Aguilar-Castro, Nallely; Castañeda-Suárez, David; Cabrera Chavez-Costa, Alejandra; Baigorrí-Santacruz, Álvaro; Eden Torres-Rojas, Yassir; Andrés Abitia-Cárdenas, Leonardo

2013-10-01

Pelagic predators such as sharks have been shown to be effective cephalopod samplers, because they have high consumption rates and swimming speeds. The stomach contents of these predators allow us to determine the distribution and abundance of cephalopods, considering the scarcity of biological information and the difficulty of catching squids and octopi using traditional methods. The silky shark (Carcharhinus falciformis), blue shark (Prionace glauca), scalloped hammerhead (Sphyrna lewini), smooth hammerhead (Sphyrna zygaena), pelagic thresher shark (Alopias pelagicus), and bigeye thresher shark (Alopias superciliosus) were caught off both coasts of Baja California Sur, Mexico, and in the Ecuadorian Pacific Ocean. Cephalopod sizes (mantle lengths, ML) were calculated based on the beak measurements to determine the size of cephalopods consumed by the sharks. We identified 21 cephalopod species based on beak items found in the shark stomachs. The most abundant cephalopods consumed by sharks in both areas were Dosidicus gigas, Ancistrocheirus lesueurii, Onychoteuthis banksii, Sthenoteuthis ovalaniensis, Argonauta spp., Abraliopsis affinis, and Mastigoteuthis dentata. The cephalopod's habitat provides information about the depth at which these sharks capture their prey. The blue shark feeds on cephalopods in epipelagic, mesopelagic, and bathypelagic waters; the silky shark feeds on cephalopods in epipelagic waters; and the scalloped hammerhead shark preys on cephalopods in neritic (bottom) and oceanic waters (epipelagic and mesopelagic). The pelagic thresher shark consumed epipelagic and neritic species; whereas the bigeye thresher shark feeds mainly on epipelagic and mesopelagic squids in Ecuadorian waters. The smooth hammerhead preys on epipelagic and mesopelagic squids off Mexico and Ecuador.

Co-operation between Polymerases and Nucleotide Synthetases in the RNA World.

PubMed

Kim, Ye Eun; Higgs, Paul G

2016-11-01

It is believed that life passed through an RNA World stage in which replication was sustained by catalytic RNAs (ribozymes). The two most obvious types of ribozymes are a polymerase, which uses a neighbouring strand as a template to make a complementary sequence to the template, and a nucleotide synthetase, which synthesizes monomers for use by the polymerase. When a chemical source of monomers is available, the polymerase can survive on its own. When the chemical supply of monomers is too low, nucleotide production by the synthetase is essential and the two ribozymes can only survive when they are together. Here we consider a computational model to investigate conditions under which coexistence and cooperation of these two types of ribozymes is possible. The model considers six types of strands: the two functional sequences, the complementary strands to these sequences (which are required as templates), and non-functional mutants of the two sequences (which act as parasites). Strands are distributed on a two-dimensional lattice. Polymerases replicate strands on neighbouring sites and synthetases produce monomers that diffuse in the local neighbourhood. We show that coexistence of unlinked polymerases and synthetases is possible in this spatial model under conditions in which neither sequence could survive alone; hence, there is a selective force for increasing complexity. Coexistence is dependent on the relative lengths of the two functional strands, the strand diffusion rate, the monomer diffusion rate, and the rate of deleterious mutations. The sensitivity of this two-ribozyme system suggests that evolution of a system of many types of ribozymes would be difficult in a purely spatial model with unlinked genes. We therefore speculate that linkage of genes onto mini-chromosomes and encapsulation of strands in protocells would have been important fairly early in the history of life as a means of enabling more complex systems to evolve.

The Role of CREB in CML

DTIC Science & Technology

2008-02-01

Feng YQ et al. Anti-beta s- ribozyme reduces beta s mRNA levels in transgenic mice: Potential application to the gene therapy of sickle cell anemia... ribozymes . RNA 2003;9:1254–1263. 13 Pace BS, Qian X, Ofori-Acquah SF. Selective inhibition of beta-globin RNA transcripts by antisense RNA molecules. Cell

Evolution in vitro of an RNA enzyme with altered metal dependence

NASA Technical Reports Server (NTRS)

Lehman, N.; Joyce, G. F.

1993-01-01

The Tetrahymena group I ribozyme catalyses a sequence-specific phosphodiester cleavage reaction on an external RNA oligonucleotide substrate in the presence of a divalent metal cation cofactor. This reaction proceeds readily with either Mg2+ or Mn2+, but no detectable reaction has been reported when other divalent cations are used as the sole cofactor. Cations such as Ca2+, Sr2+ and Ba2+ can stabilize the correct folded conformation of the ribozyme, thereby partially alleviating the Mg2+ or Mn2+ requirement. But catalysis by the ribozyme involves coordination of either Mg2+ or Mn2+ at the active site, resulting in an overall requirement for one of these two cations. Here we use an in vitro evolution process to obtain variants of the Tetrahymena ribozyme that are capable of cleaving an RNA substrate in reaction mixtures containing Ca2+ as the divalent cation. These findings extend the range of different chemical environments available to RNA enzymes and illustrate the power of in vitro evolution in generating macromolecular catalysts with desired properties.

Engineering Improvements in a Bacterial Therapeutic Delivery System for Breast Cancer

DTIC Science & Technology

2010-09-01

system comprises a nucleotide sequence encoding a toxic or 20 therapeutic RNA (e.g., mRNA, tRNA, rRNA, siRNA, ribozyme , and the like), a protein or an RNA...an RNA molecule (e.g., siRNA, ribozyme and the like, for example). The structures of such therapeutic agents are known and can be adapted to

RNA Thermodynamic Structural Entropy

PubMed Central

Garcia-Martin, Juan Antonio; Clote, Peter

2015-01-01

Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner’99 and Turner’04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

RNA Thermodynamic Structural Entropy.

PubMed

Garcia-Martin, Juan Antonio; Clote, Peter

2015-01-01

Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2012 CFR

2012-10-01

... Lemon, Negaprion brevirostris Nurse, Ginglymostoma cirratum Scalloped hammerhead, Sphyrna lewini Smooth... microlepis Lane snapper, Lutjanus synagris Lemon shark, Negaprion brevirostris Mangrove snapper, Lutjanus...

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2013 CFR

2013-10-01

... Mexico blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran Lemon... isodon Gag grouper, Mycteroperca microlepis Lane snapper, Lutjanus synagris Lemon shark, Negaprion...

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2014 CFR

2014-10-01

... Mexico blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran Lemon... isodon Gag grouper, Mycteroperca microlepis Lane snapper, Lutjanus synagris Lemon shark, Negaprion...

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such

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.

Inhibition of human papillomavirus expression using DNAzymes.

PubMed

Benítez-Hess, María Luisa; Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis Marat

2011-01-01

Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects.

In vitro selection of catalytic RNAs

NASA Technical Reports Server (NTRS)

Chapman, K. B.; Szostak, J. W.

1994-01-01

In vitro selection techniques are poised to allow a rapid expansion of the study of catalysis by RNA enzymes (ribozymes). This truly molecular version of genetics has already been applied to the study of the structures of known ribozymes and to the tailoring of their catalytic activity to meet specific requirements of substrate specificity or reaction conditions. During the past year, in vitro selection has been successfully used to isolate novel RNA catalysts from random sequence pools.

A novel mini-DNA barcoding assay to identify processed fins from internationally protected shark species.

PubMed

Fields, Andrew T; Abercrombie, Debra L; Eng, Rowena; Feldheim, Kevin; Chapman, Demian D

2015-01-01

There is a growing need to identify shark products in trade, in part due to the recent listing of five commercially important species on the Appendices of the Convention on International Trade in Endangered Species (CITES; porbeagle, Lamna nasus, oceanic whitetip, Carcharhinus longimanus scalloped hammerhead, Sphyrna lewini, smooth hammerhead, S. zygaena and great hammerhead S. mokarran) in addition to three species listed in the early part of this century (whale, Rhincodon typus, basking, Cetorhinus maximus, and white, Carcharodon carcharias). Shark fins are traded internationally to supply the Asian dried seafood market, in which they are used to make the luxury dish shark fin soup. Shark fins usually enter international trade with their skin still intact and can be identified using morphological characters or standard DNA-barcoding approaches. Once they reach Asia and are traded in this region the skin is removed and they are treated with chemicals that eliminate many key diagnostic characters and degrade their DNA ("processed fins"). Here, we present a validated mini-barcode assay based on partial sequences of the cytochrome oxidase I gene that can reliably identify the processed fins of seven of the eight CITES listed shark species. We also demonstrate that the assay can even frequently identify the species or genus of origin of shark fin soup (31 out of 50 samples).

A Novel Mini-DNA Barcoding Assay to Identify Processed Fins from Internationally Protected Shark Species

PubMed Central

Fields, Andrew T.; Abercrombie, Debra L.; Eng, Rowena; Feldheim, Kevin; Chapman, Demian D.

2015-01-01

There is a growing need to identify shark products in trade, in part due to the recent listing of five commercially important species on the Appendices of the Convention on International Trade in Endangered Species (CITES; porbeagle, Lamna nasus, oceanic whitetip, Carcharhinus longimanus scalloped hammerhead, Sphyrna lewini, smooth hammerhead, S. zygaena and great hammerhead S. mokarran) in addition to three species listed in the early part of this century (whale, Rhincodon typus, basking, Cetorhinus maximus, and white, Carcharodon carcharias). Shark fins are traded internationally to supply the Asian dried seafood market, in which they are used to make the luxury dish shark fin soup. Shark fins usually enter international trade with their skin still intact and can be identified using morphological characters or standard DNA-barcoding approaches. Once they reach Asia and are traded in this region the skin is removed and they are treated with chemicals that eliminate many key diagnostic characters and degrade their DNA (“processed fins”). Here, we present a validated mini-barcode assay based on partial sequences of the cytochrome oxidase I gene that can reliably identify the processed fins of seven of the eight CITES listed shark species. We also demonstrate that the assay can even frequently identify the species or genus of origin of shark fin soup (31 out of 50 samples). PMID:25646789

Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences.

PubMed

Pressman, Abe; Moretti, Janina E; Campbell, Gregory W; Müller, Ulrich F; Chen, Irene A

2017-08-21

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2010 CFR

2010-10-01

..., Thunnus thynnus Dolphin fish, Coryphaena hippurus Oceanic whitetip shark, Carcharhinus longimanus... Tables Table 1 of Appendix A to Part 635—Oceanic Sharks A. Large Coastal Sharks 1. Ridgeback sharks... sharks: Blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran...

50 CFR Appendix A to Part 635 - Species Tables

Code of Federal Regulations, 2011 CFR

2011-10-01

..., Thunnus thynnus Dolphin fish, Coryphaena hippurus Oceanic whitetip shark, Carcharhinus longimanus... Tables Table 1 of Appendix A to Part 635—Oceanic Sharks A. Large Coastal Sharks 1. Ridgeback sharks... sharks: Blacktip, Carcharhinus limbatus Bull, Carcharhinus leucas Great hammerhead, Sphyrna mokarran...

76 FR 23935 - Atlantic Highly Migratory Species; Atlantic Shark Management Measures

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-29

.... 110120049-1144-01] RIN 0648-BA69 Atlantic Highly Migratory Species; Atlantic Shark Management Measures... retention, transshipping, landing, storing, or selling of hammerhead sharks in the family Sphyrnidae (except for Sphyrna tiburo) and oceanic whitetip sharks (Carcharhinus longimanus) caught in association with...

Probing adenine rings and backbone linkages using base specific isotope-edited Raman spectroscopy: application to group II intron ribozyme domain V.

PubMed

Chen, Yuanyuan; Eldho, Nadukkudy V; Dayie, T Kwaku; Carey, Paul R

2010-04-27

with the differences in hydration radii. These subtle differences in electrostatic interactions may be related to observed variations in catalytic activity. For a reconstructed ribozyme comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans, cleavage of spliced exon substrates in the presence of magnesium and K(+) or Cs(+) is more efficient than that in the presence of magnesium with Na(+) or Li(+).

Application of FRET Technology to the In Vivo Evaluation of Therapeutic Nucleic Acids (ANTs)

NASA Astrophysics Data System (ADS)

Benítez-Hess, María Luisa; Alvarez-Salas, Luis Marat

2007-02-01

Developing applications for therapeutic nucleic acids (TNAs) (i.e. ribozymes, antisense oligodeoxynucleotides (AS-ODNs), siRNA and aptamers) requires a reporter system designed to rapidly evaluate their in vivo effect. To this end we designed a reporter system based on the fluorescence resonance energy transfer (FRET) engineered to release the FRET effect produced by two green fluorescent protein (GFP) variants linked by a TNA target site. Because the FRET effect occurs instantaneously when two fluorophores are very close to each other (>100nm) stimulating emission of the acceptor fluorophore by the excitation of the donor fluorophore it has been widely use to reveal interactions between molecules. The present system (FRET2) correlates the FRET effect with the in vivo activity of distinct types of TNAs based on a model consisting of RNA from human papillomavirus type 16 (HPV-16) previously shown accessible to TNAs. HPV-16 is the most common papillomavirus associated with cervical cancer, the leading cause of death by cancer in México. The FRET2 system was first tested in vitro and then used in bacteria in which transcription is linked to translation allowing controlled expression and rapid evaluation of the FRET2 protein. To assure accessibility of the target mRNA to TNAs, the FRET2 mRNA was probed by RNaseH assays prior FRET testing. The fluorescence features of the FRET2 system was tested with different FRET-producing GFP donor-acceptor pairs leading to selection of green (donor) and yellow (acceptor) variants of GFP as the most efficient. Modifications in aminoacid composition and linker length of the target sequence did not affect FRET efficiency. In vivo AS-ODN-mediated destruction of the chimerical FRET2 reporter mRNA resulted in the recovery of GFP fluorescent spectrum in a concentration and time dependent manner. Reported anti-HPV ribozymes were also tested with similar results. Therefore, we conclude that the FRET effect can be a useful tool in the

The shallow-water fish assemblage of Isla del Coco National Park, Costa Rica: Structure and patterns in an isolated, predator-dominated ecosystem

USGS Publications Warehouse

Friedlander, Alan M.; Zgliczynski, Brian J.; Ballesteros, Enric; Aburto-Oropeza, Octavio; Bolaños, Allan; Sala, Enric

2012-01-01

Fishes at Isla del Coco National Park, Costa Rica, were surveyed as part of a larger scientific expedition to the area in September 2009. The average total biomass of nearshore fishes was 7.8 tonnes per ha, among the largest observed in the tropics, with apex predators such as sharks, jacks, and groupers accounting for nearly 40% of the total biomass. The abundance of reef and pelagic sharks, particularly large aggregations of threatened species such as the scalloped hammerhead shark (up to 42 hammerheads ha-1) and large schools of jacks and snappers show the capacity for high biomass in unfished ecosystems in the Eastern Tropical Pacific. However, the abundance of hammerhead and reef whitetip sharks appears to have been declining since the late 1990s, and likely causes may include increasing fishing pressure on sharks in the region and illegal fishing inside the Park. One Galapagos shark tagged on September 20, 2009 in the Isla del Coco National Park moved 255km southeast towards Malpelo Island in Colombia, when it stopped transmitting. These results contribute to the evidence that sharks conduct large-scale movements between marine protected areas (Isla del Coco, Malpelo, Galápagos) in the Eastern tropical Pacific and emphasize the need for regional-scale management. More than half of the species and 90% of the individuals observed were endemic to the tropical eastern Pacific. These high biomass and endemicity values highlight the uniqueness of the fish assemblage at Isla del Coco and its importance as a global biodiversity hotspot.

Circularity and self-cleavage as a strategy for the emergence of a chromosome in the RNA-based protocell

PubMed Central

2013-01-01

Background It is now popularly accepted that an “RNA world” existed in early evolution. During division of RNA-based protocells, random distribution of individual genes (simultaneously as ribozymes) between offspring might have resulted in gene loss, especially when the number of gene types increased. Therefore, the emergence of a chromosome carrying linked genes was critical for the prosperity of the RNA world. However, there were quite a few immediate difficulties for this event to occur. For example, a chromosome would be much longer than individual genes, and thus more likely to degrade and less likely to replicate completely; the copying of the chromosome might start at middle sites and be only partial; and, without a complex transcription mechanism, the synthesis of distinct ribozymes would become problematic. Results Inspired by features of viroids, which have been suggested as “living fossils” of the RNA world, we supposed that these difficulties could have been overcome if the chromosome adopted a circular form and small, self-cleaving ribozymes (e.g. the hammer head ribozymes) resided at the sites between genes. Computer simulation using a Monte-Carlo method was conducted to investigate this hypothesis. The simulation shows that an RNA chromosome can spread (increase in quantity and be sustained) in the system if it is a circular one and its linear “transcripts” are readily broken at the sites between genes; the chromosome works as genetic material and ribozymes “coded” by it serve as functional molecules; and both circularity and self-cleavage are important for the spread of the chromosome. Conclusions In the RNA world, circularity and self-cleavage may have been adopted as a strategy to overcome the immediate difficulties for the emergence of a chromosome (with linked genes). The strategy suggested here is very simple and likely to have been used in this early stage of evolution. By demonstrating the possibility of the emergence of an

Renewing the Assault on mRNA

PubMed Central

McCAIN, JACK

2004-01-01

Mammalian cells dislike double-stranded RNA. They interpret it as a sign of an intruder, and they can unleash a recently discovered defensive mechanism to deal with the problem – they chop the invader into little pieces and use the remnants, called small interfering RNA, to identify and destroy the invader and its progeny. This process, known as RNA interference, may lend itself to new treatments for a wide range of diseases. RNA interference, however, resembles two therapies studied during the 1990s, antisense and ribozymes, in that the gene-silencing target is messenger RNA (mRNA). Is RNA interference really the Next Big Thing – or just a variation on an older but still intriguing theme? PMID:23372488

Catalytic Activity of a Binary Informational Macromolecule

NASA Technical Reports Server (NTRS)

Reader, John S.; Joyce, Gerald F.

2003-01-01

RNA molecules are thought to have played a prominent role in the early history of life on Earth based on their ability both to encode genetic information and to exhibit catalytic function. The modern genetic alphabet relies on two sets of complementary base pairs to store genetic information. However, due to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish. It has been suggested that the first genetic material instead contained only a single base-pairing unie'. Here we show that binary informational macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain ligase ribozymes composed of only 2,6-diaminopurine and uracil nucleotides, which catalyze the template-directed joining of two RNA molecules, one bearing a 5'-triphosphate and the other a 3'-hydroxyl. The active conformation of the fastest isolated ribozyme had a catalytic rate that was about 36,000-fold faster than the uncatalyzed rate of reaction. This ribozyme is specific for the formation of biologically relevant 3',5'-phosphodiester linkages.

A comparison of the heart and muscle total lipid and fatty acid profiles of nine large shark species from the east coast of South Africa.

PubMed

Davidson, Bruce; Sidell, Jonathan; Rhodes, Jeffrey; Cliff, Geremy

2011-03-01

We have assessed the fatty acid profiles of the hearts and different muscle tissues from nine large shark species (Carcharhinus limbatus (blacktip), Carcharhinus obscurus (dusky), Carcharhinus brevipinna (spinner), Carcharhinus leucas (Zambezi/bull), Galeocerdo cuvier (tiger), Sphyrna lewini (scalloped hammerhead), Sphyrna zygaena (smooth hammerhead), Carcharodon carcharias (great white) and Carcharias taurus (raggedtooth/grey nurse/sand tiger)) found off the east coast of South Africa. While there was generally little variation between the species, all species showed profiles rich in both n6 and n3 polyunsaturated fatty acids compared to terrestrial commercial meats that have low n3. Thus, utilizing skeletal muscle tissues from sharks caught as part of the bycatch when fishing for teleosts would avoid unnecessary wastage of a potentially valuable resource, with all the possible health benefits of high quality protein combined with balanced polyunsaturates, although contamination with high levels of metabolic wastes, such as urea, may be a negative consideration.

The effects of stabilizing and directional selection on phenotypic and genotypic variation in a population of RNA enzymes.

PubMed

Hayden, Eric J; Bratulic, Sinisa; Koenig, Iwo; Ferrada, Evandro; Wagner, Andreas

2014-02-01

The distribution of variation in a quantitative trait and its underlying distribution of genotypic diversity can both be shaped by stabilizing and directional selection. Understanding either distribution is important, because it determines a population's response to natural selection. Unfortunately, existing theory makes conflicting predictions about how selection shapes these distributions, and very little pertinent experimental evidence exists. Here we study a simple genetic system, an evolving RNA enzyme (ribozyme) in which a combination of high throughput genotyping and measurement of a biochemical phenotype allow us to address this question. We show that directional selection, compared to stabilizing selection, increases the genotypic diversity of an evolving ribozyme population. In contrast, it leaves the variance in the phenotypic trait unchanged.

[Target volume margins for lung cancer: internal target volume/clinical target volume].

PubMed

Jouin, A; Pourel, N

2013-10-01

The aim of this study was to carry out a review of margins that should be used for the delineation of target volumes in lung cancer, with a focus on margins from gross tumour volume (GTV) to clinical target volume (CTV) and internal target volume (ITV) delineation. Our review was based on a PubMed literature search with, as a cornerstone, the 2010 European Organisation for Research and Treatment of Cancer (EORTC) recommandations by De Ruysscher et al. The keywords used for the search were: radiotherapy, lung cancer, clinical target volume, internal target volume. The relevant information was categorized under the following headings: gross tumour volume definition (GTV), CTV-GTV margin (first tumoural CTV then nodal CTV definition), in field versus elective nodal irradiation, metabolic imaging role through the input of the PET scanner for tumour target volume and limitations of PET-CT imaging for nodal target volume definition, postoperative radiotherapy target volume definition, delineation of target volumes after induction chemotherapy; then the internal target volume is specified as well as tumoural mobility for lung cancer and respiratory gating techniques. Finally, a chapter is dedicated to planning target volume definition and another to small cell lung cancer. For each heading, the most relevant and recent clinical trials and publications are mentioned. Copyright © 2013. Published by Elsevier SAS.

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

PubMed

Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H

1992-01-01

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

Bacterial group II introns: not just splicing.

PubMed

Toro, Nicolás; Jiménez-Zurdo, José Ignacio; García-Rodríguez, Fernando Manuel

2007-04-01

Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.

50 CFR 635.22 - Recreational retention limits.

Code of Federal Regulations, 2014 CFR

2014-10-01

... outer boundary of the Atlantic EEZ, to a shark taken from or possessed in the Atlantic Ocean including.../Headboat permit under § 635.4(b) may not retain, possess or land oceanic whitetip sharks or scalloped, smooth, or great hammerhead sharks if swordfish, tuna, or billfish are retained or possessed on board, or...

50 CFR 635.22 - Recreational retention limits.

Code of Federal Regulations, 2013 CFR

2013-10-01

... outer boundary of the Atlantic EEZ, to a shark taken from or possessed in the Atlantic Ocean including.../Headboat permit under § 635.4(b) may not retain, possess or land oceanic whitetip sharks or scalloped, smooth, or great hammerhead sharks if swordfish, tuna, or billfish are retained or possessed on board, or...

50 CFR 635.22 - Recreational retention limits.

Code of Federal Regulations, 2012 CFR

2012-10-01

... outer boundary of the Atlantic EEZ, to a shark taken from or possessed in the Atlantic Ocean including.../Headboat permit under § 635.4(b) may not retain, possess or land oceanic whitetip sharks or scalloped, smooth, or great hammerhead sharks if swordfish, tuna, or billfish are retained or possessed on board, or...

78 FR 59878 - Atlantic Highly Migratory Species; Commercial Atlantic Aggregated Large Coastal Shark (LCS...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-30

... submitted electronically on a weekly basis through a NMFS-approved electronic reporting system by the dealer... metric tons (mt) dressed weight (dw) (149,914 lb dw) of non-blacknose SCS quota from the Atlantic region... lb dw), the Atlantic hammerhead shark management group quota is 27.1 metric tons (mt) dressed weight...

Variables and Strategies in Development of Therapeutic Post-Transcriptional Gene Silencing Agents

PubMed Central

Sullivan, Jack M.; Yau, Edwin H.; Kolniak, Tiffany A.; Sheflin, Lowell G.; Taggart, R. Thomas; Abdelmaksoud, Heba E.

2011-01-01

Post-transcriptional gene silencing (PTGS) agents such as ribozymes, RNAi and antisense have substantial potential for gene therapy of human retinal degenerations. These technologies are used to knockdown a specific target RNA and its cognate protein. The disease target mRNA may be a mutant mRNA causing an autosomal dominant retinal degeneration or a normal mRNA that is overexpressed in certain diseases. All PTGS technologies depend upon the initial critical annealing event of the PTGS ligand to the target RNA. This event requires that the PTGS agent is in a conformational state able to support hybridization and that the target have a large and accessible single-stranded platform to allow rapid annealing, although such platforms are rare. We address the biocomplexity that currently limits PTGS therapeutic development with particular emphasis on biophysical variables that influence cellular performance. We address the different strategies that can be used for development of PTGS agents intended for therapeutic translation. These issues apply generally to the development of PTGS agents for retinal, ocular, or systemic diseases. This review should assist the interested reader to rapidly appreciate critical variables in PTGS development and facilitate initial design and testing of such agents against new targets of clinical interest. PMID:21785698

Non-unity molecular heritability demonstrated by continuous evolution in vitro

NASA Technical Reports Server (NTRS)

Schmitt, T.; Lehman, N.

1999-01-01

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

Application of multiplex PCR approaches for shark molecular identification: feasibility and applications for fisheries management and conservation in the Eastern Tropical Pacific.

PubMed

Caballero, S; Cardeñosa, D; Soler, G; Hyde, J

2012-03-01

Here we describe the application of new and existing multiplex PCR methodologies for shark species molecular identification. Four multiplex systems (group ID, thresher sharks, hammerhead sharks and miscellaneous shark) were employed with primers previously described and some designed in this study, which allow for species identification after running PCR products through an agarose gel. This system was implemented for samples (bodies and fins) collected from unidentified sharks landed in the port of Buenaventura and from confiscated tissues obtained from illegal fishing around the Malpelo Island Marine Protected Area, Pacific Coast of Colombia. This method has allowed reliable identification, to date, of 407 samples to the genus and/or species levels, most of them (380) identified as the pelagic thresher shark (Alopias pelagicus). Another seven samples were identified as scalloped hammerhead sharks (Sphyrna lewini). This is an easy-to-implement and reliable identification method that could even be used locally to monitor shark captures in the main fishing ports of developed and developing countries. © 2011 Blackwell Publishing Ltd.

Efficient and Robust Paramyxoviridae Reverse Genetics Systems

PubMed Central

Beaty, Shannon M.; Won, Sohui T.; Hong, Patrick; Lyons, Michael; Vigant, Frederic; Freiberg, Alexander N.; tenOever, Benjamin R.; Duprex, W. Paul

2017-01-01

ABSTRACT The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses

3. AERIAL VIEW OF MOBILE LAUNCHER. ON TOP OF LUT ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. AERIAL VIEW OF MOBILE LAUNCHER. ON TOP OF LUT SITS A 25 TON HAMMERHEAD CRANE. STRUCTURE ON LEFT SIDE OF LAUNCH PLATFORM IS KNOWN AS A 'MILK STOOL' AND ALLOWS A SATURN 1B ROCKET TO BE USED IN PLACE OF THE SATURN V ROCKET. - Mobile Launcher One, Kennedy Space Center, Titusville, Brevard County, FL

Optimized catalytic DNA-cleaving ribozymes

NASA Technical Reports Server (NTRS)

Joyce, Gerald F. (Inventor)

1996-01-01

The present invention discloses nucleic acid enzymes capable of cleaving nucleic acid molecules, including single-stranded DNA, in a site-specific manner under physiologic conditions, as well as compositions including same. The present invention also discloses methods of making and using the disclosed enzymes and compositions.

The dawn of the RNA World: Toward functional complexity through ligation of random RNA oligomers

PubMed Central

Briones, Carlos; Stich, Michael; Manrubia, Susanna C.

2009-01-01

A main unsolved problem in the RNA World scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers obtained by random polymerization on mineral surfaces. A number of computational studies have shown that the structural repertoire yielded by that process is dominated by topologically simple structures, notably hairpin-like ones. A fraction of these could display RNA ligase activity and catalyze the assembly of larger, eventually functional RNA molecules retaining their previous modular structure: molecular complexity increases but template replication is absent. This allows us to build up a stepwise model of ligation-based, modular evolution that could pave the way to the emergence of a ribozyme with RNA replicase activity, step at which information-driven Darwinian evolution would be triggered. PMID:19318464

Design of ligand-targeted nanoparticles for enhanced cancer targeting

NASA Astrophysics Data System (ADS)

Stefanick, Jared F.

Ligand-targeted nanoparticles are increasingly used as drug delivery vehicles for cancer therapy, yet have not consistently produced successful clinical outcomes. Although these inconsistencies may arise from differences in disease models and target receptors, nanoparticle design parameters can significantly influence therapeutic efficacy. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, this work evaluates the roles of polyethylene glycol (PEG) coating, ethylene glycol (EG) peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size on tumor targeting in a systematic manner. These parameters were analyzed in multiple disease models by targeting human epidermal growth factor receptor 2 (HER2) in breast cancer and very late antigen-4 (VLA-4) in multiple myeloma to demonstrate the widespread applicability of this approach. By increasing the hydrophilicity of the targeting peptide sequence and simultaneously optimizing the EG peptide-linker length, the in vitro cellular uptake of targeted liposomes was significantly enhanced. Specifically, including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake ~80-fold using an EG6 peptide-linker compared to ~10-fold using an EG45 linker. In vivo, targeted liposomes prepared in a traditional manner lacking the oligolysine chain demonstrated similar biodistribution and tumor uptake to non-targeted liposomes. However, by including the oligolysine chain, targeted liposomes using an EG45 linker significantly improved tumor uptake ~8-fold over non-targeted liposomes, while the use of an EG6 linker decreased tumor accumulation and uptake, owing to differences in cellular uptake kinetics, clearance mechanisms, and binding site barrier effects. To further improve tumor targeting and enhance the selectivity of targeted

SuperTarget goes quantitative: update on drug–target interactions

PubMed Central

Hecker, Nikolai; Ahmed, Jessica; von Eichborn, Joachim; Dunkel, Mathias; Macha, Karel; Eckert, Andreas; Gilson, Michael K.; Bourne, Philip E.; Preissner, Robert

2012-01-01

There are at least two good reasons for the on-going interest in drug–target interactions: first, drug-effects can only be fully understood by considering a complex network of interactions to multiple targets (so-called off-target effects) including metabolic and signaling pathways; second, it is crucial to consider drug-target-pathway relations for the identification of novel targets for drug development. To address this on-going need, we have developed a web-based data warehouse named SuperTarget, which integrates drug-related information associated with medical indications, adverse drug effects, drug metabolism, pathways and Gene Ontology (GO) terms for target proteins. At present, the updated database contains >6000 target proteins, which are annotated with >330 000 relations to 196 000 compounds (including approved drugs); the vast majority of interactions include binding affinities and pointers to the respective literature sources. The user interface provides tools for drug screening and target similarity inclusion. A query interface enables the user to pose complex queries, for example, to find drugs that target a certain pathway, interacting drugs that are metabolized by the same cytochrome P450 or drugs that target proteins within a certain affinity range. SuperTarget is available at http://bioinformatics.charite.de/supertarget. PMID:22067455

Attentional Control via Parallel Target-Templates in Dual-Target Search

PubMed Central

Barrett, Doug J. K.; Zobay, Oliver

2014-01-01

Simultaneous search for two targets has been shown to be slower and less accurate than independent searches for the same two targets. Recent research suggests this ‘dual-target cost’ may be attributable to a limit in the number of target-templates than can guide search at any one time. The current study investigated this possibility by comparing behavioural responses during single- and dual-target searches for targets defined by their orientation. The results revealed an increase in reaction times for dual- compared to single-target searches that was largely independent of the number of items in the display. Response accuracy also decreased on dual- compared to single-target searches: dual-target accuracy was higher than predicted by a model restricting search guidance to a single target-template and lower than predicted by a model simulating two independent single-target searches. These results are consistent with a parallel model of dual-target search in which attentional control is exerted by more than one target-template at a time. The requirement to maintain two target-templates simultaneously, however, appears to impose a reduction in the specificity of the memory representation that guides search for each target. PMID:24489793

Genetic therapies against HIV

PubMed Central

Rossi, John J; June, Carl H; Kohn, Donald B

2015-01-01

Highly active antiretroviral therapy prolongs the life of HIV-infected individuals, but it requires lifelong treatment and results in cumulative toxicities and viral-escape mutants. Gene therapy offers the promise of preventing progressive HIV infection by sustained interference with viral replication in the absence of chronic chemotherapy. Gene-targeting strategies are being developed with RNA-based agents, such as ribozymes, antisense, RNA aptamers and small interfering RNA, and protein-based agents, such as the mutant HIV Rev protein M10, fusion inhibitors and zinc-finger nucleases. Recent advances in T-cell–based strategies include gene-modified HIV-resistant T cells, lentiviral gene delivery, CD8+ T cells, T bodies and engineered T-cell receptors. HIV-resistant hematopoietic stem cells have the potential to protect all cell types susceptible to HIV infection. The emergence of viral resistance can be addressed by therapies that use combinations of genetic agents and that inhibit both viral and host targets. Many of these strategies are being tested in ongoing and planned clinical trials. PMID:18066041

The double life of the ribosome: When its protein folding activity supports prion propagation.

PubMed

Voisset, Cécile; Blondel, Marc; Jones, Gary W; Friocourt, Gaëlle; Stahl, Guillaume; Chédin, Stéphane; Béringue, Vincent; Gillet, Reynald

2017-03-04

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

Computational design of RNA parts, devices, and transcripts with kinetic folding algorithms implemented on multiprocessor clusters.

PubMed

Thimmaiah, Tim; Voje, William E; Carothers, James M

2015-01-01

With progress toward inexpensive, large-scale DNA assembly, the demand for simulation tools that allow the rapid construction of synthetic biological devices with predictable behaviors continues to increase. By combining engineered transcript components, such as ribosome binding sites, transcriptional terminators, ligand-binding aptamers, catalytic ribozymes, and aptamer-controlled ribozymes (aptazymes), gene expression in bacteria can be fine-tuned, with many corollaries and applications in yeast and mammalian cells. The successful design of genetic constructs that implement these kinds of RNA-based control mechanisms requires modeling and analyzing kinetically determined co-transcriptional folding pathways. Transcript design methods using stochastic kinetic folding simulations to search spacer sequence libraries for motifs enabling the assembly of RNA component parts into static ribozyme- and dynamic aptazyme-regulated expression devices with quantitatively predictable functions (rREDs and aREDs, respectively) have been described (Carothers et al., Science 334:1716-1719, 2011). Here, we provide a detailed practical procedure for computational transcript design by illustrating a high throughput, multiprocessor approach for evaluating spacer sequences and generating functional rREDs. This chapter is written as a tutorial, complete with pseudo-code and step-by-step instructions for setting up a computational cluster with an Amazon, Inc. web server and performing the large numbers of kinefold-based stochastic kinetic co-transcriptional folding simulations needed to design functional rREDs and aREDs. The method described here should be broadly applicable for designing and analyzing a variety of synthetic RNA parts, devices and transcripts.

Hydrolysis of tRNA(sup Phe) on Suspensions of Amino Acids

NASA Technical Reports Server (NTRS)

Gao, Kui; Orgel, Leslie E.

2001-01-01

RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNA(sup Phe) is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and beta-glutamic acid but not by suspensions of alpha-glutamic acid, asparagine, or glutamine. The non-enzymatic hydrolysis of RNA has been studied extensively, especially because of its relevance to the mechanisms of action of ribozymes and to biotechnology and therapy. Many ribonucleases, ribozymes, and non-biological catalysts function via acid-base catalysis of an intramolecular transesterification mechanism in which the 2'-OH group attacks the adjacent phosphate group. The pentacoordinated phosphorane intermediate may collapse back to starting material, or yield isomerized or cleaved products.

Open Targets: a platform for therapeutic target identification and validation

PubMed Central

Koscielny, Gautier; An, Peter; Carvalho-Silva, Denise; Cham, Jennifer A.; Fumis, Luca; Gasparyan, Rippa; Hasan, Samiul; Karamanis, Nikiforos; Maguire, Michael; Papa, Eliseo; Pierleoni, Andrea; Pignatelli, Miguel; Platt, Theo; Rowland, Francis; Wankar, Priyanka; Bento, A. Patrícia; Burdett, Tony; Fabregat, Antonio; Forbes, Simon; Gaulton, Anna; Gonzalez, Cristina Yenyxe; Hermjakob, Henning; Hersey, Anne; Jupe, Steven; Kafkas, Şenay; Keays, Maria; Leroy, Catherine; Lopez, Francisco-Javier; Magarinos, Maria Paula; Malone, James; McEntyre, Johanna; Munoz-Pomer Fuentes, Alfonso; O'Donovan, Claire; Papatheodorou, Irene; Parkinson, Helen; Palka, Barbara; Paschall, Justin; Petryszak, Robert; Pratanwanich, Naruemon; Sarntivijal, Sirarat; Saunders, Gary; Sidiropoulos, Konstantinos; Smith, Thomas; Sondka, Zbyslaw; Stegle, Oliver; Tang, Y. Amy; Turner, Edward; Vaughan, Brendan; Vrousgou, Olga; Watkins, Xavier; Martin, Maria-Jesus; Sanseau, Philippe; Vamathevan, Jessica; Birney, Ewan; Barrett, Jeffrey; Dunham, Ian

2017-01-01

We have designed and developed a data integration and visualization platform that provides evidence about the association of known and potential drug targets with diseases. The platform is designed to support identification and prioritization of biological targets for follow-up. Each drug target is linked to a disease using integrated genome-wide data from a broad range of data sources. The platform provides either a target-centric workflow to identify diseases that may be associated with a specific target, or a disease-centric workflow to identify targets that may be associated with a specific disease. Users can easily transition between these target- and disease-centric workflows. The Open Targets Validation Platform is accessible at https://www.targetvalidation.org. PMID:27899665

Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication.

PubMed

Konopka, K; Lee, N S; Rossi, J; Düzgüneş, N

2000-09-19

We examined whether the antiviral effect of an HIV-1 Rev-binding aptamer [RBE(apt)] could be enhanced by a ribozyme directed against the HIV-1 env gene, and whether the antiviral activity was affected by different promoters. The efficacy of the aptamer and ribozyme DNAs was tested in HeLa cells co-transfected with the HIV-1 proviral clones, HXBDeltaBgl or pNL4-3, using transferrin-lipoplexes. The RBE(apt) and anti-env ribozyme genes were inserted into the pTZU6+27 plasmid, or constructed under the control of the human cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. The parental vector plasmids were used as controls. Co-transfection of the pTZU6+27 RBE(apt) plasmid with HXBDeltaBgl, or pNL4-3, at a weight ratio of 5:1, inhibited p24 production by 70 and 45%, respectively. The RSV RBE(apt) plasmid co-transfected with either HIV clone, at the same weight ratio, reduced viral production by 88%. The addition of the anti-env ribozyme to the RSV RBE(apt) did not enhance its antiviral activity. When the constructs were under the control of the CMV promoter, the expression of the HIV plasmids was very low and was independent of the presence of the RBE(apt). Thus, the effect of the RBE(apt) was strongly dependent on the promoter of the tested construct. The anti-HIV activity of the CMV RBE(apt) construct was non-specific, because co-transfection with either pCMV. SPORT-betagal or pCMVlacZ significantly suppressed HIV production from the HIV proviral clones. The reduction in p24 could not be attributed to the non-specific toxicity of the transfection procedure. Transfection of acutely HIV-infected HeLa-CD4 cells with pCMV.SPORT-betagal reduced the p24 level by 35%, while the expression of the U6 RBE(apt) did not affect p24 production. The suppression of HIV production from the HIV proviral clones by the CMV promoter constructs in the co-transfection assays may be explained by competition for transcription factors (TFs) between HIV and CMV promoters. This

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.

PubMed

Kaye, Nicholas M; Christian, Eric L; Harris, Michael E

2002-04-09

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly

Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

PubMed Central

Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

2016-01-01

The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

Literature evidence in open targets - a target validation platform.

PubMed

Kafkas, Şenay; Dunham, Ian; McEntyre, Johanna

2017-06-06

We present the Europe PMC literature component of Open Targets - a target validation platform that integrates various evidence to aid drug target identification and validation. The component identifies target-disease associations in documents and ranks the documents based on their confidence from the Europe PMC literature database, by using rules utilising expert-provided heuristic information. The confidence score of a given document represents how valuable the document is in the scope of target validation for a given target-disease association by taking into account the credibility of the association based on the properties of the text. The component serves the platform regularly with the up-to-date data since December, 2015. Currently, there are a total number of 1168365 distinct target-disease associations text mined from >26 million PubMed abstracts and >1.2 million Open Access full text articles. Our comparative analyses on the current available evidence data in the platform revealed that 850179 of these associations are exclusively identified by literature mining. This component helps the platform's users by providing the most relevant literature hits for a given target and disease. The text mining evidence along with the other types of evidence can be explored visually through https://www.targetvalidation.org and all the evidence data is available for download in json format from https://www.targetvalidation.org/downloads/data .

View southwest of 350ton crane, showing one of four castings ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

View southwest of 350-ton crane, showing one of four castings which support a stationary, tapered steel girder structure called a "tower". This tower is located within an outer rotating framework designated as the "pintle" of the 350-ton crane. The whole crane pivots around bearing at the top of this tapered support tower. - Naval Base Philadelphia-Philadelphia Naval Shipyard, 350-Ton Hammerhead Crane, League Island, Philadelphia, Philadelphia County, PA

Study of target and non-target interplay in spatial attention task.

PubMed

Sweeti; Joshi, Deepak; Panigrahi, B K; Anand, Sneh; Santhosh, Jayasree

2018-02-01

Selective visual attention is the ability to selectively pay attention to the targets while inhibiting the distractors. This paper aims to study the targets and non-targets interplay in spatial attention task while subject attends to the target object present in one visual hemifield and ignores the distractor present in another visual hemifield. This paper performs the averaged evoked response potential (ERP) analysis and time-frequency analysis. ERP analysis agrees to the left hemisphere superiority over late potentials for the targets present in right visual hemifield. Time-frequency analysis performed suggests two parameters i.e. event-related spectral perturbation (ERSP) and inter-trial coherence (ITC). These parameters show the same properties for the target present in either of the visual hemifields but show the difference while comparing the activity corresponding to the targets and non-targets. In this way, this study helps to visualise the difference between targets present in the left and right visual hemifields and, also the targets and non-targets present in the left and right visual hemifields. These results could be utilised to monitor subjects' performance in brain-computer interface (BCI) and neurorehabilitation.

LIQUID TARGET

DOEpatents

Martin, M.D.; Salsig, W.W. Jr.

1959-01-13

A liquid handling apparatus is presented for a liquid material which is to be irradiated. The apparatus consists essentially of a reservoir for the liquid, a target element, a drain tank and a drain lock chamber. The target is in the form of a looped tube, the upper end of which is adapted to be disposed in a beam of atomic particles. The lower end of the target tube is in communication with the liquid in the reservoir and a means is provided to continuously circulate the liquid material to be irradiated through the target tube. Means to heat the reservoir tank is provided in the event that a metal is to be used as the target material. The apparatus is provided with suitable valves and shielding to provide maximum safety in operation.

A catalytic metal ion interacts with the cleavage site G•U wobble in the HDV ribozyme†

PubMed Central

Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.

2009-01-01

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted > 2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335–13342.], while in crystal structures, it is well-positioned for proton transfer. However no crystallographic evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on rate and Mg2+-binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously-unobserved hydrated magnesium ion interacts with the N7 of the cleavage site G•U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data. PMID:19178151

RNAi: a potential new class of therapeutic for human genetic disease.

PubMed

Seyhan, Attila A

2011-11-01

Dominant negative genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, have been challenging since there is no cure and treatments are only to alleviate the symptoms. Current therapies involving pharmacological and biological drugs are not suitable to target mutant genes selectively due to structural indifference of the normal variant of their targets from the disease-causing mutant ones. In instances when the target contains single nucleotide polymorphism (SNP), whether it is an enzyme or structural or receptor protein are not ideal for treatment using conventional drugs due to their lack of selectivity. Therefore, there is a need to develop new approaches to accelerate targeting these previously inaccessible targets by classical therapeutics. Although there is a cooling trend by the pharmaceutical industry for the potential of RNA interference (RNAi), RNAi and other RNA targeting drugs (antisense, ribozyme, etc.) still hold their promise as the only drugs that provide an opportunity to target genes with SNP mutations found in dominant negative disorders, genes specific to pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases. Because of its exquisite specificity and potency, RNAi has attracted a considerable interest as a new class of therapeutic for genetic diseases including amyotrophic lateral sclerosis, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), spinocerebellar ataxia, dominant muscular dystrophies, and cancer. In this review, progress and challenges in developing RNAi therapeutics for genetic diseases will be discussed.

SeedVicious: Analysis of microRNA target and near-target sites.

PubMed

Marco, Antonio

2018-01-01

Here I describe seedVicious, a versatile microRNA target site prediction software that can be easily fitted into annotation pipelines and run over custom datasets. SeedVicious finds microRNA canonical sites plus other, less efficient, target sites. Among other novel features, seedVicious can compute evolutionary gains/losses of target sites using maximum parsimony, and also detect near-target sites, which have one nucleotide different from a canonical site. Near-target sites are important to study population variation in microRNA regulation. Some analyses suggest that near-target sites may also be functional sites, although there is no conclusive evidence for that, and they may actually be target alleles segregating in a population. SeedVicious does not aim to outperform but to complement existing microRNA prediction tools. For instance, the precision of TargetScan is almost doubled (from 11% to ~20%) when we filter predictions by the distance between target sites using this program. Interestingly, two adjacent canonical target sites are more likely to be present in bona fide target transcripts than pairs of target sites at slightly longer distances. The software is written in Perl and runs on 64-bit Unix computers (Linux and MacOS X). Users with no computing experience can also run the program in a dedicated web-server by uploading custom data, or browse pre-computed predictions. SeedVicious and its associated web-server and database (SeedBank) are distributed under the GPL/GNU license.

Electrically charged targets

DOEpatents

Goodman, Ronald K.; Hunt, Angus L.

1984-01-01

Electrically chargeable laser targets and method for forming such charged targets in order to improve their guidance along a predetermined desired trajectory. This is accomplished by the incorporation of a small amount of an additive to the target material which will increase the electrical conductivity thereof, and thereby enhance the charge placed upon the target material for guidance thereof by electrostatic or magnetic steering mechanisms, without adversely affecting the target when illuminated by laser energy.

Thomas R. Cech, RNA, and Ribozymes

Science.gov Websites

RSS Archive Videos XML DOE R&D Accomplishments DOE R&D Accomplishments searchQuery × Find searchQuery x Find DOE R&D Acccomplishments Navigation dropdown arrow The Basics dropdown arrow Home About Physicists Nobel Chemists Medicine Nobels Explore dropdown arrow Insights Blog Archive SC Stories Snapshots R

The drug target genes show higher evolutionary conservation than non-target genes.

PubMed

Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

2016-01-26

Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

Subcellular Localization and Rolling Circle Replication of Peach Latent Mosaic Viroid: Hallmarks of Group A Viroids

PubMed Central

Bussière, F.; Lehoux, J.; Thompson, D. A.; Skrzeczkowski, L. J.; Perreault, J.-P.

1999-01-01

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed. PMID:10400727

SECOND TARGET STATION MODERATOR PERFORMANCE WITH A ROTATING TARGET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Remec, Igor; Gallmeier, Franz X; Rennich, Mark J

2016-01-01

Oak Ridge National Laboratory manages and operates the Spallation Neutron Source and the High Flux Isotope Reactor, two of the world's most advanced neutron scattering facilities. Both facilities are funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Science, and are available to researchers from all over the world. Delivering cutting edge science requires continuous improvements and development of the facilities and instruments. The SNS was designed from the outset to accommodate an additional target station, or Second Target Station (STS), and an upgraded accelerator feeding proton beams to STS and the existing First Targetmore » Station (FTS). Upgrade of the accelerator and the design and construction of STS are being proposed. The presently considered STS configuration is driven with short (<1 s) proton pulses at 10 Hz repetition rate and 467 kW proton beam power, and is optimized for high intensity and high resolution long wavelength neutron applications. STS will allow installation of 22 beamlines and will expand and complement the current national neutron scattering capabilities. In 2015 the STS studies were performed for a compact tungsten target; first a stationary tungsten plate target was analyzed to considerable details and then dropped in favor of a rotating target. For both target options the proton beam footprint as small as acceptable from mechanical and heat removal aspects is required to arrive at a compact-volume neutron production zone in the target, which is essential for tight coupling of target and moderators and for achieving high-intensity peak neutron fluxes. This paper will present recent STS work with the emphasis on neutronics and moderator performance.« less

Advances in Integrating Autonomy with Acoustic Communications for Intelligent Networks of Marine Robots

DTIC Science & Technology

2013-02-01

Sonar AUV #Environmental Sampling Environmental AUV +name : string = OEX Ocean Explorer +name : string = Hammerhead Iver2 +name : string = Unicorn ...executable» Google Earth Bluefin 21 AUV ( Unicorn ) MOOS Computer GPS «serial» Bluefin 21 AUV (Macrura) MOOS Computer «acoustic» Micro-Modem «wired...Computer Bluefin 21 AUV ( Unicorn ) MOOS Computer NURC AUV (OEX) MOOS Computer Topside MOOS Computer «wifi» 5.0GHz WiLan «acoustic» Edgetech GPS

Robot-friendly connector. [space truss structures

NASA Technical Reports Server (NTRS)

Parma, George F. (Inventor); Vandeberghe, Mark H. (Inventor); Ruiz, Steve C. (Inventor)

1993-01-01

Robot friendly connectors, which, in one aspect, are truss joints with two parts, a receptacle and a joint, are presented. The joints have a head which is loosely inserted into the receptacle and is then tightened and aligned. In one aspect, the head is a rounded hammerhead which initially is enclosed in the receptacle with sloppy fit provided by the shape, size, and configuration of surfaces on the head and on the receptacle.

Cooperative Tertiary Interaction Network Guides RNA Folding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Behrouzi, Reza; Roh, Joon Ho; Kilburn, Duncan

2013-04-08

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends onmore » the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.« less

In vitro selection of functional nucleic acids

NASA Technical Reports Server (NTRS)

Wilson, D. S.; Szostak, J. W.

1999-01-01

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

Memory for found targets interferes with subsequent performance in multiple-target visual search.

PubMed

Cain, Matthew S; Mitroff, Stephen R

2013-10-01

Multiple-target visual searches--when more than 1 target can appear in a given search display--are commonplace in radiology, airport security screening, and the military. Whereas 1 target is often found accurately, additional targets are more likely to be missed in multiple-target searches. To better understand this decrement in 2nd-target detection, here we examined 2 potential forms of interference that can arise from finding a 1st target: interference from the perceptual salience of the 1st target (a now highly relevant distractor in a known location) and interference from a newly created memory representation for the 1st target. Here, we found that removing found targets from the display or making them salient and easily segregated color singletons improved subsequent search accuracy. However, replacing found targets with random distractor items did not improve subsequent search accuracy. Removing and highlighting found targets likely reduced both a target's visual salience and its memory load, whereas replacing a target removed its visual salience but not its representation in memory. Collectively, the current experiments suggest that the working memory load of a found target has a larger effect on subsequent search accuracy than does its perceptual salience. PsycINFO Database Record (c) 2013 APA, all rights reserved.

Internal Targeting and External Control: Phototriggered Targeting in Nanomedicine.

PubMed

Arrue, Lily; Ratjen, Lars

2017-12-07

The photochemical control of structure and reactivity bears great potential for chemistry, biology, and life sciences. A key feature of photochemistry is the spatiotemporal control over secondary events. Well-established applications of photochemistry in medicine are photodynamic therapy (PDT) and photopharmacology (PP). However, although both are highly localizable through the application of light, they lack cell- and tissue-specificity. The combination of nanomaterial-based drug delivery and targeting has the potential to overcome limitations for many established therapy concepts. Even more privileged seems the merger of nanomedicine and cell-specific targeting (internal targeting) controlled by light (external control), as it can potentially be applied to many different areas of medicine and pharmaceutical research, including the aforementioned PDT and PP. In this review a survey of the interface of photochemistry, medicine and targeted drug delivery is given, especially focusing on phototriggered targeting in nanomedicine. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

PubMed

Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

2004-03-01

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non

Dual-targeting siRNAs

PubMed Central

Tiemann, Katrin; Höhn, Britta; Ehsani, Ali; Forman, Stephen J.; Rossi, John J.; Sætrom, Pål

2010-01-01

We have developed an algorithm for the prediction of dual-targeting short interfering RNAs (siRNAs) in which both strands are deliberately designed to separately target different mRNA transcripts with complete complementarity. An advantage of this approach versus the use of two separate duplexes is that only two strands, as opposed to four, are competing for entry into the RNA-induced silencing complex. We chose to design our dual-targeting siRNAs as Dicer substrate 25/27mer siRNAs, since design features resembling pre-microRNAs (miRNAs) can be introduced for Dicer processing. Seven different dual-targeting siRNAs targeting genes that are potential targets in cancer therapy have been developed including Bcl2, Stat3, CCND1, BIRC5, and MYC. The dual-targeting siRNAs have been characterized for dual target knockdown in three different cell lines (HEK293, HCT116, and PC3), where they were as effective as their corresponding single-targeting siRNAs in target knockdown. The algorithm developed in this study should prove to be useful for predicting dual-targeting siRNAs in a variety of different targets and is available from http://demo1.interagon.com/DualTargeting/. PMID:20410240

Therapeutic nucleic acids: current clinical status

PubMed Central

Sridharan, Kannan

2016-01-01

Abstract Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand‐alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off‐target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come. PMID:27111518

SuperTarget and Matador: resources for exploring drug-target relationships.

PubMed

Günther, Stefan; Kuhn, Michael; Dunkel, Mathias; Campillos, Monica; Senger, Christian; Petsalaki, Evangelia; Ahmed, Jessica; Urdiales, Eduardo Garcia; Gewiess, Andreas; Jensen, Lars Juhl; Schneider, Reinhard; Skoblo, Roman; Russell, Robert B; Bourne, Philip E; Bork, Peer; Preissner, Robert

2008-01-01

The molecular basis of drug action is often not well understood. This is partly because the very abundant and diverse information generated in the past decades on drugs is hidden in millions of medical articles or textbooks. Therefore, we developed a one-stop data warehouse, SuperTarget that integrates drug-related information about medical indication areas, adverse drug effects, drug metabolization, pathways and Gene Ontology terms of the target proteins. An easy-to-use query interface enables the user to pose complex queries, for example to find drugs that target a certain pathway, interacting drugs that are metabolized by the same cytochrome P450 or drugs that target the same protein but are metabolized by different enzymes. Furthermore, we provide tools for 2D drug screening and sequence comparison of the targets. The database contains more than 2500 target proteins, which are annotated with about 7300 relations to 1500 drugs; the vast majority of entries have pointers to the respective literature source. A subset of these drugs has been annotated with additional binding information and indirect interactions and is available as a separate resource called Matador. SuperTarget and Matador are available at http://insilico.charite.de/supertarget and http://matador.embl.de.

About miRNAs, miRNA seeds, target genes and target pathways.

PubMed

Kehl, Tim; Backes, Christina; Kern, Fabian; Fehlmann, Tobias; Ludwig, Nicole; Meese, Eckart; Lenhof, Hans-Peter; Keller, Andreas

2017-12-05

miRNAs are typically repressing gene expression by binding to the 3' UTR, leading to degradation of the mRNA. This process is dominated by the eight-base seed region of the miRNA. Further, miRNAs are known not only to target genes but also to target significant parts of pathways. A logical line of thoughts is: miRNAs with similar (seed) sequence target similar sets of genes and thus similar sets of pathways. By calculating similarity scores for all 3.25 million pairs of 2,550 human miRNAs, we found that this pattern frequently holds, while we also observed exceptions. Respective results were obtained for both, predicted target genes as well as experimentally validated targets. We note that miRNAs target gene set similarity follows a bimodal distribution, pointing at a set of 282 miRNAs that seems to target genes with very high specificity. Further, we discuss miRNAs with different (seed) sequences that nonetheless regulate similar gene sets or pathways. Most intriguingly, we found miRNA pairs that regulate different gene sets but similar pathways such as miR-6886-5p and miR-3529-5p. These are jointly targeting different parts of the MAPK signaling cascade. The main goal of this study is to provide a general overview on the results, to highlight a selection of relevant results on miRNAs, miRNA seeds, target genes and target pathways and to raise awareness for artifacts in respective comparisons. The full set of information that allows to infer detailed results on each miRNA has been included in miRPathDB, the miRNA target pathway database (https://mpd.bioinf.uni-sb.de).

Nucleozymes

NASA Technical Reports Server (NTRS)

Yang, Jing-Hua (Inventor); Usman, Nassim (Inventor); Rich, Alexander (Inventor); Cedergren, Robert J. (Inventor); Perreault, Jean-Pierre (Inventor)

2000-01-01

Nucleozymes containing ribonucleotides and deoxyribonucleotides or nucleic acid analogues are described herein. The nucleozymes have catalytic activity and are significantly more resistant to degradation than their all-RNA ribozyme counterparts. Also described are methods for preparing the nucleozymes along with methods of using nucleozymes, e.g., as therapeutic agents.

Nucleozymes

NASA Technical Reports Server (NTRS)

Rich, Alexander (Inventor); Perreault, Jean-Pierre (Inventor); Usman, Nassim (Inventor); Cedergren, Robert J. (Inventor); Yang, Jing-Hua (Inventor)

2004-01-01

Nucleozymes containing ribonucleotides and deoxyribonucleotides or nucleic acid analogues are described herein. The nucleozymes have catalytic activity and are significantly more resistant to degradation than their all-RNA ribozyme counterparts. Also described are methods for preparing the nucleozymes along with methods of using nucleozymes, e.g., as therapeutic agents.

The Human Kinome Targeted by FDA Approved Multi-Target Drugs and Combination Products: A Comparative Study from the Drug-Target Interaction Network Perspective.

PubMed

Li, Ying Hong; Wang, Pan Pan; Li, Xiao Xu; Yu, Chun Yan; Yang, Hong; Zhou, Jin; Xue, Wei Wei; Tan, Jun; Zhu, Feng

2016-01-01

The human kinome is one of the most productive classes of drug target, and there is emerging necessity for treating complex diseases by means of polypharmacology (multi-target drugs and combination products). However, the advantages of the multi-target drugs and the combination products are still under debate. A comparative analysis between FDA approved multi-target drugs and combination products, targeting the human kinome, was conducted by mapping targets onto the phylogenetic tree of the human kinome. The approach of network medicine illustrating the drug-target interactions was applied to identify popular targets of multi-target drugs and combination products. As identified, the multi-target drugs tended to inhibit target pairs in the human kinome, especially the receptor tyrosine kinase family, while the combination products were able to against targets of distant homology relationship. This finding asked for choosing the combination products as a better solution for designing drugs aiming at targets of distant homology relationship. Moreover, sub-networks of drug-target interactions in specific disease were generated, and mechanisms shared by multi-target drugs and combination products were identified. In conclusion, this study performed an analysis between approved multi-target drugs and combination products against the human kinome, which could assist the discovery of next generation polypharmacology.

Enantioselective Effects of Chiral Pesticides on their Primary Targets and Secondary Targets.

PubMed

Yang, Ye; Zhang, Jianyun; Yao, Yijun

2017-01-01

Enantioselectivity has been well recognized in the environmental fate and effects of chiral pesticides. Enantiospecific action of the optical enantiomers on the biological molecules establishes the mechanistic basis for the enantioselective toxicity of chiral pesticides to both target and non-target organisms. We undertook a structured search of bibliographic databases for research literature concerning the enantioselective effects of chiral pesticides, including insecticides, herbicides and fungicides, on biomolecules in various species by using some key words. The results of the relevant literatures were reviewed in the text and summarized in tables. Pesticides generally exert their activity on the target organisms via disrupting the primary target biomolecules. In non-target species, effects of pesticides on the secondary targets distinguished from the primary ones make great contribution to their toxicity. Recent investigations have provided convincing evidence of enantioselective toxicity of chiral pesticides to both target and non-target species which is recognized to result from their enantiospecific action on the primary or secondary targets in organisms. This review confirms that chiral pesticides have enantiospecific effects on both primary and secondary target biomolecules in organisms. Future studies regarding toxicological effects of chiral pesticides should focus on the relationship between the enantiomeric difference in the compound-biomolecules interaction and the enantioselectivity in their toxicity.

Eye tracking a self-moved target with complex hand-target dynamics

PubMed Central

Landelle, Caroline; Montagnini, Anna; Madelain, Laurent

2016-01-01

Previous work has shown that the ability to track with the eye a moving target is substantially improved when the target is self-moved by the subject's hand compared with when being externally moved. Here, we explored a situation in which the mapping between hand movement and target motion was perturbed by simulating an elastic relationship between the hand and target. Our objective was to determine whether the predictive mechanisms driving eye-hand coordination could be updated to accommodate this complex hand-target dynamics. To fully appreciate the behavioral effects of this perturbation, we compared eye tracking performance when self-moving a target with a rigid mapping (simple) and a spring mapping as well as when the subject tracked target trajectories that he/she had previously generated when using the rigid or spring mapping. Concerning the rigid mapping, our results confirmed that smooth pursuit was more accurate when the target was self-moved than externally moved. In contrast, with the spring mapping, eye tracking had initially similar low spatial accuracy (though shorter temporal lag) in the self versus externally moved conditions. However, within ∼5 min of practice, smooth pursuit improved in the self-moved spring condition, up to a level similar to the self-moved rigid condition. Subsequently, when the mapping unexpectedly switched from spring to rigid, the eye initially followed the expected target trajectory and not the real one, thereby suggesting that subjects used an internal representation of the new hand-target dynamics. Overall, these results emphasize the stunning adaptability of smooth pursuit when self-maneuvering objects with complex dynamics. PMID:27466129

Sputter target

DOEpatents

Gates, Willard G.; Hale, Gerald J.

1980-01-01

The disclosure relates to an improved sputter target for use in the deposition of hard coatings. An exemplary target is given wherein titanium diboride is brazed to a tantalum backing plate using a gold-palladium-nickel braze alloy.

Negotiating targets with patients: choice of target in relation to occupational state.

PubMed

Robinson, Sandra M; Walker, David J

2012-02-01

Following the recent National Institute for Health and Clinical Excellence guidance on the management of RA, we were interested to see if we could negotiate targets for treatment with patients in routine clinics, how they would express this and whether staying at work would be a target. One hundred RA patients were recruited. They were consecutive within clinics, but not all clinics were used. They were asked their understanding of the DAS score and a target for treatment negotiated. Any impact of the RA on their paid employment was then explored. Four participants were unable to specify a target for their RA. Negotiated targets were expressed as restricted activities and either as maintaining an activity (70) if the disease was stable, or regaining an activity (26) if the treatment was being increased. Targets were walking a distance for 50% of patients; leisure activities for 18%; domestic activities for 17%; work for 14% and personal care for 2%. For the 21 participants currently working, maintaining work was the target for 12, with 1 wishing to regain lost hours. No patient currently not working expressed returning to work as a target. There were some differences in targets between men and women. Patients are able to negotiate a target for their treatment, expressed as maintaining or regaining a physical activity. Work ceases to be a target once it is lost. Therefore, preventing loss of occupation is likely to be more effective than trying to regain it.

Targeted Nanomaterials for Phototherapy

PubMed Central

Chitgupi, Upendra; Qin, Yiru; Lovell, Jonathan F.

2017-01-01

Phototherapies involve the irradiation of target tissues with light. To further enhance selectivity and potency, numerous molecularly targeted photosensitizers and photoactive nanoparticles have been developed. Active targeting typically involves harnessing the affinity between a ligand and a cell surface receptor for improved accumulation in the targeted tissue. Targeting ligands including peptides, proteins, aptamers and small molecules have been explored for phototherapy. In this review, recent examples of targeted nanomaterials used in phototherapy are summarized. PMID:29071178

Polarized Solid State Target

NASA Astrophysics Data System (ADS)

Dutz, Hartmut; Goertz, Stefan; Meyer, Werner

2017-01-01

The polarized solid state target is an indispensable experimental tool to study single and double polarization observables at low intensity particle beams like tagged photons. It was one of the major components of the Crystal-Barrel experiment at ELSA. Besides the operation of the 'CB frozen spin target' within the experimental program of the Crystal-Barrel collaboration both collaborative groups of the D1 project, the polarized target group of the Ruhr Universität Bochum and the Bonn polarized target group, have made significant developments in the field of polarized targets within the CRC16. The Bonn polarized target group has focused its work on the development of technically challenging polarized solid target systems towards the so called '4π continuous mode polarized target' to operate them in combination with 4π-particle detection systems. In parallel, the Bochum group has developed various highly polarized deuterated target materials and high precision NMR-systems, in the meantime used for polarization experiments at CERN, JLAB and MAMI, too.

Post-targeting strategy for ready-to-use targeted nanodelivery post cargo loading.

PubMed

Zhu, J Y; Hu, J J; Zhang, M K; Yu, W Y; Zheng, D W; Wang, X Q; Feng, J; Zhang, X Z

2017-12-14

Based on boronate formation, this study reports a post-targeting methodology capable of readily installing versatile targeting modules onto a cargo-loaded nanoplatform in aqueous mediums. This permits the targeted nanodelivery of broad-spectrum therapeutics (drug/gene) in a ready-to-use manner while overcoming the PEGylation-dilemma that frequently occurs in conventional targeting approaches.

Magnetically attached sputter targets

DOEpatents

Makowiecki, Daniel M.; McKernan, Mark A.

1994-01-01

An improved method and assembly for attaching sputtering targets to cathode assemblies of sputtering systems which includes a magnetically permeable material. The magnetically permeable material is imbedded in a target base that is brazed, welded, or soldered to the sputter target, or is mechanically retained in the target material. Target attachment to the cathode is achieved by virtue of the permanent magnets and/or the pole pieces in the cathode assembly that create magnetic flux lines adjacent to the backing plate, which strongly attract the magnetically permeable material in the target assembly.

Glioma targeting and blood-brain barrier penetration by dual-targeting doxorubincin liposomes.

PubMed

Gao, Jian-Qing; Lv, Qing; Li, Li-Ming; Tang, Xin-Jiang; Li, Fan-Zhu; Hu, Yu-Lan; Han, Min

2013-07-01

Effective chemotherapy for glioblastoma requires a carrier that can penetrate the blood-brain barrier (BBB) and subsequently target the glioma cells. Dual-targeting doxorubincin (Dox) liposomes were produced by conjugating liposomes with both folate (F) and transferrin (Tf), which were proven effective in penetrating the BBB and targeting tumors, respectively. The liposome was characterized by particle size, Dox entrapment efficiency, and in vitro release profile. Drug accumulation in cells, P-glycoprotein (P-gp) expression, and drug transport across the BBB in the dual-targeting liposome group were examined by using bEnd3 BBB models. In vivo studies demonstrated that the dual-targeting Dox liposomes could transport across the BBB and mainly distribute in the brain glioma. The anti-tumor effect of the dual-targeting liposome was also demonstrated by the increased survival time, decreased tumor volume, and results of both hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis. The dual-targeting Dox liposome could improve the therapeutic efficacy of brain glioma and were less toxic than the Dox solution, showing a dual-targeting effect. These results indicate that this dual-targeting liposome can be used as a potential carrier for glioma chemotherapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Polarized internal target apparatus

DOEpatents

Holt, Roy J.

1986-01-01

A polarized internal target apparatus with a polarized gas target of improved polarization and density achieved by mixing target gas atoms with a small amount of alkali metal gas atoms, and passing a high intensity polarized light source into the mixture to cause the alkali metal gas atoms to become polarized which interact in spin exchange collisions with target gas atoms yielding polarized target gas atoms.

Targeting an efficient target-to-target interval for P300 speller brain–computer interfaces

PubMed Central

Sellers, Eric W.; Wang, Xingyu

2013-01-01

Longer target-to-target intervals (TTI) produce greater P300 event-related potential amplitude, which can increase brain–computer interface (BCI) classification accuracy and decrease the number of flashes needed for accurate character classification. However, longer TTIs requires more time for each trial, which will decrease the information transfer rate of BCI. In this paper, a P300 BCI using a 7 × 12 matrix explored new flash patterns (16-, 18- and 21-flash pattern) with different TTIs to assess the effects of TTI on P300 BCI performance. The new flash patterns were designed to minimize TTI, decrease repetition blindness, and examine the temporal relationship between each flash of a given stimulus by placing a minimum of one (16-flash pattern), two (18-flash pattern), or three (21-flash pattern) non-target flashes between each target flashes. Online results showed that the 16-flash pattern yielded the lowest classification accuracy among the three patterns. The results also showed that the 18-flash pattern provides a significantly higher information transfer rate (ITR) than the 21-flash pattern; both patterns provide high ITR and high accuracy for all subjects. PMID:22350331

Polarized internal target apparatus

DOEpatents

Holt, R.J.

1984-10-10

A polarized internal target apparatus with a polarized gas target of improved polarization and density (achieved by mixing target gas atoms with a small amount of alkali metal gas atoms, and passing a high intensity polarized light source into the mixture to cause the alkali metal gas atoms to become polarized which interact in spin exchange collisions with target gas atoms yielding polarized target gas atoms) is described.

Integrin Targeted MR Imaging

PubMed Central

Tan, Mingqian; Lu, Zheng-Rong

2011-01-01

Magnetic resonance imaging (MRI) is a powerful medical diagnostic imaging modality for integrin targeted imaging, which uses the magnetic resonance of tissue water protons to display tissue anatomic structures with high spatial resolution. Contrast agents are often used in MRI to highlight specific regions of the body and make them easier to visualize. There are four main classes of MRI contrast agents based on their different contrast mechanisms, including T1, T2, chemical exchange saturation transfer (CEST) agents, and heteronuclear contrast agents. Integrins are an important family of heterodimeric transmembrane glycoproteins that function as mediators of cell-cell and cell-extracellular matrix interactions. The overexpressed integrins can be used as the molecular targets for designing suitable integrin targeted contrast agents for MR molecular imaging. Integrin targeted contrast agent includes a targeting agent specific to a target integrin, a paramagnetic agent and a linker connecting the targeting agent with the paramagnetic agent. Proper selection of targeting agents is critical for targeted MRI contrast agents to effectively bind to integrins for in vivo imaging. An ideal integrin targeted MR contrast agent should be non-toxic, provide strong contrast enhancement at the target sites and can be completely excreted from the body after MR imaging. An overview of integrin targeted MR contrast agents based on small molecular and macromolecular Gd(III) complexes, lipid nanoparticles and superparamagnetic nanoparticles is provided for MR molecular imaging. By using proper delivery systems for loading sufficient Gd(III) chelates or superparamagnetic nanoparticles, effective molecular imaging of integrins with MRI has been demonstrated in animal models. PMID:21547154

Magnetically attached sputter targets

DOEpatents

Makowiecki, D.M.; McKernan, M.A.

1994-02-15

An improved method and assembly for attaching sputtering targets to cathode assemblies of sputtering systems which includes a magnetically permeable material is described. The magnetically permeable material is imbedded in a target base that is brazed, welded, or soldered to the sputter target, or is mechanically retained in the target material. Target attachment to the cathode is achieved by virtue of the permanent magnets and/or the pole pieces in the cathode assembly that create magnetic flux lines adjacent to the backing plate, which strongly attract the magnetically permeable material in the target assembly. 11 figures.

Genomic Target Database (GTD): A database of potential targets in human pathogenic bacteria

PubMed Central

Barh, Debmalya; Kumar, Anil; Misra, Amarendra Narayana

2009-01-01

A Genomic Target Database (GTD) has been developed having putative genomic drug targets for human bacterial pathogens. The selected pathogens are either drug resistant or vaccines are yet to be developed against them. The drug targets have been identified using subtractive genomics approaches and these are subsequently classified into Drug targets in pathogen specific unique metabolic pathways,Drug targets in host-pathogen common metabolic pathways, andMembrane localized drug targets. HTML code is used to link each target to its various properties and other available public resources. Essential resources and tools for subtractive genomic analysis, sub-cellular localization, vaccine and drug designing are also mentioned. To the best of authors knowledge, no such database (DB) is presently available that has listed metabolic pathways and membrane specific genomic drug targets based on subtractive genomics. Listed targets in GTD are readily available resource in developing drug and vaccine against the respective pathogen, its subtypes, and other family members. Currently GTD contains 58 drug targets for four pathogens. Shortly, drug targets for six more pathogens will be listed. Availability GTD is available at IIOAB website http://www.iioab.webs.com/GTD.htm. It can also be accessed at http://www.iioabdgd.webs.com.GTD is free for academic research and non-commercial use only. Commercial use is strictly prohibited without prior permission from IIOAB. PMID:20011153

Pilots' Attention Distributions Between Chasing a Moving Target and a Stationary Target.

PubMed

Li, Wen-Chin; Yu, Chung-San; Braithwaite, Graham; Greaves, Matthew

2016-12-01

Attention plays a central role in cognitive processing; ineffective attention may induce accidents in flight operations. The objective of the current research was to examine military pilots' attention distributions between chasing a moving target and a stationary target. In the current research, 37 mission-ready F-16 pilots participated. Subjects' eye movements were collected by a portable head-mounted eye-tracker during tactical training in a flight simulator. The scenarios of chasing a moving target (air-to-air) and a stationary target (air-to-surface) consist of three operational phases: searching, aiming, and lock-on to the targets. The findings demonstrated significant differences in pilots' percentage of fixation during the searching phase between air-to-air (M = 37.57, SD = 5.72) and air-to-surface (M = 33.54, SD = 4.68). Fixation duration can indicate pilots' sustained attention to the trajectory of a dynamic target during air combat maneuvers. Aiming at the stationary target resulted in larger pupil size (M = 27,105, SD = 6565), reflecting higher cognitive loading than aiming at the dynamic target (M = 23,864, SD = 8762). Pilots' visual behavior is not only closely related to attention distribution, but also significantly associated with task characteristics. Military pilots demonstrated various visual scan patterns for searching and aiming at different types of targets based on the research settings of a flight simulator. The findings will facilitate system designers' understanding of military pilots' cognitive processes during tactical operations. They will assist human-centered interface design to improve pilots' situational awareness. The application of an eye-tracking device integrated with a flight simulator is a feasible and cost-effective intervention to improve the efficiency and safety of tactical training.Li W-C, Yu C-S, Braithwaite G, Greaves M. Pilots' attention distributions between chasing a moving target and a stationary target. Aerosp Med

TargetSpy: a supervised machine learning approach for microRNA target prediction.

PubMed

Sturm, Martin; Hackenberg, Michael; Langenberger, David; Frishman, Dmitrij

2010-05-28

Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences.In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila

TargetSpy: a supervised machine learning approach for microRNA target prediction

PubMed Central

2010-01-01

Background Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. Results We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences. In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Conclusion Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well

Structure of a group II intron in complex with its reverse transcriptase.

PubMed

Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

2016-06-01

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

Human target acquisition performance

NASA Astrophysics Data System (ADS)

Teaney, Brian P.; Du Bosq, Todd W.; Reynolds, Joseph P.; Thompson, Roger; Aghera, Sameer; Moyer, Steven K.; Flug, Eric; Espinola, Richard; Hixson, Jonathan

2012-06-01

The battlefield has shifted from armored vehicles to armed insurgents. Target acquisition (identification, recognition, and detection) range performance involving humans as targets is vital for modern warfare. The acquisition and neutralization of armed insurgents while at the same time minimizing fratricide and civilian casualties is a mounting concern. U.S. Army RDECOM CERDEC NVESD has conducted many experiments involving human targets for infrared and reflective band sensors. The target sets include human activities, hand-held objects, uniforms & armament, and other tactically relevant targets. This paper will define a set of standard task difficulty values for identification and recognition associated with human target acquisition performance.

Tumor suppressor molecules and methods of use

DOEpatents

Welch, Peter J.; Barber, Jack R.

2004-09-07

The invention provides substantially pure tumor suppressor nucleic acid molecules and tumor suppressor polypeptides. The invention also provides hairpin ribozymes and antibodies selective for these tumor suppressor molecules. Also provided are methods of detecting a neoplastic cell in a sample using detectable agents specific for the tumor suppressor nucleic acids and polypeptides.

The Species and Origin of Shark Fins in Taiwan's Fishing Ports, Markets, and Customs Detention: A DNA Barcoding Analysis.

PubMed

Chuang, Po-Shun; Hung, Tzu-Chiao; Chang, Hung-An; Huang, Chien-Kang; Shiao, Jen-Chieh

2016-01-01

The increasing consumption of shark products, along with the shark's fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources were dominated by Prionace glauca, which accounted for more than 30% of the collected samples. Over 60% of the species identified in the fin products also appeared in the port landings, suggesting the domestic-dominance of shark fin products in Taiwan. However, international trade also contributes a certain proportion of the fin product markets, as four species identified from the shark fin products are not found in Taiwan's waters, and some domestic-available species were also found in the customs-detained sample. In addition to the species identification, we also found geographical differentiation in the cox1 gene of the common thresher sharks (Alopias vulpinus), the pelagic thresher shark (A. pelagicus), the smooth hammerhead shark (Sphyrna zygaena), and the scalloped hammerhead shark (S. lewini). This result might allow fishing authorities to more effectively trace the origins as well as enforce the management and conservation of these sharks.

Target-directed catalytic metallodrugs

PubMed Central

Joyner, J.C.; Cowan, J.A.

2013-01-01

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vs metal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements. PMID:23828584

Target attribute-based false alarm rejection in small infrared target detection

NASA Astrophysics Data System (ADS)

Kim, Sungho

2012-11-01

Infrared search and track is an important research area in military applications. Although there are a lot of works on small infrared target detection methods, we cannot apply them in real field due to high false alarm rate caused by clutters. This paper presents a novel target attribute extraction and machine learning-based target discrimination method. Eight kinds of target features are extracted and analyzed statistically. Learning-based classifiers such as SVM and Adaboost are developed and compared with conventional classifiers for real infrared images. In addition, the generalization capability is also inspected for various infrared clutters.

Nuclear Security: Target Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Surinder Paul; Gibbs, Philip W.; Bultz, Garl A.

2014-03-01

This objectives of this session were to understand the basic steps of target identification; describe the SNRI targets in detail; characterize specific targets with more detail; prioritize targets based on guidance documents; understand the graded safeguards concept; identify roll up and understand why it is a concern; and recognize the category for different materials.

Unification of automatic target tracking and automatic target recognition

NASA Astrophysics Data System (ADS)

Schachter, Bruce J.

2014-06-01

The subject being addressed is how an automatic target tracker (ATT) and an automatic target recognizer (ATR) can be fused together so tightly and so well that their distinctiveness becomes lost in the merger. This has historically not been the case outside of biology and a few academic papers. The biological model of ATT∪ATR arises from dynamic patterns of activity distributed across many neural circuits and structures (including retina). The information that the brain receives from the eyes is "old news" at the time that it receives it. The eyes and brain forecast a tracked object's future position, rather than relying on received retinal position. Anticipation of the next moment - building up a consistent perception - is accomplished under difficult conditions: motion (eyes, head, body, scene background, target) and processing limitations (neural noise, delays, eye jitter, distractions). Not only does the human vision system surmount these problems, but it has innate mechanisms to exploit motion in support of target detection and classification. Biological vision doesn't normally operate on snapshots. Feature extraction, detection and recognition are spatiotemporal. When vision is viewed as a spatiotemporal process, target detection, recognition, tracking, event detection and activity recognition, do not seem as distinct as they are in current ATT and ATR designs. They appear as similar mechanism taking place at varying time scales. A framework is provided for unifying ATT and ATR.

26 CFR 1.338-1 - General principles; status of old target and new target.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 4 2013-04-01 2013-04-01 false General principles; status of old target and new... principles; status of old target and new target. (a) In general—(1) Deemed transaction. Elections are..., old target and new target, generally are considered to exist for purposes of subtitle A of the...

26 CFR 1.338-1 - General principles; status of old target and new target.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 4 2012-04-01 2012-04-01 false General principles; status of old target and new... principles; status of old target and new target. (a) In general—(1) Deemed transaction. Elections are..., old target and new target, generally are considered to exist for purposes of subtitle A of the...

26 CFR 1.338-1 - General principles; status of old target and new target.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 4 2014-04-01 2014-04-01 false General principles; status of old target and new... principles; status of old target and new target. (a) In general—(1) Deemed transaction. Elections are..., old target and new target, generally are considered to exist for purposes of subtitle A of the...

Bar coded retroreflective target

DOEpatents

Vann, Charles S.

2000-01-01

This small, inexpensive, non-contact laser sensor can detect the location of a retroreflective target in a relatively large volume and up to six degrees of position. The tracker's laser beam is formed into a plane of light which is swept across the space of interest. When the beam illuminates the retroreflector, some of the light returns to the tracker. The intensity, angle, and time of the return beam is measured to calculate the three dimensional location of the target. With three retroreflectors on the target, the locations of three points on the target are measured, enabling the calculation of all six degrees of target position. Until now, devices for three-dimensional tracking of objects in a large volume have been heavy, large, and very expensive. Because of the simplicity and unique characteristics of this tracker, it is capable of three-dimensional tracking of one to several objects in a large volume, yet it is compact, light-weight, and relatively inexpensive. Alternatively, a tracker produces a diverging laser beam which is directed towards a fixed position, and senses when a retroreflective target enters the fixed field of view. An optically bar coded target can be read by the tracker to provide information about the target. The target can be formed of a ball lens with a bar code on one end. As the target moves through the field, the ball lens causes the laser beam to scan across the bar code.

Using the Dual-Target Cost to Explore the Nature of Search Target Representations

ERIC Educational Resources Information Center

Stroud, Michael J.; Menneer, Tamaryn; Cave, Kyle R.; Donnelly, Nick

2012-01-01

Eye movements were monitored to examine search efficiency and infer how color is mentally represented to guide search for multiple targets. Observers located a single color target very efficiently by fixating colors similar to the target. However, simultaneous search for 2 colors produced a dual-target cost. In addition, as the similarity between…

Target size matters: target errors contribute to the generalization of implicit visuomotor learning.

PubMed

Reichenthal, Maayan; Avraham, Guy; Karniel, Amir; Shmuelof, Lior

2016-08-01

The process of sensorimotor adaptation is considered to be driven by errors. While sensory prediction errors, defined as the difference between the planned and the actual movement of the cursor, drive implicit learning processes, target errors (e.g., the distance of the cursor from the target) are thought to drive explicit learning mechanisms. This distinction was mainly studied in the context of arm reaching tasks where the position and the size of the target were constant. We hypothesize that in a dynamic reaching environment, where subjects have to hit moving targets and the targets' dynamic characteristics affect task success, implicit processes will benefit from target errors as well. We examine the effect of target errors on learning of an unnoticed perturbation during unconstrained reaching movements. Subjects played a Pong game, in which they had to hit a moving ball by moving a paddle controlled by their hand. During the game, the movement of the paddle was gradually rotated with respect to the hand, reaching a final rotation of 25°. Subjects were assigned to one of two groups: The high-target error group played the Pong with a small ball, and the low-target error group played with a big ball. Before and after the Pong game, subjects performed open-loop reaching movements toward static targets with no visual feedback. While both groups adapted to the rotation, the postrotation reaching movements were directionally biased only in the small-ball group. This result provides evidence that implicit adaptation is sensitive to target errors. Copyright © 2016 the American Physiological Society.

TargetCompare: A web interface to compare simultaneous miRNAs targets.

PubMed

Moreira, Fabiano Cordeiro; Dustan, Bruno; Hamoy, Igor G; Ribeiro-Dos-Santos, André M; Dos Santos, Andrea Ribeiro

2014-01-01

MicroRNAs (miRNAs) are small non-coding nucleotide sequences between 17 and 25 nucleotides in length that primarily function in the regulation of gene expression. A since miRNA has thousand of predict targets in a complex, regulatory cell signaling network. Therefore, it is of interest to study multiple target genes simultaneously. Hence, we describe a web tool (developed using Java programming language and MySQL database server) to analyse multiple targets of pre-selected miRNAs. We cross validated the tool in eight most highly expressed miRNAs in the antrum region of stomach. This helped to identify 43 potential genes that are target of at least six of the referred miRNAs. The developed tool aims to reduce the randomness and increase the chance of selecting strong candidate target genes and miRNAs responsible for playing important roles in the studied tissue. http://lghm.ufpa.br/targetcompare.

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Partitioning the Fitness Components of RNA Populations Evolving In Vitro

PubMed Central

Díaz Arenas, Carolina; Lehman, Niles

2013-01-01

All individuals in an evolving population compete for resources, and their performance is measured by a fitness metric. The performance of the individuals is relative to their abilities and to the biotic surroundings – the conditions under which they are competing – and involves many components. Molecules evolving in a test tube can also face complex environments and dynamics, and their fitness measurements should reflect the complexity of various contributing factors as well. Here, the fitnesses of a set of ligase ribozymes evolved by the continuous in vitro evolution system were measured. During these evolution cycles there are three different catalytic steps, ligation, reverse transcription, and forward transcription, each with a potential differential influence on the total fitness of each ligase. For six distinct ligase ribozyme genotypes that resulted from continuous evolution experiments, the rates of reaction were measured for each catalytic step by tracking the kinetics of enzymes reacting with their substrates. The reaction products were analyzed for the amount of product formed per time. Each catalytic step of the evolution cycle was found to have a differential incidence in the total fitness of the ligases, and therefore the total fitness of any ligase cannot be inferred from only one catalytic step of the evolution cycle. Generally, the ribozyme-directed ligation step tends to impart the largest effect on overall fitness. Yet it was found that the ligase genotypes have different absolute fitness values, and that they exploit different stages of the overall cycle to gain a net advantage. This is a new example of molecular niche partitioning that may allow for coexistence of more than one species in a population. The dissection of molecular events into multiple components of fitness provides new insights into molecular evolutionary studies in the laboratory, and has the potential to explain heretofore counterintuitive findings. PMID:24391957

TargetCompare: A web interface to compare simultaneous miRNAs targets

PubMed Central

Moreira, Fabiano Cordeiro; Dustan, Bruno; Hamoy, Igor G; Ribeiro-dos-Santos, André M; dos Santos, Ândrea Ribeiro

2014-01-01

MicroRNAs (miRNAs) are small non-coding nucleotide sequences between 17 and 25 nucleotides in length that primarily function in the regulation of gene expression. A since miRNA has thousand of predict targets in a complex, regulatory cell signaling network. Therefore, it is of interest to study multiple target genes simultaneously. Hence, we describe a web tool (developed using Java programming language and MySQL database server) to analyse multiple targets of pre-selected miRNAs. We cross validated the tool in eight most highly expressed miRNAs in the antrum region of stomach. This helped to identify 43 potential genes that are target of at least six of the referred miRNAs. The developed tool aims to reduce the randomness and increase the chance of selecting strong candidate target genes and miRNAs responsible for playing important roles in the studied tissue. Availability http://lghm.ufpa.br/targetcompare PMID:25352731

Stem-Loop V of Varkud Satellite RNA Exhibits Characteristics of the Mg2+ Bound Structure in the Presence of Monovalent Ions

PubMed Central

2015-01-01

The Varkud Satellite RNA contains a self-cleaving ribozyme that has been shown to function independently of its surroundings. This 160 nucleotide ribozyme adopts a catalytically active tertiary structure that includes a kissing hairpin complex formed by stem-loop I and stem-loop V (SLV). The five-nucleotide 5′-rUGACU loop of the isolated SLV has been shown to adopt a Mg2+-dependent U-turn structure by solution NMR. This U-turn hairpin is examined here by molecular dynamics simulations in the presence of monovalent and divalent ions. Simulations confirm on an all-atom level the hypotheses for the role of the Mg2+ ions in stabilizing the loop, as well as the role of the solvent exposed U700 base. Additionally, these simulations suggest the Mg2+-free stem-loop adopts a wide range of structures, including energetically favorable structures similar to the Mg2+-bound loop structure. We propose this structure is a “gatekeeper” or precursor to Mg2+ binding when those ions are present. PMID:26328924

Targeted Therapy for Cancer

Cancer.gov

Targeted therapy is a type of cancer treatment that targets the changes in cancer cells that help them grow, divide, and spread. Learn how targeted therapy works against cancer and about side effects that may occur.

HYDROGEN ISOTOPE TARGETS

DOEpatents

Ashley, R.W.

1958-08-12

The design of targets for use in the investigation of nuclear reactions of hydrogen isotopes by bombardment with accelerated particles is described. The target con struction eomprises a backing disc of a metal selected from the group consisting of molybdenunn and tungsten, a eoating of condensed titaniunn on the dise, and a hydrogen isotope selected from the group consisting of deuterium and tritium absorbed in the coatiag. The proeess for preparing these hydrogen isotope targets is described.

Targets and methods for target preparation for radionuclide production

DOEpatents

Zhuikov, Boris L; Konyakhin, Nicolai A; Kokhanyuk, Vladimir M; Srivastava, Suresh C

2012-10-16

The invention relates to nuclear technology, and to irradiation targets and their preparation. One embodiment of the present invention includes a method for preparation of a target containing intermetallic composition of antimony Ti--Sb, Al--Sb, Cu--Sb, or Ni--Sb in order to produce radionuclides (e.g., tin-117 m) with a beam of accelerated particles. The intermetallic compounds of antimony can be welded by means of diffusion welding to a copper backing cooled during irradiation on the beam of accelerated particles. Another target can be encapsulated into a shell made of metallic niobium, stainless steel, nickel or titanium cooled outside by water during irradiation. Titanium shell can be plated outside by nickel to avoid interaction with the cooling water.

Immunotherapy Targets in Pediatric Cancer

PubMed Central

Orentas, Rimas J.; Lee, Daniel W.; Mackall, Crystal

2011-01-01

Immunotherapy for cancer has shown increasing success and there is ample evidence to expect that progress gleaned in immune targeting of adult cancers can be translated to pediatric oncology. This manuscript reviews principles that guide selection of targets for immunotherapy of cancer, emphasizing the similarities and distinctions between oncogene-inhibition targets and immune targets. It follows with a detailed review of molecules expressed by pediatric tumors that are already under study as immune targets or are good candidates for future studies of immune targeting. Distinctions are made between cell surface antigens that can be targeted in an MHC independent manner using antibodies, antibody derivatives, or chimeric antigen receptors versus intracellular antigens which must be targeted with MHC restricted T cell therapies. Among the most advanced immune targets for childhood cancer are CD19 and CD22 on hematologic malignancies, GD2 on solid tumors, and NY-ESO-1 expressed by a majority of synovial sarcomas, but several other molecules reviewed here also have properties which suggest that they too could serve as effective targets for immunotherapy of childhood cancer. PMID:22645714

Chemical Structural Novelty: On-Targets and Off-Targets

PubMed Central

Yera, Emmanuel R.; Cleves, Ann. E.; Jain, Ajay N.

2011-01-01

Drug structures may be quantitatively compared based on 2D topological structural considerations and based on 3D characteristics directly related to binding. A framework for combining multiple similarity computations is presented along with its systematic application to 358 drugs with overlapping pharmacology. Given a new molecule along with a set of molecules sharing some biological effect, a single score based on comparison to the known set is produced, reflecting either 2D similarity, 3D similarity, or their combination. For prediction of primary targets, the benefit of 3D over 2D was relatively small, but for prediction of off-targets, the added benefit was large. In addition to assessing prediction, the relationship between chemical similarity and pharmacological novelty was studied. Drug pairs that shared high 3D similarity but low 2D similarity (i.e. a novel scaffold) were shown to be much more likely to exhibit pharmacologically relevant differences in terms of specific protein target modulation. PMID:21916467

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 5 2014-04-01 2014-04-01 false Determination of target normal cost and funding target. 1.430(d)-1 Section 1.430(d)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Determination of target normal cost and funding target. (a) In general—(1) Overview. This section sets forth...

26 CFR 1.430(d)-1 - Determination of target normal cost and funding target.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 5 2012-04-01 2011-04-01 true Determination of target normal cost and funding target. 1.430(d)-1 Section 1.430(d)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Determination of target normal cost and funding target. (a) In general—(1) Overview. This section sets forth...

Changing paradigm from one target one ligand towards multi target directed ligand design for key drug targets of Alzheimer disease: An important role of Insilco methods in multi target directed ligands design.

PubMed

Kumar, Akhil; Tiwari, Ashish; Sharma, Ashok

2018-03-15

Alzheimer disease (AD) is now considered as a multifactorial neurodegenerative disorder and rapidly increasing to an alarming situation and causing higher death rate. One target one ligand hypothesis is not able to provide complete solution of AD due to multifactorial nature of disease and one target one drug seems to fail to provide better treatment against AD. Moreover, current available treatments are limited and most of the upcoming treatments under clinical trials are based on modulating single target. So the current AD drug discovery research shifting towards new approach for better solution that simultaneously modulate more than one targets in the neurodegenerative cascade. This can be achieved by network pharmacology, multi-modal therapies, multifaceted, and/or the more recently proposed term "multi-targeted designed drugs. Drug discovery project is tedious, costly and long term project. Moreover, multi target AD drug discovery added extra challenges such as good binding affinity of ligands for multiple targets, optimal ADME/T properties, no/less off target side effect and crossing of the blood brain barrier. These hurdles may be addressed by insilico methods for efficient solution in less time and cost as computational methods successfully applied to single target drug discovery project. Here we are summarizing some of the most prominent and computationally explored single target against AD and further we discussed successful example of dual or multiple inhibitors for same targets. Moreover we focused on ligand and structure based computational approach to design MTDL against AD. However is not an easy task to balance dual activity in a single molecule but computational approach such as virtual screening docking, QSAR, simulation and free energy are useful in future MTDLs drug discovery alone or in combination with fragment based method. However, rational and logical implementations of computational drug designing methods are capable of assisting AD drug

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae.

PubMed

Deaner, Matthew; Holzman, Allison; Alper, Hal S

2018-04-16

Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts. Next, we devise and validate a scheme to allow modular construction of tRNA-sgRNA (TST) operons using an iterative Type IIs digestion/ligation extension approach, termed CRISPR-Ligation Extension of sgRNA Operons (LEGO). This approach enables facile construction of large TST operons. We demonstrate this utility by constructing a metabolic rewiring prototype for 2,3-butanediol production in 2 distinct yeast strain backgrounds. These results demonstrate that our approach can act as a surrogate for traditional genetic modification on a much shorter design-cycle timescale. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models.

PubMed

Yao, Zhi-Jiang; Dong, Jie; Che, Yu-Jing; Zhu, Min-Feng; Wen, Ming; Wang, Ning-Ning; Wang, Shan; Lu, Ai-Ping; Cao, Dong-Sheng

2016-05-01

Drug-target interactions (DTIs) are central to current drug discovery processes and public health fields. Analyzing the DTI profiling of the drugs helps to infer drug indications, adverse drug reactions, drug-drug interactions, and drug mode of actions. Therefore, it is of high importance to reliably and fast predict DTI profiling of the drugs on a genome-scale level. Here, we develop the TargetNet server, which can make real-time DTI predictions based only on molecular structures, following the spirit of multi-target SAR methodology. Naïve Bayes models together with various molecular fingerprints were employed to construct prediction models. Ensemble learning from these fingerprints was also provided to improve the prediction ability. When the user submits a molecule, the server will predict the activity of the user's molecule across 623 human proteins by the established high quality SAR model, thus generating a DTI profiling that can be used as a feature vector of chemicals for wide applications. The 623 SAR models related to 623 human proteins were strictly evaluated and validated by several model validation strategies, resulting in the AUC scores of 75-100 %. We applied the generated DTI profiling to successfully predict potential targets, toxicity classification, drug-drug interactions, and drug mode of action, which sufficiently demonstrated the wide application value of the potential DTI profiling. The TargetNet webserver is designed based on the Django framework in Python, and is freely accessible at http://targetnet.scbdd.com .

TargetNet: a web service for predicting potential drug-target interaction profiling via multi-target SAR models

NASA Astrophysics Data System (ADS)

Yao, Zhi-Jiang; Dong, Jie; Che, Yu-Jing; Zhu, Min-Feng; Wen, Ming; Wang, Ning-Ning; Wang, Shan; Lu, Ai-Ping; Cao, Dong-Sheng

2016-05-01

Drug-target interactions (DTIs) are central to current drug discovery processes and public health fields. Analyzing the DTI profiling of the drugs helps to infer drug indications, adverse drug reactions, drug-drug interactions, and drug mode of actions. Therefore, it is of high importance to reliably and fast predict DTI profiling of the drugs on a genome-scale level. Here, we develop the TargetNet server, which can make real-time DTI predictions based only on molecular structures, following the spirit of multi-target SAR methodology. Naïve Bayes models together with various molecular fingerprints were employed to construct prediction models. Ensemble learning from these fingerprints was also provided to improve the prediction ability. When the user submits a molecule, the server will predict the activity of the user's molecule across 623 human proteins by the established high quality SAR model, thus generating a DTI profiling that can be used as a feature vector of chemicals for wide applications. The 623 SAR models related to 623 human proteins were strictly evaluated and validated by several model validation strategies, resulting in the AUC scores of 75-100 %. We applied the generated DTI profiling to successfully predict potential targets, toxicity classification, drug-drug interactions, and drug mode of action, which sufficiently demonstrated the wide application value of the potential DTI profiling. The TargetNet webserver is designed based on the Django framework in Python, and is freely accessible at http://targetnet.scbdd.com.

Targeted therapies: a nursing perspective.

PubMed

Kay, Polly

2006-02-01

To review the development of targeted therapies and the biology of relevant therapeutic targets. To analyze the relevance of targeted agents as part of current clinical practice. Research articles. Several targeted agents are now available for clinical use. Their mechanisms of action are more specific against tumor cells than traditional cytotoxics. Monotherapy regimens based on targeted agents tend to be better tolerated than chemotherapy, and most combination regimens with targeted agents have proven feasible. Their availability has greatly expanded cancer treatment options, especially for chemorefractory patients. Nurses involved in the care of patients with cancer can benefit from an increased understanding of targeted therapies, including their mechanisms of action, their efficacy profile, as well as prophylaxis and management of adverse events and administration procedures.

A method for detecting small targets based on cumulative weighted value of target properties

NASA Astrophysics Data System (ADS)

Jin, Xing; Sun, Gang; Wang, Wei-hua; Liu, Fang; Chen, Zeng-ping

2015-03-01

Laser detection based on the "cat's eye effect" has become the hot research project for its initiative compared to the passivity of sound detection and infrared detection. And the target detection is one of the core technologies in this system. The paper puts forward a method for detecting small targets based on cumulative weighted value of target properties using given data. Firstly, we make a frame difference to the images, then make image processing based on Morphology Principles. Secondly, we segment images, and screen the targets; then find some interesting locations. Finally, comparing to a quantity of frames, we locate the target. We did an exam to 394 true frames, the experimental result shows that the mathod can detect small targets efficiently.

Burglar Target Selection

PubMed Central

Townsley, Michael; Bernasco, Wim; Ruiter, Stijn; Johnson, Shane D.; White, Gentry; Baum, Scott

2015-01-01

Objectives: This study builds on research undertaken by Bernasco and Nieuwbeerta and explores the generalizability of a theoretically derived offender target selection model in three cross-national study regions. Methods: Taking a discrete spatial choice approach, we estimate the impact of both environment- and offender-level factors on residential burglary placement in the Netherlands, the United Kingdom, and Australia. Combining cleared burglary data from all study regions in a single statistical model, we make statistical comparisons between environments. Results: In all three study regions, the likelihood an offender selects an area for burglary is positively influenced by proximity to their home, the proportion of easily accessible targets, and the total number of targets available. Furthermore, in two of the three study regions, juvenile offenders under the legal driving age are significantly more influenced by target proximity than adult offenders. Post hoc tests indicate the magnitudes of these impacts vary significantly between study regions. Conclusions: While burglary target selection strategies are consistent with opportunity-based explanations of offending, the impact of environmental context is significant. As such, the approach undertaken in combining observations from multiple study regions may aid criminology scholars in assessing the generalizability of observed findings across multiple environments. PMID:25866418

UniDrug-target: a computational tool to identify unique drug targets in pathogenic bacteria.

PubMed

Chanumolu, Sree Krishna; Rout, Chittaranjan; Chauhan, Rajinder S

2012-01-01

Targeting conserved proteins of bacteria through antibacterial medications has resulted in both the development of resistant strains and changes to human health by destroying beneficial microbes which eventually become breeding grounds for the evolution of resistances. Despite the availability of more than 800 genomes sequences, 430 pathways, 4743 enzymes, 9257 metabolic reactions and protein (three-dimensional) 3D structures in bacteria, no pathogen-specific computational drug target identification tool has been developed. A web server, UniDrug-Target, which combines bacterial biological information and computational methods to stringently identify pathogen-specific proteins as drug targets, has been designed. Besides predicting pathogen-specific proteins essentiality, chokepoint property, etc., three new algorithms were developed and implemented by using protein sequences, domains, structures, and metabolic reactions for construction of partial metabolic networks (PMNs), determination of conservation in critical residues, and variation analysis of residues forming similar cavities in proteins sequences. First, PMNs are constructed to determine the extent of disturbances in metabolite production by targeting a protein as drug target. Conservation of pathogen-specific protein's critical residues involved in cavity formation and biological function determined at domain-level with low-matching sequences. Last, variation analysis of residues forming similar cavities in proteins sequences from pathogenic versus non-pathogenic bacteria and humans is performed. The server is capable of predicting drug targets for any sequenced pathogenic bacteria having fasta sequences and annotated information. The utility of UniDrug-Target server was demonstrated for Mycobacterium tuberculosis (H37Rv). The UniDrug-Target identified 265 mycobacteria pathogen-specific proteins, including 17 essential proteins which can be potential drug targets. UniDrug-Target is expected to accelerate

Targeted Nanotechnology for Cancer Imaging

PubMed Central

Toy, Randall; Bauer, Lisa; Hoimes, Christopher; Ghaghada, Ketan B.; Karathanasis, Efstathios

2014-01-01

Targeted nanoparticle imaging agents provide many benefits and new opportunities to facilitate accurate diagnosis of cancer and significantly impact patient outcome. Due to the highly engineerable nature of nanotechnology, targeted nanoparticles exhibit significant advantages including increased contrast sensitivity, binding avidity and targeting specificity. Considering the various nanoparticle designs and their adjustable ability to target a specific site and generate detectable signals, nanoparticles can be optimally designed in terms of biophysical interactions (i.e., intravascular and interstitial transport) and biochemical interactions (i.e., targeting avidity towards cancer-related biomarkers) for site-specific detection of very distinct microenvironments. This review seeks to illustrate that the design of a nanoparticle dictates its in vivo journey and targeting of hard-to-reach cancer sites, facilitating early and accurate diagnosis and interrogation of the most aggressive forms of cancer. We will report various targeted nanoparticles for cancer imaging using X-ray computed tomography, ultrasound, magnetic resonance imaging, nuclear imaging and optical imaging. Finally, to realize the full potential of targeted nanotechnology for cancer imaging, we will describe the challenges and opportunities for the clinical translation and widespread adaptation of targeted nanoparticles imaging agents. PMID:25116445

High or Low Target Prevalence Increases the Dual-Target Cost in Visual Search

ERIC Educational Resources Information Center

Menneer, Tamaryn; Donnelly, Nick; Godwin, Hayward J.; Cave, Kyle R.

2010-01-01

Previous studies have demonstrated a dual-target cost in visual search. In the current study, the relationship between search for one and search for two targets was investigated to examine the effects of target prevalence and practice. Color-shape conjunction stimuli were used with response time, accuracy and signal detection measures. Performance…

RNA-Targeted Therapeutics.

PubMed

Crooke, Stanley T; Witztum, Joseph L; Bennett, C Frank; Baker, Brenda F

2018-04-03

RNA-targeted therapies represent a platform for drug discovery involving chemically modified oligonucleotides, a wide range of cellular RNAs, and a novel target-binding motif, Watson-Crick base pairing. Numerous hurdles considered by many to be impassable have been overcome. Today, four RNA-targeted therapies are approved for commercial use for indications as diverse as Spinal Muscular Atrophy (SMA) and reduction of low-density lipoprotein cholesterol (LDL-C) and by routes of administration including subcutaneous, intravitreal, and intrathecal delivery. The technology is efficient and supports approaching "undruggable" targets. Three additional agents are progressing through registration, and more are in clinical development, representing several chemical and structural classes. Moreover, progress in understanding the molecular mechanisms by which these drugs work has led to steadily better clinical performance and a wide range of mechanisms that may be exploited for therapeutic purposes. Here we summarize the progress, future challenges, and opportunities for this drug discovery platform. Copyright © 2018 Elsevier Inc. All rights reserved.

Stable RNA nanoparticles as potential new generation drugs for cancer therapy☆