SEARCH · PubMed Health

Results for “RNA Stability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources

PubMed2004

Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.

We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] mercuric chloride (APM) polyacrylamide gels, whereas millimolar concentrations of TCEP gave no difference in the fraction of thiophosphorylated RNA retained on the APM interface relative to samples containing no reductant. Ribozyme activity in TCEP, assessed by the self-thiophosphorylating Kin.46 ribozyme, was unaffected by the presence of DTT or TCEP or by the absence of reductant, as measured on APM gels and evaluated by Michaelis-Menten kinetics. Unexpectedly, TCEP more than doubled the half-life of full-length RNA at 50 and 70 degrees C, whether in 5 or 50mM MgCl(2), relative to DTT and the absence of reductant. Under these same conditions, the 5(')-thiophosphate showed negligible decay, and TCEP was more stable than DTT. TCEP thermostability was equivalent in the presence of 5 or 50mM MgCl(2) and 10mM adenosine or ATP.

Base Sequence↗

PubMed2004

Computed energetics of nucleotides in spatial ribozyme structures: an accurate identification of functional regions from structure.

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.

Catalytic Domain↗

PubMed1994

Decay of the IS10 antisense RNA by 3' exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack.

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology.

Bacterial Proteins↗

PubMed2005

Kinetic analysis of hydrolytic reaction of homo- and heterochiral adenylyl(3'-5')adenosine isomers: breaking homochirality reduces hydrolytic stability of RNA.

The hydrolytic stability of the diastereomeric isomers of ApA was compared and the results show that heterochiral ApAs are more rapidly hydrolyzed than homochiral ApAs at low temperatures, suggesting that hydrolytic selection in cold environments in conjunction with selective polymerization may have been effective in enriching the homochirality of RNA.

Dinucleoside Phosphates↗

PubMed2002

Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions.

A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of < or = 200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs.

Animals↗

PubMed2005

2Apro is a multifunctional protein that regulates the stability, translation and replication of poliovirus RNA.

Poliovirus 2A(pro) is required for the inhibition of host cell protein synthesis and efficient viral replication. We investigated the role of 2A(pro) in regulating viral RNA stability, translation and replication in HeLa S10 reactions. The protease activity of 2A(pro) or its polyprotein precursors, 2AB or P2, was required to increase the stability of viral RNA and prolong translation. Since other viral proteins were not required for the observed effects of 2A(pro), it is likely that a cellular protein(s) modified by 2A(pro) mediated these effects on stability and translation. In addition, the protease activity of 2A(pro) stimulated negative-strand initiation by approximately five-fold but had no effect on positive-strand initiation. The 2A(pro) stimulation of negative-strand synthesis was independent of its effect on stability and translation. These findings further extend the previously known functions of protein 2A(pro) to include its role in increasing RNA stability, prolonging translation and stimulating negative-strand synthesis.

Cysteine Endopeptidases↗

PubMed1986

Improved free-energy parameters for predictions of RNA duplex stability.

Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in delta GO at 37 degrees C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5 degrees C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G X U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.

Hydrogen Bonding↗

PubMed2001

Influence of N6-isopentenyladenosine (i(6)A) on thermal stability of RNA duplexes.

The thermodynamic stability of self-complementary oligoribonucleotides containing N6-isopentenyladenosine (i(6)A) or N6-isopentanyladenosine (p(6)A) was determined. The base pairs i(6)A.U and p(6)A.U were placed in either an internal (separated and tandem) and a terminal position within the duplex, or unpaired i(6)A and p(6)A as a 3'-dangling ends. The thermal unfolding of the oligomers was determined by means of UV melting profiles and the thermodynamic parameters: enthalpy (DeltaH degrees ), entropy (DeltaS degrees) and free energy (DeltaG degrees (37)) as well as the melting temperature (T(m)) were calculated. Both modified nucleosides destabilized the duplexes, however, the effect depended on the position of the modified adenosine within the duplex. The similarity of the behavior of oligomers containing i(6)A and p(6)A suggests a negligible effect of the double bond on the thermal stability. The largest destabilization was observed when derivatives of adenosine were placed in an internal position. The effect of 3'-dangling ends suggests that the presence of the N6-isopentenyl- or N6-isopentanyl substitutent affects hydrogen bonding rather than stacking within duplex.

Base Sequence↗

PubMed2002

Stabilization of RNA strands in protein synthesis by type I procollagen C-proteinase enhancer protein, a potential RNA-binding protein, in hepatic stellate cells.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.

Adipocytes↗

PubMed1999

Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas.

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5' untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5' end and near position -30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position -60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions -20 to -15), and the second, called PRB1 (positions -14 to -11), is complementary to the 3' end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5' untranslated region could play an important role in the control of initiation of psbD mRNA translation.

5' Untranslated Regions↗

PubMed2004

The DNA binding protein H-NS binds to and alters the stability of RNA in vitro and in vivo.

H-NS is an abundant prokaryotic transcription factor that preferentially binds to intrinsically bent DNA. Although H-NS has been shown to reduce the transcription of over 100 genes, evidence suggests that H-NS can also affect the translation of some genes. One such gene, rpoS, specifies a sigma factor, RpoS. The ability of H-NS to bind to the rpoS mRNA and the non-coding RNA regulator, DsrA, was tested. Electrophoretic mobility-shift assays yielded an apparent binding affinity of H-NS binding to curved DNA of approximately 1 microM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 microM. This RNA binding was not prevented by an excess of competitor yeast RNA, suggesting that H-NS specifically bound these RNAs. Footprint analysis with a single strand-specific ribonuclease was used to identify the H-NS binding site(s) on DsrA and rpoS mRNA. Surprisingly, H-NS appeared to enhance the cleavage of DsrA and rpoS mRNA. The enhanced cleavage was at sites that were predicted to be single-stranded and did not result from contaminating nucleases in the H-NS protein preparation or non-specific effects of the nuclease. Quantitative RT-PCR of RNA isolated from wild-type and hns- strains revealed that H-NS also affects the stability of DsrA in vivo. Thus H-NS appears to modulate RNA stability in vivo and in vitro.

Bacterial Proteins↗

PubMed1994

A model for the stabilities of RNA hairpins based on a study of the sequence dependence of stability for hairpins of six nucleotides.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGCXUAAUYGCC, where XY is the set of 10 possible mismatch base pairs. A nearest neighbor analysis of the data indicates the free energy for loop formation at 37 C varies from 2.9 to 4.5 kcal/mol. Thermodynamic parameters are also reported for hairpin formation by RNA sequences of the type GGXGUAAUAYCC (where XY are CG, GC, AU, UA, GU, and UG), with the common naturally occurring GA first mismatch (45% of small and large subunit rRNA loops of six). These results allow the development of a model to predict the stability of RNA hairpin loops. The model includes the size of the loop, the identity of the closing base pair, the free energy increment (delta G zero 37MM) for interaction of the closing base pair with the first mismatch, and an additional stabilization term for GA and UU first mismatches. delta G zero 37L(n) = delta G zero 37i(n) + delta G zero 37MM + 0.4 (if closed by AU or UA) -0.7 (if first mismatch is GA or UU). Here delta G zero 37i(n) is the free energy for initiating a loop of n nucleotides. delta G zero 37i(n) for n = 4-9 is 4.9, 4.4, 5.0, 5.0, 5.1, and 5.2 kcal/mol, respectively. The delta G zero 37MM is derived from measurements of model duplexes with terminal mismatches. The model gives good agreement when tested against four naturally occurring hairpin sequences.

Base Sequence↗

PubMed1999

Relationship between internucleotide linkage geometry and the stability of RNA.

The inherent chemical instability of RNA under physiological conditions is primarily due to the spontaneous cleavage of phosphodiester linkages via intramolecular transesterification reactions. Although the protonation state of the nucleophilic 2'-hydroxyl group is a critical determinant of the rate of RNA cleavage, the precise geometry of the chemical groups that comprise each internucleotide linkage also has a significant impact on cleavage activity. Specifically, transesterification is expected to be proportional to the relative in-line character of the linkage. We have examined the rates of spontaneous cleavage of various RNAs for which the secondary and tertiary structures have previously been modeled using either NMR or X-ray crystallographic data. Rate constants determined for the spontaneous cleavage of different RNA linkages vary by almost 10,000-fold, most likely reflecting the contribution that secondary and tertiary structures make towards the overall chemical stability of RNA. Moreover, a correlation is observed between RNA cleavage rate and the relative in-line fitness of each internucleotide linkage. One linkage located within an ATP-binding RNA aptamer is predicted to adopt most closely the ideal conformation for in-line attack. This linkage has a rate constant for transesterification that is approximately 12-fold greater than is observed for an unconstrained linkage and was found to be the most labile among a total of 136 different sites examined. The implications of this relationship for the chemical stability of RNA and for the mechanisms of nucleases and ribozymes are discussed.

Adenosine Triphosphate↗

PubMed1988

Characterization of RNA hairpin loop stability.

Fifteen RNA hairpins that share the same stem sequence and have homopolymer loops of A, C and U residues which vary in length from three to nine nucleotides were synthesized and their thermal stabilities determined. Tm varies as a function of loop size but is almost independent of loop composition. Loops of four or five nucleotides are found to be the most stable loop size. This is consistent with the observation that four-membered loops are the most prevalent loop size in 16S-like RNAs. The contribution of each loop to hairpin stability was calculated by subtracting the known contribution of the helical stem. These data should be useful for predicting the stability of other hairpins.

Base Composition↗

PubMed2005

Stability of RNA isolated from human trabecular bone at post-mortem and surgery.

To determine the reliability of gene expression studies in human post-mortem bone, it is important to evaluate the stability of RNA isolated from such tissues as a function of the post-mortem interval. The stability of total RNA and bone-specific mRNA species was examined in bone samples obtained from routine autopsies and at surgery. The optimal temperature for any storage and transport of the bone before RNA isolation was shown to be 4 degrees C, and RT-PCR analysis is the preferred technique for the analysis of gene expression in post-mortem bone as it tolerates partial RNA degradation. For gene expression studies in bone, post-mortem cases, with a post-mortem interval of less than 48 h, should be selected, and the time that bone is stored after retrieval at autopsy or surgery should be kept to a minimum. Overall, our findings indicate that with appropriate storage and handling, RNA can be reliably isolated from human bone obtained at post-mortem and surgery to study ex vivo the pattern of gene expression in healthy individuals and in patients with musculoskeletal diseases such as osteoporosis and osteoarthritis.

Adult↗

PubMed2000

Stabilization of RNA tertiary structure by monovalent cations.

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.

Base Sequence↗

PubMed1986

Extensive postmortem stability of RNA from rat and human brain.

The postmortem stability of brain RNA was measured in rat and human samples for up to 48 hr. Whole rat brains, cooled at a rate approximating that of human brains awaiting autopsy, were collected at intervals from 0 to 48 hr after death and frozen. These samples were compared with freshly obtained, unfrozen rat brains. Two potentially independent characteristics of these RNA populations were measured: the recovery or yield of RNA/gram tissue (quantity) and the integrity or extent of degradation (quality). Total RNA yields were similar after all postmortem delays. Hybridization of [32P]-labeled cDNA probes to nitrocellulose filter blots of electrophoretically separated rat brain RNA failed to reveal degradation of the specific rat brain mRNAs during the postmortem period. Similarly, in vitro translation of these same rat total RNA samples produced high molecular weight translation products with no differences between long and short postmortem times. Human cerebral cortex RNA was prepared by the same methods as those used for rat brain from a neurosurgical sample and four other donors with postmortem intervals from 7 to 36 hr. Typically, the yield of total RNA from human brain was 40-50% of the yield from rat brain. When analyzed by RNA gel blot hybridization studies, as for rat brain RNA, human cortical RNA appeared slightly degraded. However, the degree of apparent RNA degradation was not related to the postmortem interval. In vitro translation products of human cortical RNA revealed high molecular weight peptides at all postmortem intervals, but slightly less [35S]incorporation into these bands was found at the longer postmortem intervals relative to the shorter times. Together, these results demonstrate an extensive stability of brain RNA that invites aggressive use of molecular genetic techniques for the study of human neurodegenerative diseases.

Animals↗

PubMed1997

Improved parameters for the prediction of RNA hairpin stability.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXANmAYCC, where XY is the set of four Watson-Crick base pairs and the underlined loop sequences are three to nine nucleotides. A nearest neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C is dependent upon loop size and closing base pair. The model previously developed to predict the stability for RNA hairpin loops (n > 3) includes contributions from the size of the loop, the identity of the closing base pair, the free energy increment (deltaGo(37mm)) for the interaction of the closing base pair with the first mismatch and an additional stabilization term for GA and UU first mismatches [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289]. The results presented here allow improvements in the parameters used to predict RNA hairpin stability. For hairpin loops of n = 4-9, deltaGo(37iL)(n) is 4.9, 5.0, 5.0, 5.0, 4.9, and 5.5 kcal/mol, respectively, and the penalty for hairpin closure by AU or UA is +0.6 kcal/mol. deltaGo(37iL)(n) is the free energy for initiating a loop of n nucleotides. The model for predicting hairpin loop stability for loops larger than three becomes deltaGo(37L)(n) = deltaGo(37iL)(n) + deltaGo(37mm) + 0.6(if closed by AU or UA) - 0.7(if first mismatch is GA or UU). Hairpin loops of three are modeled as independent of loop sequence with deltaGo(37iL)(3) = 4.8 and the penalty for AU closure of +0.6 kcal/mol. Thermodynamic parameters for hairpin formation in 1 M NaCl for 11 naturally occurring RNA hairpin sequences are reported. The model provides good agreement with the measured values for both T(M) (within 10 degrees C of the measured value) and deltaGo(37) (within 0.8 kcal/mol of the measured value) for hairpin formation. In general, the nearest neighbor model allows prediction of RNA hairpin stability to within 5-10% of the experimentally measured values.

Models, Chemical↗