Stability Of RNA Duplexes Research Articles

Improved Model for Predicting the Free Energy Contribution of Dinucleotide Bulges to RNA Duplex Stability.

Predicting the secondary structure of RNA is an intermediate in predicting RNA three-dimensional structure. Commonly, determining RNA secondary structure from sequence uses free energy minimization and nearest neighbor parameters. Current algorithms utilize a sequence-independent model to predict free energy contributions of dinucleotide bulges. To determine if a sequence-dependent model would be more accurate, short RNA duplexes containing dinucleotide bulges with different sequences and nearest neighbor combinations were optically melted to derive thermodynamic parameters. These data suggested energy contributions of dinucleotide bulges were sequence-dependent, and a sequence-dependent model was derived. This model assigns free energy penalties based on the identity of nucleotides in the bulge (3.06 kcal/mol for two purines, 2.93 kcal/mol for two pyrimidines, 2.71 kcal/mol for 5'-purine-pyrimidine-3', and 2.41 kcal/mol for 5'-pyrimidine-purine-3'). The predictive model also includes a 0.45 kcal/mol penalty for an A-U pair adjacent to the bulge and a -0.28 kcal/mol bonus for a G-U pair adjacent to the bulge. The new sequence-dependent model results in predicted values within, on average, 0.17 kcal/mol of experimental values, a significant improvement over the sequence-independent model. This model and new experimental values can be incorporated into algorithms that predict RNA stability and secondary structure from sequence.

Investigation of the effect of amino acid chirality in the internucleoside linker on DNA:DNA and DNA:RNA duplex stability

Enzymatically and chemically stable amide-linked di/oligonucleosides are highly desired synthetic targets in which the phosphodiester linkages in native DNA are replaced by amide linkers of appropriate length and stereochemistry. The five-atom amide-linked dimers, synthesized from 3′-amino-3′-deoxy thymidine, α-(l/d) proline/prochiral glycine and thymidine/uridine-4′carboxylic acid derivatives, were incorporated into the DNA backbone to achieve partial replacement of selected phosphodiester linkages. The results stressed the importance of the chirality of linker amino acid. d-Proline was found to be the most compatible as an internucleoside linker in the DNA backbone to stabilize the complexes with DNA or RNA as compared to l-proline and glycine.

2-Thiouracil deprived of thiocarbonyl function preferentially base pairs with guanine rather than adenine in RNA and DNA duplexes.

2-Thiouracil-containing nucleosides are essential modified units of natural and synthetic nucleic acids. In particular, the 5-substituted-2-thiouridines (S2Us) present in tRNA play an important role in tuning the translation process through codon–anticodon interactions. The enhanced thermodynamic stability of S2U-containing RNA duplexes and the preferred S2U-A versus S2U-G base pairing are appreciated characteristics of S2U-modified molecular probes. Recently, we have demonstrated that 2-thiouridine (alone or within an RNA chain) is predominantly transformed under oxidative stress conditions to 4-pyrimidinone riboside (H2U) and not to uridine. Due to the important biological functions and various biotechnological applications for sulfur-containing nucleic acids, we compared the thermodynamic stabilities of duplexes containing desulfured products with those of 2-thiouracil-modified RNA and DNA duplexes. Differential scanning calorimetry experiments and theoretical calculations demonstrate that upon 2-thiouracil desulfuration to 4-pyrimidinone, the preferred base pairing of S2U with adenosine is lost, with preferred base pairing with guanosine observed instead. Therefore, biological processes and in vitro assays in which oxidative desulfuration of 2-thiouracil-containing components occurs may be altered. Moreover, we propose that the H2U-G base pair is a suitable model for investigation of the preferred recognition of 3′-G-ending versus A-ending codons by tRNA wobble nucleosides, which may adopt a 4-pyrimidinone-type structural motif.

Dangling ends perturb the stability of RNA duplexes responsive to surrounding conditions.

Unpaired terminal nucleotides (dangling ends) occur in various biologically important RNA structures. We studied the thermal stability of RNA duplexes with dangling ends under conditions that mimic those in cells. Dangling ends of one or two nucleotides stabilized a duplex up to approximately 2.7 kcal mol(-1) in the absence of cosolutes. RNA duplexes with dangling purine nucleotides were more stable than those with pyrimidine nucleotides. Interestingly, in the presence of various cosolutes, RNA duplexes with purine dangling ends were significantly destabilized, although those with pyrimidine dangling ends were destabilized slightly. For example, in 30 wt % poly(ethylene glycol), stabilization resulting from adenine dangling ends was reduced by 1.4 kcal mol(-1) . Our quantitative analyses also showed that the number of water molecules bound to the dangling ends in an aqueous solution was independent of the nucleotide type but dependent on the stability of the dangling-end region. It has been considered that dangling ends stabilize helices; however, our results suggest that the stabilization is responsive to the surrounding conditions.

Improved model to predict the free energy contribution of trinucleotide bulges to RNA duplex stability.

Trinucleotide bulges in RNA commonly occur in nature. Yet, little data exists concerning the thermodynamic parameters of this motif. Algorithms that predict RNA secondary structure from sequence currently attribute a constant free energy value of 3.2 kcal/mol to all trinucleotide bulges, regardless of bulge sequence. To test the accuracy of this model, RNA duplexes that contain frequent naturally occurring trinucleotide bulges were optically melted, and their thermodynamic parameters—enthalpy, entropy, free energy, and melting temperature—were determined. The thermodynamic data were used to derive a new model to predict the free energy contribution of trinucleotide bulges to RNA duplex stability: ΔG°37, trint bulge = ΔG°37, bulge + ΔG°37, AU + ΔG°37, GU. The parameter ΔG°37, bulge is variable depending upon the purine and pyrimidine composition of the bulge, ΔG°37, AU is a 0.49 kcal/mol penalty for an A-U closing pair, and ΔG° 37, GU is a −0.56 kcal/mol bonus for a G-U closing pair. With both closing pair and bulge sequence taken into account, this new model predicts free energy values within 0.30 kcal/mol of the experimental value. The new model can be used by algorithms that predict RNA free energies as well as algorithms that use free energy minimization to predict RNA secondary structure from sequence.

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

The mixture of 2'-5' and 3'-5' linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2'-5' linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2'-5' linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2'-linked sugars were in the expected 2'-endo conformation, some were partially or fully in the 3'-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2'-5' linkages. Our results contribute to the view that a low level of 2'-5' substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.

Effect of Sodium Ions on RNA Duplex Stability

The standard sodium concentration for RNA optical melting experiments is 1.021 M. Algorithms that predict Tm, ΔG°37, and secondary structure from sequence generally rely on parameters derived from optical melting experiments performed in 1.021 M sodium. Physiological monovalent cation concentrations are much lower than 1.021 M. In fact, many molecular biology techniques require buffers containing monovalent cation concentrations other than 1.021 M. Predictions based on the 1.021 M Na(+) parameters may not be accurate when the monovalent cation concentration is not 1.021 M. Here, we report thermodynamic data from optical melting experiments for a set of 18 RNA duplexes, each melted over a wide range of sodium ion concentrations (71, 121, 221, and 621 mM). Using these data and previously published data for the same sequences melted in 1.021 M Na(+), we report Tm and ΔG°37 correction factors to scale the standard 1.021 M Na(+) RNA parameters to other sodium ion concentrations. The recommended Tm correction factor predicts the melting temperature within 0.7 °C, and the recommended ΔG°37 correction factor predicts the free energy within 0.14 kcal/mol. These correction factors can be incorporated into prediction algorithms that predict RNA secondary structure from sequence and provide Tm and ΔG°37 values for RNA duplexes.

Effects of Salt, Polyethylene Glycol, and Locked Nucleic Acids on the Thermodynamic Stabilities of Consecutive Terminal Adenosine Mismatches in RNA Duplexes

Consecutive terminal mismatches add thermodynamic stability to RNA duplexes and occur frequently in microRNA-mRNA interactions. Accurate thermodynamic stabilities of consecutive terminal mismatches contribute to the development of specific, high-affinity siRNA therapeutics. Consecutive terminal adenosine mismatches (TAMS) are studied at different salt concentrations, with polyethylene glycol cosolutes, and with locked nucleic acid (LNA) substitutions. These measurements provide benchmarks for the application of thermodynamic predictions to different physiological or therapeutic conditions. The salt dependence for RNA duplex stability is similar for TAMS, internal loops, and Watson-Crick duplexes. A unified model for predicting the free energy of an RNA duplex with or without loops and mismatches at lower sodium concentrations is presented. The destabilizing effects of PEG 200 are larger for TAMS than internal loops or Watson-Crick duplexes, which may result from different base stacking conformations, dynamics, and water hydration. In contrast, LNA substitutions stabilize internal loops much more than TAMS. Surprisingly, the average per adenosine increase in stability for LNA substitutions in internal loops is -1.82 kcal/mol and only -0.20 kcal/mol for TAMS. The stabilities of TAMS and internal loops with LNA substitutions have similar favorable free energies. Thus, the unfavorable free energy of adenosine internal loops is largely an entropic effect. The favorable stabilities of TAMS result mainly from base stacking. The ability of RNA duplexes to form extended terminal mismatches in the absence of proteins such as argonaute and identifying the enthalpic contributions to terminal mismatch stabilities provide insight into the physical basis of microRNA-mRNA molecular recognition and specificity.

Effect of Guanine to Inosine Substitution on Stability of Canonical DNA and RNA Duplexes: Molecular Dynamics Thermodynamics Integration Study

Guanine to inosine (G → I) substitution has often been used to study various properties of nucleic acids. Inosine differs from guanine only by loss of the N2 amino group, while both bases have similar electrostatic potentials. Therefore, G → I substitution appears to be optimally suited to probe structural and thermodynamics effects of single H-bonds and atomic groups. However, recent experiments have revealed substantial difference in free energy impact of G → I substitution in the context of B-DNA and A-RNA canonical helices, suggesting that the free energy changes reflect context-dependent balance of energy contributions rather than intrinsic strength of a single H-bond. In the present study, we complement the experiments by free energy computations using thermodynamics integration method based on extended explicit solvent molecular dynamics simulations. The computations successfully reproduce the basic qualitative difference in free energy impact of G → I substitution in B-DNA and A-RNA helices although the magnitude of the effect is somewhat underestimated. The computations, however, do not reproduce the salt dependence of the free energy changes. We tentatively suggest that the different effect of G → I substitution in A-RNA and B-DNA may be related to different topologies of these helices, which affect the electrostatic interactions between the base pairs and the negatively charged backbone. Limitations of the computations are briefly discussed.

2′-modified oligoribonucleotides containing 1,2-diol and aldehyde groups. Synthesis and properties

1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.

Non-specific binding of Na + and Mg 2+ to RNA determined by force spectroscopy methods

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na+]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg2+ salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

Synthesis of N2-Alkyl-8-oxo-7,8-dihydro-2′-deoxyguanosine Derivatives and Effects of These Modifications on RNA Duplex Stability

N(2)-alkyl analogues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N(2)-alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA-protein interactions in the minor vs major grooves.

Positional and Neighboring Base Pair Effects on the Thermodynamic Stability of RNA Single Mismatches

Many naturally occurring RNA structures contain single mismatches, many of which occur near the ends of helices. However, previous thermodynamic studies have focused their efforts on thermodynamically characterizing centrally placed single mismatches. Additionally, algorithms currently used to predict secondary structure from sequence are based on two assumptions for predicting the stability of RNA duplexes containing this motif. It has been assumed that the thermodynamic contribution of small RNA motifs is independent of both its position in the duplex and the identity of the non-nearest neighbors. Thermodynamically characterizing single mismatches three nucleotides from both the 3' and 5' ends (i.e., off-center) of an RNA duplex and comparing these results to those of the same single mismatch-nearest neighbor combination centrally located have allowed for the investigation of these effects. The thermodynamic contributions of 13 single mismatch-nearest neighbor combinations are reported, but only nine combinations are studied at all three duplex positions and are used to determine trends and patterns. In general, the 5'- and 3'-shifted single mismatches are relatively similar, on average, and more favorable in free energy than centrally placed single mismatches. However, close examination and comparison shows there are several associated idiosyncrasies with these identified general trends. These peculiarities may be due, in part, to the identities of the single mismatch, the nearest neighbors, and the non-nearest neighbors, along with the effects of the single mismatch position in the duplex. The prediction algorithm recently proposed by Davis and Znosko [Davis, A. R., and Znosko, B. M. (2008) Biochemistry 47, 10178-10187] is used to predict the thermodynamic parameters of single mismatch contribution, and those values are compared to the measured values presented here. This comparison suggests the proposed model is a good approximation but could be improved by the addition of parameters that account for positional and/or non-nearest neighbor effects. However, more data are required to improve our understanding of these effects and to accurately account for them.

Optimization of Duplex Stability and Terminal Asymmetry for shRNA Design

Prediction of efficient oligonucleotides for RNA interference presents a serious challenge, especially for the development of genome-wide RNAi libraries which encounter difficulties and limitations due to ambiguities in the results and the requirement for significant computational resources. Here we present a fast and practical algorithm for shRNA design based on the thermodynamic parameters. In order to identify shRNA and siRNA features universally associated with high silencing efficiency, we analyzed structure-activity relationships in thousands of individual RNAi experiments from publicly available databases (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/). Using this statistical analysis, we found free energy ranges for the terminal duplex asymmetry and for fully paired duplex stability, such that shRNAs or siRNAs falling in both ranges have a high probability of being efficient. When combined, these two parameters yield a ∼72% success rate on shRNAs from the siRecords database, with the target RNA levels reduced to below 20% of the control. Two other parameters correlate well with silencing efficiency: the stability of target RNA and the antisense strand secondary structure. Both parameters also correlate with the short RNA duplex stability; as a consequence, adding these parameters to our prediction scheme did not substantially improve classification accuracy. To test the validity of our predictions, we designed 83 shRNAs with optimal terminal asymmetry, and experimentally verified that small shifts in duplex stability strongly affected silencing efficiency. We showed that shRNAs with short fully paired stems could be successfully selected by optimizing only two parameters: terminal duplex asymmetry and duplex stability of the hypothetical cleavage product, which also relates to the specificity of mRNA target recognition. Our approach performs at the level of the best currently utilized algorithms that take into account prediction of the secondary structure of the target and antisense RNAs, but at significantly lower computational costs. Based on this study, we created the si-shRNA Selector program that predicts both highly efficient shRNAs and functional siRNAs (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/).

Contributions of Stacking, Preorganization, and Hydrogen Bonding to the Thermodynamic Stability of Duplexes between RNA and 2′-O-Methyl RNA with Locked Nucleic Acids

Locked nucleic acids (LNA) considerably enhance the thermodynamic stability of DNA and RNA duplexes. We report the thermodynamic stabilities of LNA-2'-O-methyl RNA/RNA duplexes designed to provide insight into the contributions of stacking and hydrogen bonding interactions to the enhanced stability. The results show that hydrogen bonding of LNA nucleotides is similar to that of 2'-O-methyl RNA nucleotides, whereas the 3'-stacking interactions are on average approximately 0.7 kcal/mol more favorable at 37 degrees C than for 2'-O-methyl or RNA nucleotides. Moreover, NMR spectra suggest helical preorganization of the single-stranded tetramer, C(L)A(M)A(L)U(M), probably due to restriction of some torsion angles. Thus, enhanced stacking interactions and helical preorganization of single-stranded oligonucleotides contribute to the extraordinary stabilization of duplexes by LNA nucleotides.

Probing the Transcription Mechanisms of Reovirus Cores with Molecules That Alter RNA Duplex Stability

The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps.

Establishing a Mechanistic Basis for the Large Kinetic Steps of the NS3 Helicase

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to approximately 11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.

A locked derivative of 8-aza-7-deazaadenosine

The title compound [systematic name: (1S,3S,4R,7S)-3-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-hydroxymethyl-2,5-dioxabicyclo[2.2.1]heptan-7-ol], C(11)H(13)N(5)O(4), belongs to a family of nucleosides with modifications in both the sugar and nucleobase moieties: these modifications are known to increase the thermodynamic stability of DNA and RNA duplexes. There are two symmetry-independent molecules in the asymmetric unit that differ significantly in conformation, and both exhibit a high-anti conformation about the N-glycosidic bond, with chi torsion angles of -85.4 (3) and -87.4 (3) degrees . The sugar C atom attached to the nucleobase N atom is -0.201 (4) and 0.209 (4) A from the 8-aza-7-deazaadenine skeleton plane in the two molecules. The molecules are assembled into layers via hydrogen bonds and pi-pi stacking interactions between the modified nucleobases.

Different Strategies for the Synthesis of 2′- O -Aminoethyl Adenosine Building Blocks

The chemical modification of the 2′-O-position of nucleosides proved to be of great importance for the RNA stability. Greater stability of RNA duplexes allows a longer half life in the cell and, therefore, a better effect of RNA Interference. Here we investigated the synthesis of 2′-O-aminoethyl adenosine as a cationic modified building block.