15-mer DNA duplexes containing an abasic site are thermodynamically more stable with adjacent purines than with pyrimidines

Abasic site (AP)-containing duplexes, with flanking adenine (A) or cytosine (C) bases, were shown to be more stable with flanking A than with C bases [Sági, J., Hang, B., and Singer, B. (1999) Chem. Res. Toxicol. 12, 917-923]. We investigated whether the lower-magnitude destabilization by an AP site, with A neighbors, is a general effect of the purine versus the pyrimidine neighbors. Duplex stability, as compared to that of the corresponding control duplexes, was markedly decreased by the incorporation of the AP site (x) opposite any of the four bases. However, for the duplexes containing T, A, or C opposite the AP site, replacement of the symmetric doublet flanking pyrimidine bases with purines resulted in a smaller destabilization effect. The average stabilizing effect of the symmetric doublet purine neighbors of an AP site opposite T, A, or C bases was 3.2 degrees C (DeltaT(m)) and 1.3 kcal/mol (DeltaDeltaG degrees (37)) compared to those of pyrimidine neighbors. In contrast, a G.AP pair reduced or eliminated the differential effect of the neighbors. Using unrestrained molecular dynamics, it was shown that for the duplexes containing T opposite the AP site, with doublet pyrimidine neighbors, there was a larger magnitude of curvature around the lesion site than for the duplexes with the purines flanking the AP site. Purines flanking the AP site tend to shift toward each other, creating overlap, in contrast to the flanking pyrimidines. This indicates the possibility of stacking between purine bases at the AP site and can be the reason for the observed smaller thermodynamic destabilization of the duplexes with the AAxAA and GGxGG central sequences, as compared to those with TTxTT and CCxCC sequences. This work showed that for an AP site the GC content is not the only determinant of duplex stability, but rather is influenced more by whether purines or pyrimidines flank the AP site.     

The conformational study of two carbocyclic nucleosides: why carbocyclic nucleic acids (CarNAs) form more stable duplexes with RNA than DNA does

Replacement of the furanose moiety of DNA with a cyclopentane ring produces a modified sugar analogue: Carbocyclic nucleic acid (CarNA). UV melting-temperature experiments demonstrate that the incorporation of 2'-deoxycarbaguanosine ((c)G) and 2'-deoxyaristeromycin ((c)A) of carbocyclic nucleosides into a DNA strand increases the stability of the CarNA/RNA hybrid. Circular Dichroism (CD) study indicates that the CarNA/RNA hybrid adopts an A-like conformation. To elucidate the molecular basis of the increased stability of the CarNA/RNA, the conformation of (c)G and (c)A were examined by (1)H NMR conformational analysis of (3)J(HH) coupling constants and ab initio molecular orbital (MO) calculations. These results show that the populations of N-type of (c)G and (c)A are higher than those of dG and dA, respectively, at different temperatures [For example, 37% (N%) of (c)G vs. 28%of dG, 36% (N%) of (c)A vs. 25% of dA at 278 K], which suggest that the cyclopentane rings of (c)G and (c)A prefer the N-type conformation in two-state N-S pseudorotional equilibrium in comparison with the furanose rings of dG and dA. The DeltaH degrees of (c)G (DeltaH degrees = - 0.43 kcal mol(-1)) and (c)A (DeltaH degrees = - 0.41kcal mol(-1)) are lower than that of dG (dG = - 1.8 kcal mol(-1)) and dA (dA = - 1.0 kcal mol(-1)), respectively, which suggest that the gauche effect in the (c)A and (c)G driving N-S pseudorotional equilibrium to S-type is reduced by replacement of the 4'-oxygen by a CH(2) group. These results suggest that the preferred N-type of the (c)G and (c)A leads to the A-like conformation, which contributes to the stability of CarNA/RNA hybrid.     

Derivation of nearest-neighbor properties from data on nucleic acid oligomers. II. Thermodynamic parameters of DNA.RNA hybrids and DNA duplexes

Using nearest-neighbor models consisting of independent short sequence combinations of nearest neighbors (ISS models), values of thermodynamic parameters for sets of independent sequences are derived from published oligomer data for DNA.RNA hybrids [N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi, M. Yoneyama, and M. Sasaki (1995) Biochemistry, Vol. 34, pp. 11211-11216] and dsDNA duplexes [J. SantaLucia, Jr., H. T. Allawi, and P. A. Seneviratne (1996) Biochemistry, Vol. 35, pp. 3555-3562]. The results are compared with those from models that assign values of thermodynamic parameters to individual nearest neighbors (INN models). Differences in the use of ISS and INN models are also illustrated in an appendix, which shows examples of analyses for values of a fictitious nearest-neighbor property. INN models that include an initiation parameter contain an implicit assumption that combinations of end neighbors have the same value of a property. It is found that combinations of end neighbors (e.g., base pairs neighboring solvent) in oligomers can have significant and different apparent values of thermodynamic properties, so that the assumption inherent in INN models is not always correct. Even though ISS models do not allow the assignment of values to individual nearest neighbors, except for the like neighbors [such as d(AA)/r(UU), etc., for hybrids and d(AA)/d(TT) and d(GG)/d(CC) for DNA duplexes], they do provide physically meaningful values for the like neighbors, for sequence combinations, and for specified combinations of end neighbors.     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.     

Thermodynamic study of internal loops in oligoribonucleotides: symmetric loops are more stable than asymmetric loops.

Thermodynamic parameters for internal loops of unpaired adenosines in oligoribonucleotides have been measured by optical melting studies. Comparisons are made between helices containing symmetric and asymmetric loops. Asymmetric loops destabilize a helix more than symmetric loops. The differences in free energy between symmetric and asymmetric loops are roughly half the magnitude suggested from a study of parameters required to give accurate predictions of RNA secondary structure [Papanicolaou, C., Gouy, M., & Ninio, J. (1984) Nucleic Acids Res. 12, 31-44]. Circular dichroism spectra indicate no major structural difference between helices containing symmetric and asymmetric loops. The measured sequence dependence of internal loop stability is not consistent with approximations used in current algorithms for predicting RNA secondary structure.     

DNA gelation in concentrated solutions

Sonicated calf-thymus DNA (200 ± 30 base pairs) spontaneously forms viscoelastic gels over a wide range of concentration, temperature, and buffer conditions. Quasielastic light scattering (QLS) can be used to monitor this process, because the ratio of dynamic-to-static scattering intensity decreases dramatically as gelation occurs. Using QLS, we have explored the effects of DNA concentration and mono- and divalent cations on the thermal stability of DNA gels. We found that the gel–sol transition temperature (T_gel) varies linearly with [DNA] from 7.5 to 17 mg/mL. Both Na⁺ and Mg²⁺ strongly stabilize the gel state. The sharpness of the transition increases with increasing ionic and DNA concentrations. Analysis of the Na⁺-dependent gelation indicates that the process requires the association of one Na⁺ per 118 base pairs. Mg²⁺ effectively stabilizes the gel at concentrations 10-fold below those required for Na⁺. The unexpectedly large effect of Mg²⁺ suggests that ion-specific interactions may play an important role in determining gel stability.     

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen‐associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral‐derived sequences efficiently induce pro‐inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88‐dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.     

Structural studies of heterochiral DNA/DNA, RNA/RNA, and DNA/RNA duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation.

Nuclear magnetic resonance experiments reveal that the base H8/H6 protons of oligoribonucleotides (RNA) have T1 relaxation times that are distinctly longer than those of oligodeoxyribonucleotides (DNA). Similarly, the T1 values for the RNA H1' protons are approximately twice those of the corresponding DNA H1' protons. These relaxation differences persist in single duplexes containing covalently linked RNA and DNA segments and cause serious overestimation of distances involving RNA protons in typical NOESY spectra collected with a duty cycle of 2-3 s. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. The difference in local proton density is the major cause of the longer nonselective T1s of RNA compared to DNA, although small differences in internal motion cannot be completely ruled out. Fortunately, any internal motion differences that might exist are shown to be too small to affect cross-relaxation rates, and therefore reliable distance data can be obtained from time-dependent NOESY data sets provided an adequately long relaxation delay is used. In hybrid or chimeric RNA-DNA duplexes, if the longer RNA relaxation times are not taken into account in the recycle delay of NOESY pulse sequences, serious errors in measuring RNA proton distances are introduced.     

Alpha are more stable than beta anomer oligonucleotides in 3T3 cellular extracts

We have compared the stability of alpha and beta anomeric oligonucleotides in NIH 3T3 cellular extracts. We had already shown that alpha are much more resistant than beta oligonucleotides towards purified nucleases. This result is confirmed when using cellular extracts although the difference is smaller. When alpha molecules are combined with an intercalating agent binding in the 3' position a synergistic increase of resistance to degradation is observed.     

Very stable mismatch duplexes: structural and thermodynamic studies on tandem G.A mismatches in DNA.

We have used ultraviolet melting techniques to compare the stability of several DNA duplexes containing tandem G.A mismatches to similar duplexes containing tandem A.G, I.A, and T.A base pairs. We have found that tandem G.A mismatches in 5'-Y-G-A-R-3' duplexes are more stable than their I.A counterparts and that they are sometimes more stable than tandem 5'-Y-T-A-R-3' sequences. This is not the case for tandem G.A mismatches in other base stacking environments, and it suggests that tandem G.A mismatches in 5'-Y-G-A-R-3' sequences have a unique configuration. In contrast to tandem 5'-G-A-3' mismatches, tandem 5'-A-G-3' mismatches were found to be unstable in all cases examined.     

Holliday junctions are formed in concentrated solutions of purified products of DNA amplification

Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.     

Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide.

The thermal stabilities of RNA:DNA hybrids are substantially greater than those of DNA:DNA duplexes in aqueous electrolyte solutions containing high concentrations of formamide. Association rates to form DNA:DNA duplexes and DNA:RNA hybrids have been measured in these solvents. There is a temperature range in which DNA:DNA rates are negligible and RNA:DNA rates close to optimal.     

RNA polymerases B and C are more closely related to each other than to RNA polymerase A

Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements.     

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.     

Initiated complexes of RNA polymerase II are concentrated in the nuclear skeleton associated DNA

Several preparations of nuclear matrices containing varying amounts of DNA were obtained from mouse plasmocytoma P3-X63-Ag8.653 cells and tested for the presence of RNA polymerase II activity. It has been demonstrated that about 25% of RNA polymerase II activity detected in the original nuclei can be recovered in isolated nuclear matrices. Only DNA-bound RNA polymerase II was found in the isolated matrices, while both free and DNA-bound RNA polymerase II activities were detected in the original nuclei. RNA polymerase II activity found in the isolated matrices did not depend on the portion of DNA recovered in the nuclear matrices in a large interval between 91 and 1.5% of DNA content in the original nuclei. The conclusion has been drawn that initiated RNA polymerase II molecules are non-randomly distributed along DNA loops. They are concentrated near the points of DNA attachment to the nuclear skeleton.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.