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.     

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 hybridization properties of heterochiral nucleic acids

Heterochiral DNA and RNA heptamers, which contained an unnatural L-nucleotide, were synthesized, and thermodynamic analyses of their hybridization properties with complementary DNA and RNA strands were systematically conducted by UV melting experiments. The results clearly demonstrated that the incorporation of an L-ribonucleotide into the RNA strand leads to more significant destabilization of the duplexes than that of an L-deoxyribonucleotide into the DNA strand, regardless of whether the complementary strand is DNA or RNA. The destabilization of the duplexes by the substitution of D-thymidine with L-thymidine in the DNA strand is entropically driven, whereas that by the substitution of D-uridine with L-uridine in the RNA strand is enthalpically driven. The thermodynamic characteristic that the stability of homochiral duplex is far superior to that of heterochiral duplex is much more remarkable in RNA than in DNA. Thus, RNA might have been a self-replicating system superior to DNA to exclude the chiral antipode.     

Stereodefined phosphorothioate analogues of DNA: relative thermodynamic stability of the model PS-DNA/DNA and PS-DNA/RNA complexes

Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo-defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS-DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively.     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.     

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.     

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2′-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.     

Antisense recognition of the HER-2 mRNA: effects of phosphorothioate substitution and polyamines on DNA.RNA, RNA.RNA, and DNA.DNA duplex stability

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.     

Hydration of short DNA, RNA and 2'-OMe oligonucleotides determined by osmotic stressing

Studies on hydration are important for better understanding of structure and function of nucleic acids. We compared the hydration of self-complementary DNA, RNA and 2′-O-methyl (2′-OMe) oligonucleotides GCGAAUUCGC, (UA)₆ and (CG)₃ using the osmotic stressing method. The number of water molecules released upon melting of oligonucleotide duplexes, Δn_W, was calculated from the dependence of melting temperature on water activity and the enthalpy, both measured with UV thermal melting experiments. The water activity was changed by addition of ethylene glycol, glycerol and acetamide as small organic co-solutes. The Δn_W was 3–4 for RNA duplexes and 2–3 for DNA and 2′-OMe duplexes. Thus, the RNA duplexes were hydrated more than the DNA and the 2′-OMe oligonucleotide duplexes by approximately one to two water molecules depending on the sequence. Consistent with previous studies, GC base pairs were hydrated more than AU pairs in RNA, whereas in DNA and 2′-OMe oligonucleotides the difference in hydration between these two base pairs was relatively small. Our data suggest that the better hydration of RNA contributes to the increased enthalpic stability of RNA duplexes compared with DNA duplexes.     

Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region

The structural properties of a DNA/RNA duplex having a pyrene residue at the 5′ end of DNA and a G-rich single strand region at the 3′ end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration.     

CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution.

Circular dichroism (CD) spectra and melting temperature (Tm) data for five duplexes containing phosphorothioate linkages were compared with data for four unmodified duplexes to assess the effect of phosphorothioate modification on the structure and stability of DNA. DNA and DNA.RNA duplexes. Nine duplexes were formed by mixing oligomers 24 nt long in 0.15 M K+(phosphate buffer), pH 7.0. Unmodified DNA.DNA and RNA.RNA duplexes were used as reference B-form and A-form structures. The CD spectra of the modified hybrids S-d(AC)12.r(GU)12 and r(AC)12.S-d(GT)12 differed from each other but were essentially the same as the spectra of the respective unmodified hybrids. They were more A-form than B-form in character. CD spectra of duplexes S-d(AC)12.d(GT)12 and d(AC)12.S-d(GT)12 were similar to that of d(AC)12.d(GT)12, except for a reduced long wavelength CD band. Sulfur modifications on both strands of the DNA duplex caused a pronounced effect on its CD spectrum. The order of thermal stability was: RNA.RNA > DNA.DNA > DNA.RNA > S-DNA.DNA > S-DNA. RNA > S-DNA.S-DNA. Phosphorothioation of one strand decreased the melting temperature by 7.8+/-0.6 degrees C, regardless of whether the substitution was in a hybrid or DNA duplex. Thermodynamic parameters were obtained from a multistate analysis of the thermal melting profiles. Interestingly, the destabilizing effect of the phosphorothioate substitution appears to arise from a difference in the entropy upon forming the DNA.DNA duplexes, while the destabilizing effect in the DNA.RNA hybrids appears to come from a difference in enthalpy.     

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.     

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.     

Recognition of nucleic acid double helices by homopyrimidine 2', 5'-linked RNA.

We have studied the effect of a 2',5'-RNA third strand backbone on the stability of triple helices with a 'pyrimidine motif' targeting the polypurine strand of duplex DNA, duplex RNA and DNA/RNA hybrids. Comparative experiments were run in parallel with DNA and the regioisomeric RNA as third strands adopting the experimental design of Roberts and Crothers. The results reveal that 2',5'-RNA is indeed able to recognize double helical DNA (DD) and DNA (purine):RNA (pyrimidine) hybrids (DR). However, when the duplex purine strand is RNA and the duplex pyrimidine strand is DNA or RNA (i.e. RD or RR), triplex formation is not observed. These results exactly parallel what is observed for DNA third strands. Based on T m data, the affinities of 2',5'-RNA and DNA third strands towards DD and DR duplexes were similar. The RNA third strand formed triplexes with all four hairpins, as previously demonstrated. In analogy to the arabinose and 2'-deoxyribose third strands, the possible C2'- endo pucker of 2',5'-linked riboses together with the lack of an alpha-2'-OH group are believed to be responsible for the selective binding of 2',5'-RNA to DD and DR duplexes, over RR and RD duplexes. These studies indicate that the use of other oligonucleotide analogues will prove extremely useful in dissecting the contributions of backbone and/or sugar puckering to the recognition of nucleic acid duplexes.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.     

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

Relative flexibility of DNA and RNA: a molecular dynamics study

State of the art molecular dynamics simulations are used to study the structure, dynamics, molecular interaction properties and flexibility of DNA and RNA duplexes in aqueous solution. Special attention is paid to the deformability of both types of structures, revisiting concepts on the relative flexibility of DNA and RNA duplexes. Our simulations strongly suggest that the concepts of flexibility, rigidity and deformability are much more complex than usually believed, and that it is not always true that DNA is more flexible than RNA.