Thermodynamic and structural characterization of 2'-nitrogen-modified RNA duplexes

2′-aminonucleosides are commonly used as sites of post-synthetic chemical modification within nucleic acids. As part of a larger cross-linking strategy, we appended alkyl groups onto the N2′ position of 2′-amino-modified RNAs via 2′-ureido and 2′-amido linkages. We have characterized the thermodynamics of 2′-amino, 2′-alkylamido and 2′-alkylureido-modified RNA duplexes and show that 2′-ureido-modified RNAs are significantly more stable than analogous 2′-amido-modified RNAs. Using NMR spectroscopy and NMR-based molecular modeling of 2′-modified RNA duplexes, we examined the effects that 2′-nitrogen modifications have on RNA helices. Our data suggest that the 2′-ureido group forms a specific intra-nucleoside interaction that cannot occur within 2′-amido-modified helices. These results indicate that 2′-ureido modifications are superior to analogous 2′-amido ones for applications that require stable base pairing.     

DNA, RNA and hybrid RNA-DNA oligomers of identical sequence: structural and dynamic differences

A 27-mer sequence was synthesised as DNA duplex (DD), RNA duplex (RR), and RNA-DNA (RD) hybrid in order to characterise their structural and dynamic features. The hydrodynamic radius (Rh) and the rise (b) values of the three samples were consistent with the conformations predicted by CD analysis. The value of the torsional constant (alpha) of the samples containing RNA was approximately twice that of the DD sample and followed the order: DD < RD < RR. The same order was observed in the thermodynamic stability and in the reduction of the electrophoretic mobility. gamma-Ray footprinting analysis was carried out to resolve the individual strand conformation in the hybrid. The RNA strand preserved its conformation, while the DNA strand showed local deformations mainly at TA and TG steps.     

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.     

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.     

Local base dynamics and local structural features in RNA and DNA duplexes

Local base motion and local structural base information are derived with a simple motional model from site specifically spin-labeled polyribo- and polydeoxyribonucleotides. The model was developed earlier for some nucleic acids and has now been applied to analyze 22 different nucleic acid systems. We conclude that the base motion of the spin-labeled nucleotide in single-stranded RNA, DNA, or non-base-paired bases in duplexes is of the order of 1 ns and that its base mobility decreases by about a factor of 4 upon base pairing. Also, the tether motion of the probe is slower in an RNA than in a DNA duplex.     

Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes.

The electrophoretic behavior of defined DNA and RNA oligonucleotide duplexes from 10 to 20 bp in length has been investigated as a function of salt conditions, gel concentration, and temperature. The RNA oligomers migrated much more slowly than the DNA oligomers of the same sequence under all conditions. From sedimentation equilibrium and velocity measurements, the apparent partial specific volume in 0.1 M KCI, 20 mM NaPi, pH 7, was determined as 0.56 +/- 0.015 ml g(-1) for DNA and 0.508 ml g(-1) for RNA. The translational friction coefficients were determined and compared with the values calculated for cylinders. Taking into account the shape factors, the solution density, and partial specific volumes, the effective degree of hydration was estimated as 0.8-1 g g(-1) DNA. There was no significant difference in the frictional coefficients of the DNA and RNA oligomers, indicating that the effective sizes of DNA and RNA are very similar in solution. The differential electrophoretic mobility of DNA and RNA must arise from the differences in interaction with counterions, which is probably a global property of the oligonucleotides.     

The sequence dependence of circular dichroism spectra of DNA duplexes.

The three satellite DNAs of Drosophila virilis, that approximate to poly d(CAAACTA)-poly d(TAGTTTG), poly d(TAAACTA)-poly d(TAGTTTA), poly d(CAAATTA)-poly d(TAATTTG), the satellite DNA of Drosophila melanogaster that approximates to poly d(AATAT)-poly d(ATATT), the synthetic DNA duplexes, poly dG-poly dC, poly d(AT)-poly d(AT), poly d(AAT)-poly d(ATT), poly d(AAC)-poly d(GTT), poly d(TAC)-poly d(GTA) and the block copolymer d(C15A15)-d(T15G15) all have circular dichroism spectra consistent with the propositions that they have the same molecular geometry in solution and that it is the kind and frequency of nucleotide triplet sequences that determines their spectral characteristics. Poly dA-poly dT is apparently an exception.     

Thermodynamic and kinetic properties of short RNA helices: the oligomer sequence AnGCUn

We studied the thermodynamic, kinetic and optical properties of the double helices formed by the series of self-complementary oligonucleotides, (AP)_nGpC(pU)_n, 2 ≤ n ≤ 4, and found that the shortest helix, containing just 6 base pairs, is less stable than would be predicted from the properties of the larger molecules. It also shows a markedly smaller hyperchromism on melting than expected. These anomalous properties of a short helix indicate that one cannot always assume that base pair free energies and extinction coefficient changes are independent of helix size.     

Thermodynamic, thermomechanical, and structural properties of a hydrated asymmetric phosphatidylcholine.

1-Behenyl-2-lauryl-sn-glycero-3-phosphocholine (22/12 PC) belongs to a unique group of phospholipids in which the molecule has one acyl chain almost twice as long as the other. The temperature-composition phase diagram for this lipid in the range of 25-65 degrees C, and 0 to 84.3% (w/w) water has been constructed by using the isoplethal method in the heating direction and x-ray diffraction for phase identification and structure characterization. At water contents between 10.3 and 34% (w/w) and at temperatures below 43 degrees C, a single mixed interdigitated lamellar gel phase (Lm beta, [symbol: see text]) of the type described by Hui et al. (1984. Biochemistry. 23:5570-5577) and McIntosh et al. (1984. Biochemistry. 23:4038-4044) was found. A second phase consisting of bulk aqueous solution coexists with the Lm beta phase at hydration levels above 34% (w/w) water in the temperature range between 25 and 43 degrees C. Above 43 degrees C, a partially interdigitated lamellar liquid crystalline (Lp alpha) phase ([symbol: see text]) is seen in the water concentration range extending from 0 to 84.3% (w/w). The pure Lp alpha phase is found below 43% (w/w) water, while coexistence of the Lp alpha phase and the bulk aqueous solution is observed above this water concentration which marks the hydration boundary. Interestingly, the latter boundary for both Lm beta and Lp alpha phases is nearly vertical in the temperature range studied. Furthermore, the lamellar chain-melting transition temperature appears to be relatively insensitive to hydration in the range 0-85% (w/w) water. We have confirmed the identify of the Lm beta phase by constructing a 5.7-A resolution electron density profile on oriented samples by the swelling method. Temperature-induced chain melting effects an increase in lipid bilayer thickness suggesting that the Lp alpha phase has chains packed in the partially as opposed to the mixed interdigitated configuration. Unlike the symmetric phosphatidylcholines a ripple (P beta') phase was not found as an intermediate between the low and high temperature lamellar phases of 22/12 PC. The specific volume of 22/12 PC is 940 (+/- 1) microliter/g and 946 (+/- 1) microliter/g in the hydrated lamellar gel state at 28 (+/- 2) and 40 (+/- 2) degrees C, respectively, from neutral buoyancy experiments. Based on measurements of the temperature dependence of the various lattice parameters of the different phases encountered in this study the corresponding lattice thermal expansion coefficients have been measured. These are discussed and their dependence on lipid hydration is reported.     

SUBCUR: Visualization of structural differences between DNA duplexes

A computer program, SUBCUR, is described which permits analysis and rapid identification of geometrical differences and patterns of variance between two DNA duplexes. The program is compatible with the CURVES 3.1 package² and allows graphical visualization of the structural differences. Examples are provided which illustrate the applicability of the program in analyzing the different backbone conformations of two helices and the different curvatures of two helices.     

Effect of DNA flanking sequence on charge transport in short DNA duplexes

Several factors can influence charge transport (CT)-mediated DNA, such as sequence, distance, base stacking, base pair mismatch, conformation, tether length, etc. However, the DNA context effect or how flanking sequences influence redox active drugs in the DNA CT reaction and later in DNA enzymatic repair and synthesis is still not well understood. The set of seven DNA molecules in this study have been characterized well for the study of flanking sequence effects. These DNA duplexes are formed from self-complementary strands and contain the common central four-base sequence 5'-A-G-C-T-3', flanked on both sides by either (AT)(n) or (AA)(n) (n = 2, 3, or 4) or AA(AT)(2). UV-vis, fluorescence, UV melting, circular dichroism, and cyclic voltammetry experiments were used to study the flanking sequence effect on CT-mediated DNA by using daunomycin or adriamycin cross-linked with these seven DNA molecules. Our results showed that charge transport was related to the flanking sequence, DNA melting free energy, and ionic strength. For (AA)(n) or (AT)(n) species of the same length, (AA)(n) series were more stable and more efficient CT was observed through the (AA)(n) series. The same trend was observed for (AA)(n)() and (AT)(n) series at different ionic strengths, further supporting the idea that flanking sequence can result in different base stacking and modulate charge transport through these seven DNA molecules.     

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.     

PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.     

Thermodynamic stability and drug-binding properties of oligodeoxyribonucleotide duplexes containing 3-deazaadenine:thymine base pairs.

We have used ultraviolet melting techniques to study the effect on stability of incorporating the nucleoside analogue 2'-deoxy-3-deazaadenosine (d3cA) into the duplex 5'-d(CGCAATCG)-3'-d(GCGTTAGC). Our results demonstrate that the successive replacement of dA by d3CA increasingly destabilises the duplex. The destabilising effect of this analogue is considerably enhanced as the pH is lowered and the results are consistent with protonation of 3-deazaadenine (probably at N-1) contributing to duplex destablisation. Surprisingly, the incorporation of d3CA does not significantly affect the binding of distamycin-A.     

Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.     

Effect of environment, conformation, sequence and base substituents on the imino proton exchange rates in guanine and inosine-containing DNA, RNA, and DNA-RNA duplexes

High-resolution 1H nuclear magnetic resonance in H2O has been used to study the effect of sequence, conformation, environmental factors and base substituents on the exchange behavior of the hydrogen-bonded imino protons of guainine X cytosine and inosine X cytosine base-pairs in DNA, RNA, and DNA-RNA duplexes. The exchange rates were determined by measurement of the spin-lattice relaxation rates of the imino protons as a function of temperature. The exchange was not altered by the presence of high concentrations of salt, and the inability of phosphate to catalyze the exchange indicates that the exchange is limited by formation of a solvent-accessible "open" state. The exchange behavior depends on the duplex conformation and sequence. Exchange from the Z form polymers was orders of magnitude slower than the corresponding duplexes in the B conformation, and the A form RNA duplexes exchanged more slowly than the B form DNA polymers with the same sequence. The exchange behavior of the DNA-RNA hybrids was dependent on whether the purine or the pyrimidine strand contained the deoxyribose sugar. For both the guanine and inosine-containing duplexes, the homopolymer duplexes exchange more slowly than the more stable alternating copolymers. For the alternating duplexes, substitution of cytosine with 5-bromo- or 5-methylcytosine slowed the exchange and increased the activation energy for exchange. The inosine-containing duplexes exchanged more rapidly than the guanosine-containing duplexes, but both showed similar changes in exchange behavior in response to changes in sequence and base substituents. The activation energies for base-pair opening in B form DNA are correlated with the van der Waals contribution to the base-base interaction energy, suggesting that the purine base is partially unstacked in the open state. Using the relaxation measurements to set an upper limit on the exchange rate in poly(dG-dC) and the tritium exchange behavior at low temperature, we find that even though Z-DNA exchanges very slowly, the activation energy is similar to that observed in the A and B form duplexes, suggesting that exchange occurs from a similar open state.     

RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions.

Rates of formation of RNA:DNA hybrids have been measured as a function of temperature and compared to DNA:RNA duplex denaturation temperatures in 4 M sodium perchlorate, 4 M NaClO4-6 M urea, and 3 M rubidium trichloracetate solvents. The usual bell shaped curves of reaction rate versus temperature were observed. The optimal temperatures for the RNA:DNA association reaction are 5 degrees to 12 degrees greater than the Tm's for DNA:DNA denaturation in these solvents, just as in formamide. R-loops of phi80d3ilv DNA with E. coli rRNA can be formed at high efficiency in these solvents.