Cyclohexyl "base pairs" stabilize duplexes and intensify pyrene fluorescence by shielding it from natural base pairs

In this study, we investigated the stability and structure of artificial base pairs that contain cyclohexyl rings. The introduction of a single pair of isopropylcyclohexanes into the middle of DNA slightly destabilized the duplex. Interestingly, as the number of the "base pairs" increased, the duplex was remarkably stabilized. A duplex with six base pairs was even more stable than one containing six A-T pairs. Thermodynamic analysis revealed that changes in entropy and not enthalpy contributed to duplex stability, demonstrating that hydrophobic interactions between isopropyl groups facilitated the base pairing, and thus stabilized the duplex. NOESY of a duplex containing an isopropylcyclohexane-methylcyclohexane pair unambiguously demonstrated its "pairing" in the duplex because distinct NOEs between the protons of cyclohexyl moieties and imino protons of both of the neighboring natural base pairs were observed. CD spectra of duplexes tethering cyclohexyl moieties also showed a positive-negative couplet that is characteristic of the B-form DNA duplex. Taken together, these results showed that cyclohexyl moieties formed base pairs in the DNA duplex without severely disturbing the helical structure of natural DNA. Next, we introduced cyclohexyl base pairs between pyrene and nucleobases as an "insulator" that suppresses electron transfer between them. We found a massive increase in the quantum yield of pyrene due to the efficient shielding of pyrene from nucleobases. The cyclohexyl base pairs reported here have the potential to prepare highly fluorescent labeling agents by multiplying fluorophores and insulators alternately into DNA duplexes.     

Metal incorporation in modified PNA duplexes

Substitution of bipyridine for a nucleobase leads to modified peptide nucleic acid (PNA) single strands that are bridged in the presence of Ni2+ into a duplex containing a combination of hydrogen and coordinative bonds. CD experiments demonstrate that the duplex adopts a structure similar to that of an unmodified 10-bp PNA duplex, and UV melting experiments show a very sensitive dependence of the duplex stability on the substitution of a nucleobase pair with a pair of ligands or a metal-ligand alternative base pair.     

Sequence specificity of hydrogen-bonded molecular duplexes

Hydrogen-bonded molecular duplexes, 1.3 and 1.4, each of which contains a mismatched binding site (acceptor-to-acceptor in 1.3, and donor-to-donor in 1.4), were designed and synthesized based on duplex 1.2. One- and two-dimensional NMR studies demonstrated that, despite their single mismatched binding sites, the backbones of duplexes 1.3 and 1.4 still stayed in register through the formation of the remaining five H-bonds. The backbones of 1.3 and 1.4 adjusted to the presence of the mismatched binding sites by slightly twisting around these sites, which alleviate any head-on repulsive interactions between two H-bond donors (amide O) or between two acceptors (amide H). After 1 equiv of single strand 2, which forms a perfectly matched duplex 1.2 with single strand 1, was added into the solution of either 1.3 or 1.4, only 1.2 and single strand 3 or 4, were detected. Isothermal titration calorimetry (ITC, in chloroform containing 5% DMSO) indicated that duplexes 1.3 and 1.4 were significantly (>40 times) less stable than the corresponding perfectly hydrogen-bonded duplex 1.2. These NMR and ITC results indicate that the pairing of two complementary single strands is not affected by another very similar single strand that contains only one wrong H-bond donor or acceptor, which demonstrates that the self-assembly of this class of H-bonded duplexes is a highly sequence-specific process. The role of these H-bonded duplexes as predictable and programmable molecular recognition units for directing intermolecular interactions has thus been established.     

Aromatic oligomers that form hetero duplexes in aqueous solution

The electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (Ndi) and electron-rich 1,5-dialkoxynaphthalene (Dan) have been shown to complex strongly with each other in water due to the hydrophobic effect as modulated through the electrostatic complementarity of the stacked dimer. Previously, oligomers of alternating Ndi and Dan units, termed aedamers, were the first foldamers to employ intramolecular aromatic stacking to effect the formation of secondary structure of nonnatural chains in aqueous solution. Described here is the use of this aromatic-aromatic (or pi-pi) interaction, this time in an intermolecular format, to demonstrate the self-assembly of stable hetero duplexes from a set of molecular strands (1a-4a) and (1b-4b) incorporating Ndi and Dan units, respectively. A 1-to-1 binding stoichiometry was determined from NMR and isothermal titration calorimetry (ITC) investigations, and these experiments indicated that association is enthalpically favored with the tetra-Ndi (4a) and tetra-Dan (4b) strands forming hetero duplexes (4a:4b) with a stability constant of 350 000 M-1 at T = 318 K. Polyacrylamide gel electrophoresis (PAGE) also illustrated the strong interaction between 4a and 4b and support a 1-to-1 binding mode even when one component is in slight excess. Overall, this system is the first to utilize complementary aromatic units to drive discrete self-assembly in aqueous solution. This new approach for designing assemblies is encouraging for future development of duplex systems with highly programmable modes of binding in solution or on surfaces.     

Additional base-pair formation in DNA duplexes by a double-headed nucleotide

We have designed and synthesised a double-headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson-Crick base pairs with two complementary adenosines in a Watson-Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double-headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double-headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B-type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double-headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required.     

Self-assembly of dipeptidyl ureas: a new class of hydrogen-bonded molecular duplexes

The dipeptidyl urea 1 composed of two dipeptide chains bearing the C-terminal pyridyl moiety (-L-Ala-L-Pro-NHPy) was prepared. Two molecules of 1 are revealed to be held together by six intermolecular hydrogen bonds to form a hydrogen-bonded duplex by the single-crystal X-ray structure determination. Proton magnetic resonance nuclear Overhauser effect (NOE) study indicates the hydrogen-bonded duplex even in solution. Furthermore, a shuttle-like molecular dynamics based on recombination of the hydrogen bonds was observed. The dipeptidyl urea composed of two dipeptide chains bearing the C-terminal pyrenyl moiety (-L-Ala-L-Pro-NHCH(2)Pyr) exhibited both monomer and eximer emissions in the fluorescence spectra, supporting the formation of a duplex. A combination of the C-terminal amide NH function in each side and the designed sequence of hydrogen-bonding sites are considered to be a crucial factor for the duplex formation.     

Molecular architecture: construction of self-assembled organophosphonate duplexes and their electrochemical characterization

Self-assembled monolayers of phosphonates (SAMPs) of 11-hydroxyundecylphosphonic acid, 2,6-diphosphonoanthracene, 9,10-diphenyl-2,6-diphosphonoanthracene, and 10,10'-diphosphono-9,9'-bianthracene and a novel self-assembled organophosphonate duplex ensemble were synthesized on nanometer-thick SiO(2)-coated, highly doped silicon electrodes. The duplex ensemble was synthesized by first treating the SAMP prepared from an aromatic diphosphonic acid to form a titanium complex-terminated one; this was followed by addition of a second equivalent of the aromatic diphosphonic acid. SAMP homogeneity, roughness, and thickness were evaluated by AFM; SAMP film thickness and the structural contributions of each unit in the duplex were measured by X-ray reflection (XRR). The duplex was compared with the aliphatic and aromatic monolayer SAMPs to determine the effect of stacking on electrochemical properties; these were measured by impedance spectroscopy using aqueous electrolytes in the frequency range 20 Hz to 100 kHz, and data were analyzed using resistance-capacitance network based equivalent circuits. For the 11-hydroxyundecylphosphonate SAMP, C(SAMP) = 2.6 ± 0.2 μF/cm(2), consistent with its measured layer thickness (ca. 1.1 nm). For the anthracene-based SAMPs, C(SAMP) = 6-10 μF/cm(2), which is attributed primarily to a higher effective dielectric constant for the aromatic moieties (ε = 5-10) compared to the aliphatic one; impedance spectroscopy measured the additional capacitance of the second aromatic monolayer in the duplex (2ndSAMP) to be C(Ti/2ndSAMP) = 6.8 ± 0.7 μF/cm(2), in series with the first.     

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.     

Theoretical studies of d(A:T)-based parallel-stranded DNA duplexes.

Poly d(A:T) parallel-stranded DNA duplexes based on the Hoogsteen and reverse Watson-Crick hydrogen bond pairing are studied by means of extensive molecular dynamics (MD) simulations and molecular mechanics coupled to Poisson-Boltzmann (MM-PB/SA) calculations. The structural, flexibility, and reactivity characteristics of Hoogsteen and reverse Watson-Crick parallel duplexes are described from the analysis of the trajectories. Theoretical calculations show that the two parallel duplexes are less stable than the antiparallel Watson-Crick duplex. The difference in stability between antiparallel and parallel duplexes increases steadily as the length of the duplex increases. The reverse Watson-Crick arrangement is slightly more stable than the Hoogsteen duplex, the difference being also increased linearly with the length of the duplex. A subtle balance of intramolecular and solvation terms is responsible for the preference of a given helical structure.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.     

Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes

Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences and have investigated the properties of the duplexes formed from these strands upon binding of Cu(2+). Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu(2+), duplexes are formed even from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations demonstrate that at any temperature, one Cu(2+) ion binds a pair of Q-PNA strands that each contain one 8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate effect, which prevents the transformation in the presence of excess Cu(2+) of the 1:2 Cu(2+):Q-PNA complexes into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ(2)] moieties that are similar to the corresponding square planar synthetic complex formed between Cu(2+) and 8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a C-terminal l-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy. The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic acid nanostructures.     

Stabilizing or destabilizing oligodeoxynucleotide duplexes containing single 2'-deoxyuridine residues with 5-alkynyl substituents

The 5-position of pyrimidines in DNA duplexes offers a site for introducing alkynyl substituents that protrude into the major groove and thus do not sterically interfere with helix formation. Substituents introduced at the 5-position of the deoxyuridine residue of dU:dA base pairs may stabilize duplexes and reinforce helices weakened by a low G/C content, which would otherwise lead to false negative results in DNA chip experiments. Here we report on a method for preparing oligonucleotides with a 5-alkynyl substituent at a 2'-deoxyuridine residue by on-support Sonogashira coupling involving the fully assembled oligonucleotide. A total of 25 oligonucleotides with 5-alkynyl substituents were prepared. The substituents either decrease the UV melting point of the duplex with the complementary strand or increase it by up to 7.1 degrees C, compared with that of the unmodified control duplex. The most duplex-stabilizing substituent, a pyrenylbutyramidopropyne moiety, is likely to intercalate but does not prevent sequence-specific base pairing of the modified deoxyuridine residue or the neighboring nucleotides. It also increases the signal for a target strand when employed on a small oligonucleotide microarray. The ability to tune the melting point of a DNA dodecamer duplex with a single side chain over a temperature range of >11 degrees C may prove useful when developing DNA sequences for biomedical applications.     

Strong and selective binding of amiloride to an abasic site in RNA duplexes: thermodynamic characterization and microRNA detection

Firmly tied: The binding affinity of amiloride for an abasic (AP) site-containing RNA duplex is two orders of magnitude superior to the affinity of the corresponding AP site-containing DNA duplex. The observed high binding affinity for the RNA duplex arises from a favorable enthalpy gain. The binding-induced fluorescence response of amiloride is applicable to microRNA detection.     

Duplex structure of a minimal nucleic acid

The crystal structure of an 8-mer (S)-GNA duplex is presented. As a tool for phasing, the anomalous diffraction of two copper(II) ions within two artificial metallo-base pairs was employed. The duplex structure confirms a canonical Watson-Crick base pairing scheme of GNA with antiparallel strands. The duplex secondary structure is distinct from canonical A- and B-form nucleic acids and can be described as a right-handed helical ribbon wrapped around the helix axis, resulting in a large hollow core. Most intriguingly, neighboring base pairs slide strongly against each other, resulting in extensive interstrand base-base hydrophobic interactions along with unusual hydrophobic intrastrand interactions of nucleobases with their backbone. These results reveal how a minimal nucleic acid backbone can support highly stable Watson-Crick-like duplex formation.     

Theoretical study of the guanine --> 6-thioguanine substitution in duplexes, triplexes, and tetraplexes

Molecular dynamics and thermodynamic integration calculations have been carried out on a set of G-rich single-strand, duplex, triplex, and quadruplex DNAs to study the structural and stability changes connected with the guanine --> 6-thioguanine (G --> S) mutation. The presence of 6-thioguanine leads to a shift of the geometry from the B/A intermediate to the pure B-form in duplex DNA. The G --> S mutation does not largely affect the structure of the antiparallel triplex when it is located at the reverse-Hoogsteen position, but leads to a non-negligible local distortion in the structure when it is located at the Watson-Crick position. The G --> S mutation leads to destabilization of all studied structures: the lowest effect has been observed for the G --> S mutation in the reverse-Hoogsteen strand of the triplex, a medium effect has been observed in the Watson-Crick strand of the triplex and duplex, and the highest influence of the G -->S mutation has been found for the quadruplex structures.     

Stabilization of DNA duplexes by covalently-linked peptides

The hybridization properties of various peptide-oligonucleotide hybrids were assessed from UV thermal denaturation experiments. Analysis of these and other published data suggests that peptide chains, both hydrophobic and positively charged, generally have a clear stabilizing effect on short-chain oligonucleotide duplexes.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

Salt-dependent heat capacity changes for RNA duplex formation

Two short non-self-complementary RNA duplexes have been probed extensively to assess whether the heat capacity change (DeltaCP) for RNA duplex formation is sensitive to the solution ionic strength. Isothermal titration calorimetry data for duplex formation were collected as a function of temperature to obtain the DeltaCP values under each condition. Ionic strength was varied by repeating the measurements in the presence of 0.1-1.5 M added NaCl. In both cases, the DeltaCP for duplex formation was shown to have a significant dependence on the solution ionic strength, varying linearly with the log of the added NaCl. In each case, the DeltaCP became more negative at high ionic strength. Heat capacity changes have been much better studied in the area of protein folding where DeltaCP is not strongly dependent upon ionic strength but instead depends primarily on the burial of hydrophobic surface. Whereas hydrophobic burial is almost certainly involved in RNA duplex formation as well, the polyanionic nature of the backbone and the associated ion condensation effects are almost certainly a contributor to the observed ionic strength dependence of DeltaCPs for duplex formation. These data also hint at the possibility that nearest neighbor effects may be evident in the heat capacity changes associated with nucleic acid folding.