13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.     

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.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of A.C and A.O4meT pairing in dodecanucleotide duplexes

Structural features of A.C mismatches and A.O4meT pairs in the interior of oligodeoxynucleotide duplexes have been investigated by high-resolution two-dimensional proton NMR spectroscopy on the self-complementary d(C-G-C-A-A-G-C-T-C-G-C-G) duplex (designated A.C 12-mer) and and the self-complementary d(C-G-C-A-A-G-C-T-O4meT-G-C-G) duplex (designated A.O4meT 12-mer) containing A.C and A.O4meT pairs at identical positions four base pairs in from either end of and A.O4meT pairs at identical positions four base pairs in from either end of the duplex. Proton NMR shows that there are similar pH-dependent changes in the structure in the A.C 12-mer and A.O4meT 12-mer duplexes. Our studies have focused on the low-pH (pH 5.5) conformation where high-quality two-dimensional NOESY data sets were collected from the exchangeable and nonexchangeable protons in these duplexes. The spectral parameters for the A.C 12-mer and the A.O4meT 12-mer duplexes were very similar, indicating that they must have similar structures at this pH in aqueous solution. Both structures are right-handed double helices with all the bases adopting the normal anti configuration about the glycosidic bond. The same atoms are involved in hydrogen-bond pairing for the A.C mismatch and the A.O4meT pair, and these pairs have a similar spatial relationship to flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of imperfect DNA duplexes containing unpaired bases and non-Watson-Crick base pairs.

Synthetic duplex DNAs of repeating sequence, such as poly d(TTC).poly d(GAA), were separated into their individual single strands. The various single strands complexed not only, as expected, with their complementary strands, but also with other non-complementary strands. Characterization of such complexes with respect to stoichiometry, Tm values and the dependence of Tm on NaCl concentration showed that a variety of unusual structures could be inferred at physiological salt concentrations. These included extrahelical thymines, G.T oppositions, A.C oppositions and T.C oppositions.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite acyclic lesions

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated APP 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated APE 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the APP 9-mer and APE 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution between -5 and 5 degrees C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and E14) and stacks between the flanking G4.C15 and G6.C13 Watson-Crick base pairs in both the APP 9-mer and APE 9-mer duplexes. The helix is right-handed at and adjacent to the abasic site, and all glycosidic torsion angles are anti in both 9-mer duplexes. Proton NMR parameters for the APP 9-mer and APE 9-mer duplexes are similar to those reported previously for the APF 9-mer duplex (F = furan) in which a cyclic analogue of deoxyribose was embedded in an otherwise identical DNA sequence [Kalnik, M. W., Chang, C. N., Grollman, A. P., & Patel, D. J. (1988) Biochemistry 27, 924-931]. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the APP 9-mer and APE 9-mer duplexes (5 degrees C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles. These same torsion angles are also distorted in the APF 9-mer but assume a different conformation than those in the APP 9-mer and APE 9-mer duplexes.     

Methionine-90-spin-labeled bovine alpha-lactalbumin: electron spin resonance and NMR distance measurements

The unique methionine residue of bovine alpha-lactalbumin was modified by irreversible alkylation with the bromoacetamido nitroxide spin-label 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl. The line shape of the electron spin resonance (ESR) spectrum was indicative of a fairly mobile spin-label and was sensitive to the calcium-induced conformational change. Paramagnetic broadening of the spin-label ESR lines by a Gd(III) ion substituted at the high-affinity calcium site of the protein yielded a distance between the spin-label and the metal-binding site of 8.0 +/- 1.0 A. The extent of the paramagnetic line broadening by the covalently attached nitroxide spin-label on the proton resonances of several amino acid residues of the protein at 500 MHz allowed estimation of intramolecular distances between the methionine-90 residue and several resolvable protons.     

NMR studies of the interaction of chromomycin A3 with small DNA duplexes I

1H and 31P NMR spectral analysis of a chromomycin/d(ATGCAT)2 complex provides strong evidence for a nonintercalative mode of drug binding. Investigation of the imino proton region of the duplex suggests a protection of one of the two guanine imino protons from fast exchange with the bulk water up to at least 45 degrees C by the drug. Subsequent one-dimensional nuclear Overhauser enhancement experiments place the exchangeable chromomycin chromophoric hydroxyl proton less than 0.45 nm from this guanine imino proton and the chromophore 7-methyl less than 0.45 from the internal thymine 6-proton and/or the guanine 8-proton. 1H two-dimensional NMR reveals that the duplex retains a right-handed B conformation but there are distortions at the TGC region of one chain and large deviations in the chemical shift of protons relative to the uncomplexed duplex in the other chain in the same TGC region. The data suggest that the chromomycin chromophore is oriented such that the hydrophilic side of the ring system is proximal to the helix center in the major groove near the TG region while the aromatic side of the ring is oriented away from the helix but is partially protected from the solvent by the aliphatic chain, which bends back over the two aromatic protons. Changes in the 31P spectrum of the duplex on binding of the drug are different from the effect of either actinomycin or netropsin on nucleic acid fragments.     

Equilenin-derived DNA adducts to cytosine in DNA duplexes: structures and thermodynamics

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite the cyclic analogue of 2-deoxyribose

Proton and phosphorus NMR studies are reported for the complementary d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-F-C-A-T-G) nonanucleotide duplex (designated APF 9-mer duplex) which contains a stable abasic site analogue, F, in the center of the helix. This oligodeoxynucleotide contains a modified tetrahydrofuran moiety, isosteric with 2-deoxyribofuranose, which serves as a structural analogue of a natural apurinic/apyrimidinic site [Takeshita, M., Chang, C.N., Johnson, F., Will, S., & Grollman, A.P. (1987) J. Biol. Chem. 262, 10171-10179]. Exchangeable and nonexchangeable base and sugar protons, including those located at the abasic site, have been assigned in the complementary APF 9-mer duplex by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution at low temperature (0 degrees C). These studies indicate that A5 inserts into the helix opposite the abasic site F14 and stacks with flanking G4.C15 and G6.C13 Watson-Crick base pairs. Base-sugar proton NOE connectivities were measured through G4-A5-G6 on the unmodified strand and between the base protons of C15 and the sugar protons of the 5'-flanking residue F14 on the modified strand. These studies establish that all glycosidic torsion angles are anti and that the helix is right-handed at and adjacent to the abasic site in the APF 9-mer duplex. Two of the 16 phosphodiester groups exhibit phosphorus resonances outside the normal spectral dispersion indicative of altered torsion angles at two of the phosphate groups in the backbone of the APF 9-mer duplex.     

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.     

Dependence of DNA condensation on the correlation distance between condensed counterions

Based on the ground state of counterions condensed on a DNA molecule, a model has been developed to successfully detect the process of DNA condensation. Through further investigation, the process of DNA condensation strongly depends on the correlation distance between condensed counterions on DNA molecules. Generally, there are two routes. The process of DNA condensation with the correlation distance between condensed counterions being 2 nm or 4 nm is different from the one with the correlation distance between condensed counterions being 3 nm or 5 nm. Effects of ionic strength on the diameter of toroidal condensates originate from the increase of correlation distance between condensed counterions.     

Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin- spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of –170 ± 15ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived m values for proteins. The near identity of observed T2 and T1 values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine m values. It is also noted that the reliable determination of m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as m increases.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.C pairing in dodecanucleotide duplexes

The pairing of O6etG with C located four base pairs in from either end of the self-complementary d(C1-G2-C3-O6etG4-A5-G6-C7-T8-C9-G10-C11-G12) duplex (designated O6etG.C 12-mer) has been investigated from an analysis of proton and phosphorus two-dimensional NMR experiments. The structural consequences of increasing the alkyl group size were elucidated from a comparative study of the pairing of O6meG4 with C9 in a related sequence (designated O6meG.C 12-mer). The NMR parameters for both O6alkG-containing dodecanucleotides are also compared with those of the control sequence containing G4.C9 base pairs (designated G.C 12-mer). The NOE cross-peaks detected in the two-dimensional NOESY spectra of the O6alkG.C 12-mer duplexes in H2O solution establish that the O6etG4/O6meG4 and C9 bases at the lesion site stack into the helix between the flanking C3.G10 and A5.T8 Watson-Crick base pairs. The amino protons of C9 at the O6alkG4-C9 lesion site resonate as an average resonance at 7.78 and 7.63 ppm in the O6etG.C 12-mer and O6meG.C 12-mer duplexes, respectively. The observed NOEs between the amino protons of C9 and the CH3 protons of O6alkG4 establish a syn orientation of the O6-alkyl group with respect to the N1 of alkylated guanine. A wobble alignment of the O6alkG4.C9 base pair stablized by two hydrogen bonds, one between the amino group of C9 and N1 of O6alkG and the other between the amino group of O6alkG and N3 of C9, is tentatively proposed on the basis of the NOEs between the amino protons of C9 at the lesion site and the imino protons of flanking Watson-Crick base pairs. The proton and phosphorus chemical shift differences between the O6etG.C 12-mer and O6meG.C 12-mer duplexes are small compared to the differences between these O6alkG-containing duplexes and the control G.C 12-mer duplex.     

Inducing the replacement of PNA in DNA.PNA duplexes by DNA

The uncharged DNA-analogue peptide nucleic acid (PNA) can invade into dsDNA by displacing the non-complementary DNA strand. The formed strand displacement complexes can create a sterical hindrance to block access of enzymes such as nucleases and polymerases. Due to the high stability of DNA.PNA duplexes it is usually not possible to displace the PNA strand by ssDNA or ssRNA. We herein report that the polycationic, comb-type copolymer alphaPLL-g-Dex can induce such a replacement of PNA in DNA.PNA duplexes by ssDNA. The influence of the copolymer on strand exchange highly depends on the nature of the oligonucleotides. Acceleration has only been observed when both the starting duplex and the single-stranded exchanger strand were negatively charged. The presented approach should allow the withdrawal of PNA induced sterical hindrance of DNA by rehybridisation with ssDNA.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.T pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies are reported on the self-complementary d-(C1-G2-C3-O6etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O6etG.T 12-mer) containing two symmetrically related O6etG.T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O6meG.T lesion sites (designated O6meG.T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G.T mismatch sites (designated G.T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O6etG.T 12-mer, O6meG.T 12-mer, and G.T 12-mer duplexes in H2O and D2O solutions. The distance connectivities observed in the NOESY spectra of the O6alkG.T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O6alkG4 and T9 bases at the lesion site. The imino proton of T9 at the O6alkG.T lesion sites resonates at 8.85 ppm in the O6etG.T 12-mer duplex and at 9.47 ppm in the O6meG.T 12-mer duplex. The large upfield shift of the T9 imino proton resonance at the O6alkG4.T9 lesion site relative to that of the same proton in the G4.T9 wobble pair (11.99 ppm) and the A4.T9 Watson-Crick pair (13.95 ppm) in related sequences establishes that the hydrogen bonding of the imino proton of T9 to O6alkG4 is either very weak or absent. The imino proton of T9 develops NOEs to the CH3 protons of the O6etG and O6meG alkyl groups across the base pair, as well as to the imino and H5 protons of the flanking C3.G10 base pair and the imino and CH3 protons of the flanking A5.T8 base pair in the O6alkG.T 12-mer duplexes. These observations establish that the O6alkG4 and T9 residues are stacked into the duplex and that the O6CH3 and O6CH2CH3 groups of O6alkG4 adopt a syn orientation with respect to the N1 of the alkylated guanine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unique properties of purine/pyrimidine asymmetric PNA.DNA duplexes: differential stabilization of PNA.DNA duplexes by purines in the PNA strand

PNA.DNA duplexes are significantly stabilized by purine nucleobases in the PNA strand. To elucidate and understand the effect of switching the backbone in a nucleic acid duplex, we now report a thermodynamics study along with a solution conformations study of two purine/pyrimidine strand asymmetric duplexes and a strand symmetrical control by comparing the behavior of all four possible PNA/DNA combinations. In essence, we are comparing an identical basepair stack connected by either an aminoethyl glycine PNA or a deoxyribose DNA backbone. We show that the PNA.DNA duplexes containing purine-rich PNA strands are stabilized with regard to the thermal melting temperature and free energy as well as enthalpy (and concomitantly relatively less entropically disfavored). Based on our data, we find it unlikely that differences in counterion binding (identical ionic-strength dependence was observed), hydration (identical and insignificant water release was observed), or single-strand conformation can be responsible for the difference in duplex stability. The only consistent difference observed between the purine-rich PNA versus the pyrimidine-rich PNA in isosequential PNA.DNA duplexes is the significant increase in both binding enthalpy and entropy for the PNA.DNA duplexes containing pyrimidine-rich PNA in organic solvent, which would indicate that these duplexes are relatively enthalpically disfavored in water. Although our results so far do not allow us to identify the origin of the different stabilities of homopurine/homopyrimidine PNA.DNA duplexes, the evidence does point to a significant structural component, which involves enthalpic contributions both within the duplex structure and also from bound water molecules.