UV resonance Raman and circular dichroism studies of a DNA duplex containing an A(3)T(3) tract: evidence for a premelting transition and three-centered H-bonds.

The presence of A(n) and A(n)T(n) tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B'-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A(n) tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B'- to B-DNA. Structural features of A(n) and A(n)T(n) tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)(2) (A(3)T(3)) and d(CGCATATATGCG)(2) [(AT)(3)]. These studies address the presence of three-centered H-bonds in the B' conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A(3)T(3) duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A(3)T(3) duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar-phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH(2) and dT C4=O vibrational modes suggest that the A(3)T(3) duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)(3) duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4=O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.     

Solution structure of an oncogenic DNA duplex containing a G.A mismatch

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.     

Solution structure of DAPI selectively bound in the minor groove of a DNA T.T mismatch-containing site: NMR and molecular dynamics studies.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer [d(GCGATTCGC)]2, containing a central T.T mismatch, has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics and molecular dynamics computations including relaxation matrix refinement. The results show that the DAPI molecule binds in the minor groove of the central region 5'-ATT-3' of the DNA oligomer, which predominantly adopts a duplex structure with a global right-handed B-like conformation. In the final models of the complex, the DAPI molecule is located nearly isohelical with its NH indole proton oriented towards the DNA helix axis and forming a bifurcated hydrogen bond with the carbonyl O2 groups of a mismatched T5 and the T6 residue of the opposite strand. Mismatched thymines adopt a wobble base pair conformation and are found stacked between the flanking base pairs, inducing only minor local conformational changes in global duplex structure. In addition, no other binding mechanisms were observed, showing that minor groove binding of DAPI to the mismatch-containing site is favoured in comparison with any other previously reported interaction with G.C sequences.     

The hydrogen-bonding structure in parallel-stranded duplex DNA is reverse Watson-Crick

Raman spectra of the parallel-stranded duplex formed from the deoxyoligonucleotides 5'-d-[(A)10TAATTTTAAATATTT]-3' (D1) and 5'-d[(T)10ATTAAAATTTATAAA]-3' (D2) in H2O and D2O have been acquired. The spectra of the parallel-stranded DNA are then compared to the spectra of the antiparallel double helix formed from the deoxyoligonucleotides D1 and 5'-d(AAATATTTAAAATTA-(T)10]-3' (D3). The Raman spectra of the antiparallel-stranded (aps) duplex are reminiscent of the spectra of poly[d(A)].poly[d(T)] and a B-form structure similar to that adopted by the homopolymer duplex is assigned to the antiparallel double helix. The spectra of the parallel-stranded (ps) and antiparallel-stranded duplexes differ significantly due to changes in helical organization, i.e., base pairing, base stacking, and backbone conformation. Large changes observed in the carbonyl stretching region (1600-1700 cm-1) implicate the involvement of the C(2) carbonyl of thymine in base pairing. The interaction of adenine with the C(2) carbonyl of thymine is consistent wtih formation of reverse Watson-Crick base pairing in parallel-stranded DNA. Phosphate-furanose vibrations similar to those observed for B-form DNA of heterogenous sequence and high A,T content are observed at 843 and 1092 cm-1 in the spectra of the parallel-stranded duplex. The 843-cm-1 band is due to the presence of a sizable population of furanose rings in the C2'-endo conformation. Significant changes observed in the regions from 1150 to 1250 cm-1 and from 1340 to 1400 cm-1 in the spectra of the parallel-stranded duplex are attributed to variations in backbone torsional and glycosidic angles and base stacking.     

An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification.

The backbone modification amide-3, in which -CH2-NH-CO-CH2- replaces -C5'H2-O5'-PO2-O3'-, is studied in the duplex d(G1-C2-G3-T4.T5-G6-C7-G8)*mr(C9-G10-C11-A12-A13-C14-G15+ ++-C16) where . indicates the backbone modification and mr indicates the 2'-OMe RNA strand. The majority of the exchangeable and non-exchangeable resonances have been assigned. The assignment procedure differs from standard methods. The methyl substituent of the 2'-OMe position of the RNA strand can be used as a tool in the interpretation. The duplex structure is a right-handed double helix. The sugar conformations of the 2'-OMe RNA strand are predominantly N-type and the 2'-OMe is positioned at the surface of the minor groove. In the complementary strand, only the sugar of residue T4 is found exclusively in N-type conformation. The incorporation of the amide modification does not effect very strongly the duplex structure. All bases are involved in Watson-Crick base pairs.     

Hybrid characteristics of oligonucleotides consisting of isonucleoside 2',5'-anhydro-3'-deoxy-3'-(thymin-1-yl)-D-mannitol with different linkage modes

Oligonucleotides consisting of the isonucleoside repeating unit 2',5'-anhydro-3'-deoxy-3'-(thymin-1-yl)-D-mannitol (4) were synthesized with the monomeric unit 4 incorporated into oligonucleotides as 1'-->4' linkage 4a (oligomer I) or 6'-->4' linkage 4b (oligomer II). The hybrid properties of the two oligonucleotides I and II with their complementary strands were investigated by thermal denaturation and CD spectra. Oligonucleotide I (4a) formed a stable duplex with d(A)(14) with a slightly reduced T(m) value of 36.6 degrees C, relative to 38.2 degrees C for the control duplex d(T)(14)/d(A)(14), but oligomer II (4b) failed to hybridize with a DNA complementary single strand. The spectrum of the duplex oligomer I/d(A)(14) showed a positive CD band at 217 nm and a negative CD band at 248 nm attributable to a B-like conformation. Molecular modeling showed that in the case of oligomer I: the C6' hydroxy group of each unit could be located in the groove area when hybridized to the DNA single strand, which might contribute additional hydrogen bonding to the stability of duplex formation.     

NMR studies of DNA duplexes singly cross-linked by different synthetic linkers.

Molecular modelling studies resulted in the design of a variety of non-nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand and the 5'-end of the (-)-strand in DNA duplexes. Three of these linkers were synthesized and used to prepare singly cross-linked duplexes d(GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene-, a phosphate- and a second propylene-group and is thought to mimic the backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of the cross-linked duplexes are 65 degrees C for I and 73 degrees C for II and III, as compared with 36 degrees C for the corresponding non-linked nonadeoxynucleotide duplex. The three cross-linked duplexes were structurally characterized by nuclear magnetic resonance spectroscopy. The 1H and 31P resonance assignments in the DNA stem were obtained using standard methods. For the resonance assignment of the linker protons, two-dimensional 1H-31P heteronuclear COSY and two-quantum-experiments were used. Distance geometry calculations with NOE-derived distance constraints were performed and the resulting structures were energy-minimized. In duplex I, the nucleotides flanking the propylene-phosphate-propylene-linker do not form a Watson-Crick base pair, whereas in duplexes II and III the entire DNA stem is in a B-type double helix conformation.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

Structure of DNA sequence d-TGATCA by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics

The 5' d-TpG 3' element is a part of DNA sequences involved in regulation of gene expression and is also a site for intercalation of several anticancer drugs. Solution conformation of DNA duplex d-TGATCA containing this element has been investigated by two-dimensional NMR spectroscopy. Using a total of 12 torsional angles and 121 distance constraints, structural refinement has been carried out by restrained molecular dynamics (rMDs) in vacuum up to 100 ps. The structure is characterized by a large positive roll at TpG/CpA base pair step and large negative propeller twist for AT and TA base pairs. The backbone torsional angle, gamma(O5'-C5'-C4'-C3'), of T1 residue adopts a trans-conformation which is corroborated by short intra nucleotide T1H6-T1H5' (3.7A) distance in nuclear overhauser effect spectroscopy (NOESY) spectra while the backbone torsional angle, beta(P-O5'-C5'-C4'), exists in trans as well as gauche state for T1 and C5 residues. There is evidence of significant flexibility of the sugar-phosphate backbone with rapid inter-conversion between two different conformers at TpG/CpA base pair step. The base sequence dependent variations and local structural heterogeneity have important implications in specific recognition of DNA by ligands.     

DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization

The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7°C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5′-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 ± 0.17 Å. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.     

Comparative analysis of inosine-substituted duplex DNA by circular dichroism and X-ray crystallography

Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from ‘electrostatic’ or ‘base stacking’ influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I?C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I?C and two A?T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1′-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2′-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I?C pairs has a geometry similar to that of the reference duplex with eight G?C and two A?T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.     

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.     

NMR study of a synthetic DNA hairpin

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.     

Mismatch-induced DNA unbending upon duplex opening

A DNA duplex can be torn open at a specific position by introducing a branch or bulge to create an asymmetric three-way junction (TWJ). The opened duplex manifests a bent conformation (bending angle approximately 60 degrees , relative to the unopened form), which leads to a dramatic decrease in gel electrophoretic mobility. In the presence of a basepair mismatch at the opening position, the DNA backbone becomes less bent and assumes a distorted T-shaped structure, resulting in an increase in polyacrylamide gel electrophoretic mobility. Both conformational changes are confirmed using fluorescence resonance energy transfer experiments and found to be similar to the signature conformational changes of DNA duplex upon MutS protein binding. Our results imply that some structural rearrangements essential for mismatch recognition are achievable without protein interference. The gel electrophoretic mobility data for DNA TWJs with and without base mismatches correlates well with rotational diffusivity, computed by taking into account the conformational change of TWJ induced by base mismatch.     

Structural equilibrium of DNA represented with different force fields.

We have recently indicated preliminary evidence of different equilibrium average structures with the CHARMM and AMBER force fields in explicit solvent molecular dynamics simulations on the DNA duplex d(C5T5) . d(A5G5) (Feig, M. and B.M. Pettitt, 1997, Experiment vs. Force Fields: DNA conformation from molecular dynamics simulations. J. Phys. Chem. B. (101:7361-7363). This paper presents a detailed comparison of DNA structure and dynamics for both force fields from extended simulation times of 10 ns each. Average structures display an A-DNA base geometry with the CHARMM force field and a base geometry that is intermediate between A- and B-DNA with the AMBER force field. The backbone assumes B form on both strands with the AMBER force field, while the CHARMM force field produces heterogeneous structures with the purine strand in A form and the pyrimidine strand in dynamical equilibrium between A and B conformations. The results compare well with experimental data for the cytosine/guanine part but fail to fully reproduce an overall B conformation in the thymine/adenine tract expected from crystallographic data, particularly with the CHARMM force field. Fluctuations between A and B conformations are observed on the nanosecond time scale in both simulations, particularly with the AMBER force field. Different dynamical behavior during the first 4 ns indicates that convergence times of several nanoseconds are necessary to fully establish a dynamical equilibrium in all structural quantities on the time scale of the simulations presented here.     

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Nmr studies of an extrahelical cytosine in an A·T rich region of a deoxyribodecanucleotide

One-dimensional and two-dimensional (2D) nmr experiments were carried out on an oligonucleotide duplex that contains an unpaired cytosine, d(GCGAAC AAGCG)·d(CGCTTTTCGC), which will be referred to as the C-bulge decamer. Evidence from one-dimensional nuclear Overhauser effect (NOE) experiments on the exchangeable protons indicates that the unpaired cytosine is extrahelical. This conclusion is also supported by numerous cross-peaks in the 2D NOE spectroscopy (NOESY) spectrum of the nonexchangeable protons. The assignments for all of the resonances, with the exception of the H5′ and H5″ resonances, have been made through the use of 2D NOESY, correlated spectroscopy (COSY), and relayed COSY experiments. The temperature dependence of the C(H6) resonance chemical shifts indicates that the unpaired cytosine shows unusual behavior compared to other cytosines in the duplex. A comparison of chemical shifts for all, the assigned resonances of the duplexes with and without the unpaired cytosine suggests that the majority of the structural perturbation is localized in the A·T tract surrounding the unpaired base. The behavior of the imino resonances as a function of temperature also indicates that the perturbation to the duplex is localized and destabilizes the A·T base pairs adjacent to the unpaired base. © 1993 John Wiley & Sons, Inc.     

Structural features and hydration of a dodecamer duplex containing two C.A mispairs.

X-ray diffraction techniques have been used to characterise the crystal and molecular structure of the deoxyoligomer d(C-G-C-A-A-A-T-T-C-G-C-G) at 2.5A resolution. The final R factor is 0.19 with the location of 78 solvent molecules. The oligomer crystallises in a B-DNA type conformation with two strands coiled about each other to produce a duplex. This double helix consists of four A.T and six G.C Watson-Crick base pairs and two C.A mispairs. The mismatched base pairs adopt a "wobble" type structure with the cytosine displaced laterally into the major groove, the adenine into the minor groove. We have proposed that the two close contacts observed in the C.A pairing represent two hydrogen bonds one of which results from protonation of adenine. The mispairs are accommodated in the double helix with small adjustments in the conformation of the sugar-phosphate backbone. Details of the backbone conformation, base stacking interactions, thermal parameters and the hydration are now presented and compared with those of the native oligomer d(C-G-C-G-A-A-T-T-C-G-C-G) and with variations of this sequence containing G.T and G.A mispairs.     

Unusual conformation of a 3′-thioformacetal linkage in a DNA duplex

Summary The DNA·DNA duplex ·d(GCGCAAAACGCG) (designated duplex III) containing a 3-thioformacetal (3-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH₂O) linkage at the central T^T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3-TFMA-modified T6 sugar adopts an O4-endo form (an intermediate structure between the usual C2-endo and C3-endo forms). This change is accompanied by a change in the (C4–C3–S3–CH₂) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6–T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3-TFMA linkages is merely in the substitution of the T6(O3) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.Supplementary material available from the authors: One table containing J₁₂, J₁₂ and J₃₄ of duplexes I, II and III.