[show abstract][hide abstract] ABSTRACT: Scorpion primers can be used to detect PCR products in homogeneous solution. Their structure promotes a unimolecular probing mechanism. We compare their performance with that of the same probe sequence forced to act in a bimolecular manner. The data suggest that Scorpions indeed probe by a unimolecular mechanism which is faster and more efficient than the bimolecular mechanism. This mechanism is not dependent on enzymatic cleavage of the probe. A direct comparison between Scorpions, TaqMan and Molecular Beacons on a Roche LightCycler indicates that Scorpions perform better, particularly under fast cycling conditions. Development of a cystic fibrosis mutation detection assay shows that Scorpion primers are selective enough to detect single base mutations and give good sensitivity in all cases. Simultaneous detection of both normal and mutant alleles in a single reaction is possible by combining two Scorpions in a multiplex reaction. Such favourable properties of Scorpion primers should make the technology ideal in numerous applications.
Nucleic Acids Research 11/2000; 28(19):3752-61. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Vsr mismatch endonuclease recognises the sequence CTWGG (W = A or T) in which the underlined thymine is paired with guanine and nicks the DNA backbone on the 5'-side of the mispaired thymine. By using base analogues of G and T we have explored the functional groups on the mismatch pair which are recognised by the enzyme. Removal of the thymine 5-methyl group causes a 60% reduction in activity, while removing the 2-amino group of guanine reduces cleavage by 90%. Placing 2-amino-purine or nebularine opposite T generates mis-matches which are cut at a much lower rate (0.1%). When either base is removed, generating a pseudoabasic site (1', 2'-dideoxyribose), the enzyme still produces site-specific cleavage, but at only 1% of the original rate. Although TT and CT mismatches at this position are cleaved at a low rate (approximately 1%), mismatches with other bases (such as GA and AC) and Watson-Crick base pairs are not cleaved by the enzyme. There is also no cleavage when the mismatched T is replaced with difluorotoluene.
Nucleic Acids Research 08/2000; 28(13):2535-40. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have prepared oligonucleotides with a naphthylquinoline triplex-binding ligand covalently tethered to the 5'-end and have used UV-melting and DNase I footprinting to examine the stability of intra- and inter-molecular triplexes containing this modification. We find that covalent attachment of the ligand increases the melting temperature of intramolecular 6-mer triplexes by about 14 K, and increases the binding of 9-mer oligonucleotides to their duplex target sites by about 60-fold.
Biochimica et Biophysica Acta 11/1999; 1447(2-3):137-45. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G x TA triplets, while CG base pairs are targeted with thymine, forming T x CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1-propargylamino)dU (U(P)). For the target sequence (AAAT)(5)AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex-binding ligand, though stable triplexes were detected at the target site (AAAAAT)(3)AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)(5)AA and (ATGA)(5)AA. In contrast, placing C(+) x GC triplets on the 5'-side of G x TA, using the target (AGTA)(5)TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)(5)TT forms a more stable complex with target (AAAT)(5)AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)(5)AA is improved by using the modified oligonucleotide (TCGU(P))(5)TT, generating a complex in which the charged triplets C(+) x GC and U(P) x AT alternate with uncharged triplets. In contrast, placing U(P) x AT triplets adjacent to C(+) x GC, using the third strand oligonucleotide (U(P)CGT)(5)TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))(5)TT, produces a complex with intermediate stability. It appears that, although adjacent U(P) x AT triplets form stable triplexes, placing U(P) x AT adjacent to C(+) x GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)(3)AA were only detected in the presence of a triplex-binding ligand. Placing C(+) x GC on the 5'-side of T x CG triplets also reduces triplex formation, while a 3'-C(+) x GC produces complexes with increased stability.
[show abstract][hide abstract] ABSTRACT: We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent triplex A6-L-T6-L-T6at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by approximately 12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds approximately 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.
Nucleic Acids Research 05/1999; 27(8):1802-9. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) has been solved and refined at 2.5 A resolution. The refinement procedure converged at R = 0.181 for all reflections in the range 20.0-2.5 A. In the crystal, the RNA/DNA hybrid duplex has an A' conformation with all but one of the nucleotide sugar moieties adopting a C3'- endo (N) conformation. Both strands in the double helix adopt a global conformation close to the A-form and the width of the minor groove is typical of that found in the crystal structures of other A-form duplexes. However, differences are observed between the RNA and DNA strands that make up the hybrid at the local level. In the central portion of the duplex, the RNA strand has backbone alpha, beta and gamma torsion angles that alternate between the normal gauche -/ trans / gauche + conformation and an unusual trans / trans / trans conformation. Coupled with this so-called 'alpha/gamma flipping' of the backbone torsion angles, the distance between adjacent phosphorous atoms on the RNA strand systematically varies. Neither of these phenomena are observed on the DNA strand. The structure of the RNA/DNA hybrid presented here differs significantly from that found in solution for this and other sequences. Possible reasons for these differences and their implications for the current model of RNase H activity are discussed.
Nucleic Acids Research 02/1999; 27(2):555-61. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The solution conformations of the intramolecular triple helices d(AGAAGA-X-TCTTCT-X-TC+TTC+T) and d(AAGGAA-X-TTCCTT-X-TTC+C+TT) (X = non-nucleotide linker) have been determined by NMR.1H NMR spectra in H2O showed that the third strand cytosine residues are fully paired with the guanine residues, each using two Hoogsteen hydrogen bonds. Determination of the13C chemical shifts of the cytosine C6 and C5 and their one-bond coupling constants (1 J CH) conclusively showed that the Hoogsteen cytosine residues are protonated at N3. The global conformations of the two molecules determined with >19 restraints per residue are very similar (RMSD = 0.96 A). However, some differences in local conformation and dynamics were observed for the central two base triplets of the two molecules. The C N3H were less labile in adjacent CG.C+triplets than in non-adjacent ones, indicating that the adjacent charge does not kinetically destabilize these triplets. The sugar conformations of the two adjacent cytosine residues were different and the 5'-residue was atypical of protonated cytosine. Hence, there are subtle effects of the interaction between two adjacent cytosine residues. The central two purines in each sequence showed non-standard backbone conformations, averaging between gamma approximately 60 degrees and gamma approximately 180 degrees. This may be related to the difference in the dependence of the thermodynamic stability on pH observed for these two sequences.
Nucleic Acids Research 08/1998; 26(16):3677-86. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The solution properties of the parallel intramolecular DNA triplex d(GAGAGA-oct-TCTCTC-oct-CTCTCT) (oct = -O-(CH2)8-O-PO2-O-(CH2)8-O-PO2-) and the duplex d(GAGAGA-oct-TCTCTC) have been examined by UV melting and high-resolution nuclear magnetic resonance spectroscopy (NMR). All nucleotides were primarily in the S conformation (i.e. near C2'-endo) in both the duplex and the triplex. However, the sugars of the Hoogsteen pyrimidine strand had a lower fraction of the S state than the Watson-Crick strands. Glycosidic torsion angles derived from nuclear Overhauser effect (NOE) build-up curves were found in the range -103 degrees to -133 degrees, with a clear alternation in magnitude along the GAGAGA strand in the triplex, whereas the glycosidic torsion angles were more similar in the duplex. Internucleotide NOEs were also consistent with an overall B-like geometry, rather than the A family. However, particularly in the Hoogsteen strand, some sequential NOE intensities were intermediate between those of the B and A forms. Distance and torsion constraints derived from NMR experiments were used to generate structures and were refined by restrained molecular dynamics. Extensive chemical shift differences between residues in the triplex and duplex were found for the purine strand, and there were remarkable differences in the pattern of shift differences for the A and G residues that correlated with differences in glycosidic torsion angles. Although there are differences in structure between the free duplex and that in the triplex, they are in important respects similar, indicating that only small conformational adjustments are needed to make parallel triple helices.
[show abstract][hide abstract] ABSTRACT: Intercalating ligands may improve both the stability and sequence specificity of triple helices. Numerous intercalating drugs have been described, including coralyne, which preferentially binds triple helices, though its sequence specificity has been reported to be low [Lee,J.S., Latimer,L.J.P. and Hampel,K.J. (1993) Biochemistry , 32, 5591-5597]. In order to analyse the sequence preferences of coralyne we have used a combination of DNase I footprinting, UV melting, UV-visible spectrophotometry, circular dichroism and NMR spectroscopy to examine defined intermolecular triplexes and intramolecular triplexes linked either by hexaethylene glycol chains or by octandiol chains. DNase I footprinting demonstrated that coralyne has a moderate preference for triplexes over duplexes, but a substantial preference for TA.T triplets compared with CG. C+triplets. The drug was found to have essentially no effect on the melting temperatures of duplexes of the kind d(A)n.d(T)n or d(GA)n.d(TC)n. In contrast, it increased the T m for triplexes of the kind d(T)nd(A)n.dTn, but had little effect on the stability of d(TC)nd(GA).d(CT)n at either low or high pH. On binding to DNA triplexes, there is a large change in the absorption spectrum of coralyne and also a substantial fluorescence quenching that can be attributed to intercalation. The changes in the optical spectra have been used for direct titration with DNA. For triplexes d(T)6d(A)6.d(T)6, the Kd at 298 K was 0.5-0.8 microM. In contrast, the affinity for d(TC) nd(GA)n.d(CT)n triplexes was 6- to 10-fold lower and was characterized by smaller changes in the absorption and CD spectra. This indicates a preference for intercalation between TAT triples over CG.C+/TA.T triples. NMR studies confirmed interaction by intercalation. However, a single, secondary binding was observed at high concentrations of ligand to the triplex d(AGAAGA-L-TCTTCT-L-TCTTCT), presumably owing to the relatively low difference in affinity between the TA.T site and the competing, neighbouring sites.
Nucleic Acids Research 06/1997; 25(10):1890-6. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have developed a sensitive continuous assay for nucleases using proton release. The assay has been applied to the determination of the kinetics of DNase I acting on short, defined deoxyoligonucleotides. The dependence of Kcat/K(m) on sequence and structure of short oligonucleotide substrates has been measured: increasing lengths of AnTn sequences decrease the rate of cleavage. G.A mismatches in which the bases pair using imino protons are cleaved quite effectively by DNase I. In contrast, tandem G.A mismatches which use amino pairing and have BII phosphodiesters, are refractory to DNase I. Also, the DNA strands of DNA.RNA hybrid duplexes are not cleaved by DNase I. These results show that the global conformation of a duplex and the details of its minor groove affect the cleavage efficiency by DNase I. The assay has also been used to measure the inhibition constant of the minor-groove-binding ligand propamidine. A value of 3 microM has been determined for binding to the sequence d(CGCGAATTCGCG)2, showing that dissociation constants can be determined even when there are no convenient optical signals for titrations.
[show abstract][hide abstract] ABSTRACT: The so-called spine of hydration in the minor groove of AnTn tracts in DNA is thought to stabilise the structure, and kinetically bound water detected in the minor groove of such DNA species by NMR has been attributed to a narrow minor groove [Liepinsh, E., Leupin, W. and Otting, G. (1994) Nucleic Acids Res. 22, 2249-2254]. We report here an NMR study of hydration of an RNA dodecamer which has a wide, shallow minor groove. Complete assignments of exchangeable protons, and a large number of non-exchangeable protons in r(CGCAAAUUUGCG)2 have been obtained. In addition, ribose C2'-OH resonances have been detected, which are probably involved in hydrogen bonds. Hydration at different sites in the dodecamer has been measured using ROESY and NOESY experiments at 11.75 and 14.1 T. Base protons in both the major and minor grooves are in contact with water, with effective correlation times for the interaction of approximately 0.5 ns, indicating weak hydration, in contrast to the hydration of adenine C2H in the homologous DNA sequence. NOEs to H1' in the minor groove are consistent with hydration water present that is not observed in the analogous DNA sequence. Hydration kinetics in nucleic acids may be determined by chemical factors such as hydrogen-bonding more than by simple conformational factors such as groove width.
Nucleic Acids Research 11/1996; 24(19):3693-9. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4'-thio-2'-deoxythymidine, has converged at R=0.201 for 2605 reflections with F > 2sigma(F) in the resolution range 8.0-2.4 A for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3'-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1'-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4'-thio-2'-deoxynucleotides.
Nucleic Acids Research 04/1996; 24(5):951-61. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.
Nucleic Acids Research 06/1995; 23(10):1710-6. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The possible formation of inter-base C-H.O hydrogen bonds in A.T, A.U and certain non-Watson-Crick base pairs is examined. A geometrical analysis in conjunction with implications for the thermodynamic stability of the base pairs suggests that C-H.O hydrogen bonds could form in nucleic acid base pairs. They may alleviate destabilizing interactions that would arise if an unsatisfied hydrogen-bond acceptor were present and mediate secondary hydrogen-bonding effects in these base pairs.
[show abstract][hide abstract] ABSTRACT: G.A mismatches form a variety of hydrogen-bonded structures in DNA, most of which destabilise the duplex. Tandem G.A mismatches in the context YGAR (Y = pyrimidine, R = purine), however, form base pairs using the amino group of the guanine residue [Li, Y., Zon. G. & Wilson, W.D. (1991) Proc. Natl Acad. Sci. USA 88, 26-30], which permits extensive base-base stacking, leading to a slight stabilisation of the helix [Ebel, S., Lane, A. N. & Brown, T. (1992) Biochemistry 31, 12083-12086]. We have measured the thermodynamic stability of several RNA and RNA.DNA hybrid duplexes containing tandem G.A mismatches. The RNA duplexes are intrinsically much more stable than the corresponding DNA duplexes and the mutations are destabilising in all cases. NOE and coupling-constant data show that all of the sugars are in the C3'-endo range of conformations, and glycosidic torsion angles are in the range -160 degrees to -180 degrees in [sequence: see text]. Both sequential NOE intensities and circular-dichroism measurements indicate that the global conformation of the mismatched RNA is A-like. The N1H group of the mismatched guanine residue is not involved in hydrogen bonding with the adenine residue, indicating the presence of the amino-pairing scheme. Determination of the structure using 'loose' NMR-derived constraints shows that the potential energies of the imino-paired and amino-paired forms are similar, but substantially higher than energy-minimised RNA. Using tighter constraints derived from more extensive analysis of one-dimensional and two-dimensional NOE data showed that the amino-paired structure agrees with the constraint data better than the imino-paired structure, and also accounts for unusual chemical shifts and the lack of hydrogen bonding of the guanine N1H group. Resulting molecular models show that the amino-paired mismatches are not as extensively stacked on the neighbouring part of the duplex as in the B-DNA analogues, largely accounting for the lower thermodynamic stability in the RNA duplexes.
European Journal of Biochemistry 03/1994; 220(3):703-15. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nearly complete assignments of the 1H-NMR spectrum of the DNA.RNA hybrid d(GTGAACTT).r(AAGUUCAC) have been obtained by two-dimensional methods. Three-bond coupling constants measured from cross-sections of two-dimensional NOE spectra and double quantum-filtered correlation spectra showed that the sugars in the DNA strand are predominantly in the S domain of puckers, whereas the ribofuranoses are mainly C3'-endo. Analysis of time-dependent NOE intensities from one- and two-dimensional experiments showed that the glycosidic torsion angles in the DNA strand are near -120 degrees, whereas those in the RNA strand are in the range- 140 degrees to -160 degrees. These nucleotide conformations correspond to those typically found in B-DNA and A-RNA, respectively. The circular dichroism of the duplex is similar to that of A-form RNA, consistent with a global A-like conformation. A large number of duplex structures was generated in which the nucleotides were fixed in the experimentally determined conformations. A subset of these structures was found that satisfied the internucleotide NOE intensities, backbone constraints and had acceptable Lennard-Jones energies. The base pairs in the duplex were found to have positive inclinations, a translation (Dx) of about 0.4 nm from the helix axis, and more than 10 bp/turn on average. This implies a helical structure in the A family of conformations.
European Journal of Biochemistry 08/1993; 215(2):297-306. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The crystal structure of the DNA hexamer d(TGATCA) complexed with the anthracycline antibiotic idarubicin has been determined at 1.6 A resolution. The asymmetric unit consists of a single hexamer oligonucleotide strand, one drug molecule and 35 water molecules. The complex crystallizes in the tetragonal space group P4(1)2(1)2, Z = 8 with lattice dimensions of a = b = 28.19 (3), c = 52.77 (4) A, V = 41 935 A(3). The structure is isomorphous with a series of hexamer-anthracycline complexes and was solved by molecular replacement. Restrained least-squares methods interspersed with computer-graphics map inspection and model manipulation were used to refine the structure. The R factor is 0.22 for 2032 reflections with F >/= 3sigma(F) in the resolution range 8.0-1.6 A. The self-complementary DNA forms a distorted B-DNA double helix with two idarubicin molecules intercalated in the d(TpG) steps of the duplex. The duplex is formed by utilization of a crystallographic twofold axis of symmetry. The idarubicin chromophore is oriented at right angles to the long axis of the DNA base pairs with the anthracycline amino-sugar moiety positioned in the minor groove. Our structure determination allows for comparison with a d(CGATCG)-idarubicin complex recently reported. Despite the sequence alteration at the intercalation step, the structures are very similar. The geometry of the intercalation and the nature of the interactions are conserved irrespective of the DNA sequence involved in the binding.
[show abstract][hide abstract] ABSTRACT: We have used ultraviolet melting techniques to compare the stability of several DNA duplexes containing tandem G.A mismatches to similar duplexes containing tandem A.G, I.A, and T.A base pairs. We have found that tandem G.A mismatches in 5'-Y-G-A-R-3' duplexes are more stable than their I.A counterparts and that they are sometimes more stable than tandem 5'-Y-T-A-R-3' sequences. This is not the case for tandem G.A mismatches in other base stacking environments, and it suggests that tandem G.A mismatches in 5'-Y-G-A-R-3' sequences have a unique configuration. In contrast to tandem 5'-G-A-3' mismatches, tandem 5'-A-G-3' mismatches were found to be unstable in all cases examined.
[show abstract][hide abstract] ABSTRACT: The structure of the complex between d(TGATCA) and the anthracycline 4'-epiadriamycin has been determined by crystallographic methods. The crystals are tetragonal, space group P4(1)2(1)2 with unit cell dimensions of a = 28.01, c = 52.95A. The asymmetric unit consists of one strand of hexanucleotide, one molecule of 4'-epiadriamycin and 34 waters. The R-factor is 20.2% for 1694 reflections with F greater than or equal to 2 sigma F to 1.7A. Two asymmetric units associate to generate a duplex complexed with two drug molecules at the d(TpG) steps of the duplex. The chromophore intercalates between these base pairs with the anthracycline amino-sugar positioned in the minor groove. The double helix is a distorted B-DNA type structure. Our structure determination of d(TGATCA) complexed to 4'-epiadriamycin allows for comparison with the previously reported structures of 4'-epiadriamycin bound to d(TGTACA) and to d(CGATCG). The three complexes are similar in gross features and the intercalation geometry is the same irrespective of whether a d(CpG) or d(TpG) sequence is involved. However, the orientation of the amino-sugar displays a dependence on the sequence adjacent to the intercalation site. The flexibility of this amino-sugar may help explain why this class of antibiotics displays a relative insensitivity to base sequence when they bind to DNA.
Nucleic Acids Research 08/1992; 20(14):3561-6. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.