Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide lesion

One- and two-dimensional NMR spectroscopy has been used combined with molecular dynamics to determine the fine structure of the DNA duplex 5'-d(AGGAGCCACG).d(CGTGGFTCCT) where F is the N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue which is a ring fragmentation product of thymine. The formamide deoxyribose exists as two isomers with respect to the orientation about the peptide bond. The two isomers (trans and cis) are observed in a ratio 3:2 in solution. For both species, the oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. The formamide residue, whatever the isomer, is intrahelical and can pair with the guanine on the opposite strand with one hydrogen bond. For the cis isomer, the residue adopts a syn orientation and is able to form a second hydrogen bond with the guanine on the 5' side on the same strand. Off-resonance ROESY experiments have been used to investigate the chemical exchange observed at low temperature of the duplex. Conformational exchange has only been found for the oligonucleotide with the formamide residue in the trans conformation.     

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.     

Structural study of DNA duplex containing an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide frameshift by NMR and restrained molecular dynamics

The presence of an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide (F) residue, a ring fragmentation product of thymine, in a frameshift context in the sequence 5'-d-(AGGACCACG)*d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. Two-dimensional NMR studies show that the formamide residue, whether the cis or trans isomer, is rotated out of the helix and that the bases on either side of the formamide residue in the sequence, G14 and T16, are stacked over each other in a way similar to normal B-DNA. The cis and trans isomers were observed in the ratio 3:2 in solution. Information extracted from 31P NMR data reveal a modification of the phosphodiester backbone conformation at the extrahelical site, which is also observed during the molecular dynamics simulations.     

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.     

Extra thymidine stacks into the d(CTGGTGCGG).d(CCGCCCAG) duplex. An NMR and model-building study.

NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented.     

An NMR study of d(CTACTGCTTTAG).d(CTAAAGCAGTAG) showing hydration water molecules in the minor groove of a TpA step

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.     

A d(GpG)-platinated decanucleotide duplex is kinked. An extended NMR and molecular mechanics study

A conformational study of the double-stranded decanucleotide d(GCCG*G*ATCGC).d(GCGATCCGGC), with the G* guanines chelating a cis-Pt(NH3)2 moiety, has been accomplished using 1H and 31P NMR, and molecular mechanics. Correlation of the NMR data with molecular models has disclosed an equilibrium between several kinked conformations and has ruled out an unkinked structure. The deformation is localized at the CG*G*.CCG trinucleotide where the helix is kinked by approximately 60 degrees towards the major groove and unwound by 12-19 degrees. The models revealed an unexpected mobility of the cytosine complementary to the 5'-G*. This cytosine can stack on either branch of the kinked complementary strand. The energy barrier between the two positions has been calculated to be less than or equal to 12 kJ/mol. The NMR data are in support of rapid flip-flopping of this cytosine. An explanation for the strong downfield shift observed in the 31P resonance of the G*pG* phosphate is given.     

Characterization of a unique tomaymycin-d(CICGAATTCICG)2 adduct containing two drug molecules per duplex by NMR, fluorescence, and molecular modeling studies

Tomaymycin is a member of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. This antibiotic is proposed to react with the exocyclic 2-amino group (N2) of guanine to form a covalent adduct that lies snugly within the minor groove of DNA. While DNA-footprinting experiments using methidiumpropyl-EDTA have revealed the favored bonding sequences for tomaymycin and related drugs on DNA, the stereochemistry at the covalent bonding site (C-11) and orientation in the minor groove were not established by these experiments. In previous studies using a combined fluorescence, high-field NMR, and molecular modeling approach, we have shown that for tomaymycin there are two diastereomeric species (11R and 11S) on both calf thymus DNA and d(ATGCAT)2. Although we were able to infer the identity (stereochemistry at C-11 and orientation in the minor groove) of the two species on d(ATGCAT)2 by high-field NMR and fluorescence studies, in combination with molecular mechanics calculations, definitive experimental evidence was lacking. We have designed and synthesized a self-complementary 12-mer [d(CICGAATTCICG)2] based on the Dickerson dodecamer [d(CGCGAATTCGCG)2] that bonds identically two tomaymycin molecules, each having a defined orientation and stereochemistry. Thus the bis(tomaymycin)-12-mer adduct maintains the self-complementarity of the original duplex molecule. Two-dimensional proton J-correlated spectroscopy (COSY) of the bis(tomaymycin)-d(CICGAATTCICG)2 adduct (I = inosine) unequivocally shows that C-11 of tomaymycin covalently bonds through N2 of guanine with an 11S stereochemistry in the sequence 5'-CGA-3'.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational energy calculations for dinucleotide molecules: a study of the nucleotide coenzyme nicotinamide adenine dinucleotide (NAD+).

A study of the conformational states of the dinucleotide coenzyme NAD⁺ has been made using semiempirical energy calculations. Taking low-energy mononucleotide structures as starting conformations, energy minimizations have been performed. The lowest energy states are stacked structures, with interactions between the adenine and nicotinamide rings. Some structures show stabilization gained from electrostatic attractions between the positively charged nicotinamide and negatively charged phosphate oxygens. These predictions correlate well with the available experimental data.     

Solution structures of a DNA dodecamer duplex with and without a cisplatin 1,2-d(GG) intrastrand cross-link: comparison with the same DNA duplex containing an oxaliplatin 1,2-d(GG) intrastrand cross-link

Proteins that discriminate between cisplatin-DNA adducts and oxaliplatin-DNA adducts are thought to be responsible for the differences in tumor range, toxicity, and mutagenicity of these two important chemotherapeutic agents. However, the structural basis for differential protein recognition of these adducts has not been determined and could be important for the design of more effective platinum anticancer agents. We have determined high-resolution NMR structures for cisplatin-GG and undamaged DNA dodecamers in the AGGC sequence context and have compared these structures with the oxaliplatin-GG structure in the same sequence context determined previously in our laboratory. This structural study allows the first direct comparison of cisplatin-GG DNA and oxaliplatin-GG DNA solution structures referenced to undamaged DNA in the same sequence context. Non-hydrogen atom rmsds of 0.81 and 1.21 were determined for the 15 lowest-energy structures for cisplatin-GG DNA and undamaged DNA, respectively, indicating good structural convergence. The theoretical NOESY spectra obtained by back-calculation from the final average structures showed excellent agreement with the experimental data, indicating that the final structures are consistent with the NMR data. Several significant conformational differences were observed between the cisplatin-GG adduct and the oxaliplatin-GG adduct, including buckle at the 5' G6.C19 base pair, opening at the 3' G7.C18 base pair, twist at the A5G6.T20C19 base pair step, slide, twist, and roll at the G6G7.C19C18 base pair step, slide at the G7C8.C18G17 base pair step, G6G7 dihedral angle, and overall bend angle. We hypothesize that these conformational differences may be related to the ability of various DNA repair proteins, DNA binding proteins, and DNA polymerases to discriminate between cisplatin-GG and oxaliplatin-GG adducts.     

NMR studies of a deoxyribodecanucleotide containing an extrahelical thymidine surrounded by an oligo(dA).oligo(dT) tract

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.     

Conformations of an adenine bulge in a DNA octamer and its influence on DNA structure from molecular dynamics simulations.

Molecular dynamics simulations have been applied to the DNA octamer d(GCGCA-GAAC). d(GTTCGCGC), which has an adenine bulge at the center to determine the pathway for interconversion between the stacked and extended forms. These forms are known to be important in the molecular recognition of bulges. From a total of ~35 ns of simulation time with the most recent CHARMM27 force field a variety of distinct conformations and subconformations are found. Stacked and fully looped-out forms are in excellent agreement with experimental data from NMR and x-ray crystallography. Furthermore, in a number of conformations the bulge base associates with the minor groove to varying degrees. Transitions between many of the conformations are observed in the simulations and used to propose a complete transition pathway between the stacked and fully extended conformations. The effect on the surrounding DNA sequence is investigated and biological implications of the accessible conformational space and the suggested transition pathway are discussed, in particular for the interaction of the MS2 replicase operator RNA with its coat protein.     

NMR structure of an antisense DNA.RNA hybrid duplex containing a 3'-CH(2)N(CH(3))-O-5' or an MMI backbone linker.

The solution structure of an antisense DNA.RNA hybrid duplex, d(CGCGTT-MMI-TTGCGC).r(GCGCAAAACGCG) (designated R4), containing an MMI backbone linker [3'-CH(2)N(CH(3))-O5'], is elucidated. The structural details of the MMI linker, its structural effects on the neighboring residues, and the molecular basis of the MMI effects are examined. The lipophilic N-methyl group of MMI is peripheral to the helix, assuming a conformation that is most stable with regard to the N-O torsion angle. The MMI linker promotes a 3'-endo conformation for the sugar moieties at both 3'- and 5'-adjacent positions and a backbone kink involving distant residues along the 3'-direction. Comparison of R4 with other analogous hybrid duplexes previously studied in this laboratory reveals a new family of low-energy helical conformations that can be accommodated in stable duplexes and a common feature of C3'-modified sugars for adopting a C3'-endo pucker. The results of these studies emphasize the interplay of several factors that govern the formation of stable hybrid duplexes and provide a basis for the understanding of the biological role of the MMI modifications, which are important building blocks for a family of promising chimeric antisense oligonucleotides.     

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)<==>(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)<==>(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A6, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in alpha, sigma and gamma torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.     

The solution structure of an oligonucleotide duplex containing a 2'-deoxyadenosine-3-(2-hydroxyethyl)- 2'-deoxyuridine base pair determined by NMR and molecular dynamics studies

Determination of the solution structure of the duplex d(GCAAGTC(HE)AAAACG)·d(CGTTTTAGACTTGC) containing a 3-(2-hydroxyethyl)-2′-deoxyuridine·deoxyadenine (HE·A) base pair is reported. The three-dimensional solution structure, determined starting from 512 models via restrained molecular mechanics using inter-proton distances and torsion angles, converged to two final families of structures. For both families the HE and the opposite A residues are intrahelical and in the anti conformation. The hydroxyethyl chain lies close to the helix axis and for one family the hydroxyl group is above the HE·A plane and in the other case it is below. These two models were used to start molecular dynamic calculations with explicit solvent to explore the hydrogen bonding possibilities of the HE·A base pair. The dynamics calculations converge finally to one model structure in which two hydrogen bonds are formed. The first is formed all the time and is between HEO4 and the amino group of A, and the second, an intermittent one, is between the hydroxyl group and the N1 of A. When this second hydrogen bond is not formed a weak interaction CH···N is possible between HEC7H2 and N1A21. All the best structures show an increase in the C1′–C1′ distance relative to a Watson–Crick base pair.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of an 11-mer duplex containing the 3, N(4)-ethenocytosine adduct opposite 2'-deoxycytidine: implications for the recognition of exocyclic lesions by DNA glycosylases

Lipid peroxidation products, as well as the metabolic products of vinyl chloride, react with cellular DNA producing the mutagenic adduct 3,N(4)-etheno-2'-deoxycytidine (epsilondC), along with several other exocyclic derivatives. High-resolution NMR spectroscopy and restrained molecular dynamics simulations were used to establish the solution structure of an 11-mer duplex containing an epsilondC.dC base-pair at its center. The NMR data suggested a regular right-handed helical structure having all residues in the anti orientation around the glycosydic torsion angle and Watson-Crick alignments for all canonical base-pairs of the duplex. Restrained molecular dynamics generated a three-dimensional model in excellent agreement with the spectroscopic data. The (epsilondC. dC)-duplex structure is a regular right-handed helix with a slight bend at the lesion site and no severe distortions of the sugar-phosphate backbone. The epsilondC adduct and its partner dC were displaced towards opposite grooves of the helix, resulting in a lesion-containing base-pair that was highly sheared but stabilized to some degree by the formation of a single hydrogen bond. Such a sheared base-pair alignment at the lesion site was previously observed for epsilondC.dG and epsilondC.T duplexes, and was also present in the crystal structures of duplexes containing dG.T and dG. U mismatches. These observations suggest the existence of a substrate structural motif that may be recognized by specific DNA glycosylases during the process of base excision repair.     

Precursor mRNA from erythroid-enriched bone-marrow cells of the rabbit. Electron microscope investigation of precursor mRNA molecules, molecular weight about 1.7 X 10(7), containing mRNA-like structures at one end.

Precursor mRNA (pre-mRNA) molecules, sedimenting at greater than 45 S, from erythroid-enriched bone marrow cells of the rabbit and hemoglobin mRNA molecules from rabbit reticulocytes were investigated by electron microscopy. Four of 98 measured pre-mRNA molecules had a length between 15 and 17.1 mum. In some of the pre-mRNA molecules a characteristic condensed structure was observed at one end, strikingly resembling the structure of the mRNA molecules.     

Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.     

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.