Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Oligonucleotide--minor groove binder 1:2 conjugates: side by side parallel minor groove binder motif in stabilization of DNA duplex

Synthetic polycarboxamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove when they form hairpin structures with side-by-side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide-linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5'-CGTTTATTp-3'/5'-AATAAACG-3' melts at 20 degrees C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46 degrees C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60 degrees C. It is even higher than in case of "classic" octapyrrole hairpin ligand (Tm = 58 degrees C). Sequence-specific character of stabilization by two conjugated ligands was demonstrated for G:C-containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and beta units according to established recognition rules (deltaTm = 20 degrees C). The two-strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site-specific ligands recognizing single- and double-stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

Position-specific suppression and enhancement of HIV-1 integrase reactions by minor groove benzo[a]pyrene diol epoxide deoxyguanine adducts: implications for molecular interactions between integrase and substrates

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.     

Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.     

The width of the minor groove affects the binding of the bisquaternary heterocycle SN-6999 to duplex DNA.

A complex between d(GGGAAAAACGG).d(CCGTTTTTCCC) and the minor groove binding drug SN-6999 has been studied by 1H nuclear magnetic resonance spectroscopy. The drug is found to bind in the d(A)5 tract, but with interactions extending one residue in the 3'-direction along each strand. Doubling of resonances in the complex indicates slow to intermediate exchange between two binding modes. An orientational preference (7:3) is found, the first such example in an SN-6999 complex. Furthermore, the upper limit of the lifetime for the major species is longer than was found for SN-6999 with other DNA duplexes. The preferred orientation of SN-6999 has the pyridinium ring near the 5'-end of the (+) strand; the minor binding mode has the reverse orientation. The orientational preference and slower exchange rate relative to other SN-6999 complexes is attributed to increased stabilization from van der Waals interactions due to better shape complementarity between the DNA duplex and ligand. The comparison of these results with studies of SN-6999 complexed to other DNA duplexes reveals the sensitivity of the binding properties to the delicate interplay between the molecular structure of the ligand and the specific characteristics of the DNA minor groove.     

NMR structural studies of a 15-mer DNA sequence from a ras protooncogene, modified at the first base of codon 61 with the carcinogen 4-aminobiphenyl.

Proton NMR studies were conducted on the complementary 15-mer duplex d(5'-TACTCTTCTTGACCT).(5'-AGGTCAAGAAGAGTA) (designated as unmodified 15-mer duplex) spanning a portion of the mouse c-Ha-ras protooncogene centered around codon 61. Identical studies were carried out on the same sequence, after specific modification with a reactive derivative of the carcinogen 4-aminobiphenyl (ABP), which resulted in incorporation of a single N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct in the noncoding strand (designated as ABP-modified 15-mer duplex). The adduct was located at the position corresponding to the first base of codon 61. The NMR data for the unmodified 15-mer duplex were fully consistent with a standard right-handed B-type DNA duplex conformation, with the possible exception of the frayed terminal base pairs. The ABP-modified 15-mer duplex was found to adopt one major conformation, although at least one additional conformation could be detected especially near room temperature. The major form, which exhibited strikingly similar NOE patterns as to those of the parent oligomer, both in H2O and D2O spectra, assumed a standard Watson-Crick base pairing throughout the entire length of the duplex, including the modification site and its flanking base pairs. Although some local perturbation of the helix could be detected in the vicinity of the modified guanosine, the NOE distance constraints established that the helix was globally right-handed and that the glycosidic torsion angles had the normal anti orientation, both at the modified base and its partner cytidine. Furthermore, the absence of strong NOE interactions between protons in the ABP moiety, which was rapidly rotating, and the nucleic acid protons was consistent with positioning of the arylamine moiety in the major groove of a weakly distorted double-helical structure. Although insufficient data prevented a detailed characterization of the minor conformer(s), the observation of significant shieldings for all the arylamine protons indicated a different orientation at the modified site in the minor contributor(s), possibly with extensive stacking between the ABP fragment and the neighboring bases.     

Oligonucleotide—Minor Groove Binder 1:2 Conjugates: Side by Side Parallel Minor Groove Binder Motif in Stabilization of DNA Duplex

Synthetic polycarboxamides consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove when they form hairpin structures with side‐by‐side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide‐linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5′‐CGTTTATTp‐3′/5′‐AATAAACG‐3′ melts at 20°C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46°C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60°C. It is even higher than in case of “classic” octapyrrole hairpin ligand (Tm = 58°C). Sequence‐specific character of stabilization by two conjugated ligands was demonstrated for G:C‐containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and β units according to established recognition rules (ΔTm = 20°C). The two‐strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site‐specific ligands recognizing single‐ and double‐stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.     

A molecular model for RecA-promoted strand exchange via parallel triple-stranded helices.

A number of studies have concluded that strand exchange between a RecA-complexed DNA single strand and a homologous DNA duplex occurs via a single-strand invasion of the minor groove of the duplex. Using molecular modeling, we have previously demonstrated the possibility of forming a parallel triple helix in which the single strand interacts with the intact duplex in the minor groove, via novel base interactions (Bertucat et al., J. Biomol. Struct. Dynam. 16:535-546). This triplex is stabilized by the stretching and unwinding imposed by RecA. In the present study, we show that the bases within this triplex are appropriately placed to undergo strand exchange. Strand exchange is found to be exothermic and to result in a triple helix in which the new single strand occupies the major groove. This structure, which can be equated to so-called R-form DNA, can be further stabilized by compression and rewinding. We are consequently able to propose a detailed, atomic-scale model of RecA-promoted strand exchange. This model, which is supported by a variety of experimental data, suggests that the role of RecA is principally to prepare the single strand for its future interactions, to guide a minor groove attack on duplex DNA, and to stabilize the resulting, stretched triplex, which intrinsically favors strand exchange. We also discuss how this mechanism can incorporate homologous recognition.     

Minor groove DNA alkylation directed by major groove triplex forming oligodeoxyribonucleotides.

We describe sequence-specific alkylation in the minor groove of double-stranded DNA by a hybridization-triggered reactive group conjugated to a triplex forming oligodeoxyribonucleotide (TFO) that binds in the major groove. The 24 nt TFOs (G/A motif) were designed to form triplexes with a homopurine tract within a 65 bp target duplex. They were conjugated to an N 5-methyl-cyclopropapyrroloindole (MCPI) residue, a structural analog of cyclopropapyrroloindole (CPI), the reactive subunit of the potent antibiotic CC-1065. These moieties react in the DNA minor groove, alkylating adenines at their N3 position. In order to optimize alkylation efficiency, linkers between the TFO and the MCPI were varied both in length and composition. Quantitative alkylation of target DNA was achieved when the dihydropyrroloindole (DPI) subunit of CC-1065 was incorporated between an octa(propylene phosphate) linker and MCPI. The required long linker traversed one strand of the target duplex from the major groove-bound TFO to deliver the reactive group to the minor groove. Alkylation was directed by relative positioning of the TFOs. Sites in the minor groove within 4-8 nt from the end of the TFO bearing the reactive group were selectively alkylated.     

NF-kappa B binding mechanism: a nuclear magnetic resonance and modeling study of a GGG --> CTC mutation

We present the solution structure of the nonpalindromic 16 bp DNA 5'd(CTGCTCACTTTCCAGG)3'. 5'd(CCTGGAAAGTGAGCAG)3' containing a mutated kappaB site for which the mutation of a highly conserved GGG tract of the native kappaB HIV-1 site to CTC abolishes NF-kappaB binding. 1H and 31P NMR spectroscopies have been used together with molecular modeling to determine the fine structure of the duplex. NMR data show evidence for a BI-BII equilibrium of the CpA.TpG steps at the 3'-end of the oligomer. Models for the extreme conformations reached by the mutated duplex (denoted 16M) are proposed in agreement with the NMR data. Since the distribution of BII sites is changed in the mutated duplex compared to that of the native duplex (denoted 16N), large differences are induced in the intrinsic structural properties of both duplexes. In particular, in BII structures, 16M shows a kink located at the 3'-end of the duplex, and in contrast, 16N exhibits an intrinsic global curvature toward the major groove. Whereas 16N can reach a conformation very favorable for the interaction with NF-kappaB, 16M cannot mimic such a conformation and, moreover, its deeper and narrower major groove could hinder the DNA-protein interactions.     

Structure of a triple helical DNA with a triplex-duplex junction

Extended purine sequences on a DNA strand can lead to the formation of triplex DNA in which the third strand runs parallel to the purine strand. Triplex DNA structures have been proposed to play a role in gene expression and recombination and also have potential application as antisense inhibitors of gene expression. Triplex structures have been studied in solution by NMR, but have hitherto resisted attempts at crystallization. Here, we report a novel design of DNA sequences, which allows the first crystallographic study of DNA segment containing triplexes and its junction with a duplex. In the 1.8 A resolution structure, the sugar-phosphate backbone of the third strand is parallel to the purine-rich strand. The bases of the third strand associate with the Watson and Crick duplex via Hoogsteen-type interactions, resulting in three consecutive C(+).GC, BU.ABU (BU = 5-bromouracil), and C(+).GC triplets. The overall conformation of the DNA triplex has some similarity to the B-form, but is distinct from both A- and B-forms. There are large changes in the phosphate backbone torsion angles (particularly gamma) of the purine strand, probably due to the electrostatic interactions between the phosphate groups and the protonated cytosine. These changes narrow the minor groove width of the purine-Hoogsteen strands and may represent sequence-specific structural variations of the DNA triplex.     

Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of a covalent CPI-CDPI2-oligodeoxyribonucleotide decamer complex

CPI-CDPI2 is a synthetic analogue of CC-1065, which is a naturally occurring antitumor antibiotic. Assignment of the 1H NMR spectra of a CPI-CDPI2-oligodeoxyribonucleotide decamer, d-(CGCTTAAGCG)2, complex has been made by two-dimensional 1H/1H spectroscopy. The solution structure of the complex was calculated by an iterative hybrid relaxation matrix method combined with NOESY distance restrained molecular dynamics. Refinement proceeded in two steps in which the decamer was initially refined alone and then CPI-CDPI2 was added to the structure to allow initial estimates of drug-DNA contacts. A hybrid matrix/MD refinement was used to better take into account problems associated with spin diffusion. Thus the distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experimental spectrum and those calculated from an initial structure. The hybrid matrix derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. The efficacy of CC-1065 has been attributed to its minor groove binding and alkylation to the N3 position of adenosine. CPI-CDPI2 appears to bind to the decamer in a similar manner. The effect of CPI-CDPI2 on the decamer's 1H and 31P spectrum was consistent with a minor groove binding motif with the drug alkylating at A17 with the CDPI rings oriented toward the 5'-end of the alkylated strand. In addition, the NMR data support one major adduct but also indicate the presence of a minor adduct. The latter could represent a drug alkylation of the DNA at a secondary site (or alternative orientation of the rings).     

A model for parallel triple helix formation by RecA: single-single association with a homologous duplex via the minor groove

The nucleoproteic filaments of RecA polymerized on single stranded DNA are able to integrate double stranded DNA in a coaxial arrangement (with DNA stretched by a factor 1.5), to recognize homologous sequences in the duplex and to perform strand exchange between the single stranded and double stranded molecules. While experimental results favor the hypothesis of an invasion of the minor groove of the duplex by the single strand, parallel minor groove triple helices have never been isolated or even modeled, the minor groove offering little space for a third strand to interact. Based on an internal coordinate modeling study, we show here that such a structure is perfectly conceivable when the two interacting oligomers are stretched by a factor 1.5, in order to open the minor groove of the duplex. The model helix presents characteristics that coincide with known experimental data on unwinding, base pair inclination and inter-proton distances. Moreover, we show that extension and unwinding stabilize the triple helix. New patterns of triplet interaction via the minor groove are presented.     

A Model for Parallel Triple Helix Formation by Ree A: Single-Strand Association with a Homologous Duple via the Minor Groove

Abstract

The nucleoproteic filaments of RecA polymerized on single stranded DNA are able to integrate double stranded DNA in a coaxial arrangement (with DNA stretched by a factor 1.5), to recognize homologous sequences in the duplex and to perform strand exchange between the single stranded and double stranded molecules. While experimental results favor the hypothesis of an invasion of the minor groove of the duplex by the single strand, parallel minor groove triple helices have never been isolated or even modeled, the minor groove offering little space for a third strand to interact. Based on an internal coordinate modeling study, we show here that such a structure is perfectly conceivable when the two interacting oligomers are stretched by a factor 1.5, in order to open the minor groove of the duplex. The model helix presents characteristics that coincide with known experimental data on unwinding, base pair inclination and inter-proton distances. Moreover, we show that extension and unwinding stabilize the triple helix. New patterns of triplet interaction via the minor groove are presented.     

The solution structure of a locked nucleic acid (LNA) hybridized to DNA

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Solution structure of an oligodeoxynucleotide containing the malondialdehyde deoxyguanosine adduct N2-(3-oxo-1-propenyl)-dG (ring-opened M1G) positioned in a (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene.

The refined solution structure for the ring-opened N2-(3-oxo-1-propenyl)-dG derivative of the malondialdehyde deoxyguanosine adduct M(1)G [3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1, 2-a]purin-10(3H)-one] in d(ATCGCXCGGCATG) x d(CATGCCGCGCGAT) [X being N(2)-(3-oxo-1-propenyl)-dG], containing the d(CpG)(3) frameshift hotspot of the Salmonella typhimurium hisD3052 gene, is presented. When inserted into this duplex, M(1)G underwent spontaneous ring opening to N2-(3-oxo-1-propenyl)-dG. NMR analysis revealed that N2-(3-oxo-1-propenyl)-dG induced minor structural perturbations in the hisD3052 oligodeoxynucleotide. However, the stability of the duplex DNA was reduced; the N2-(3-oxo-1-propenyl)-dG-modified hisD3052 oligodeoxynucleotide exhibited a 14 degrees C decrease in T(m) relative to that of the native oligodeoxynucleotide. The modified guanine maintained stacking interactions with neighboring bases but was not Watson-Crick hydrogen bonded. A total of 13 NOEs were observed from the 3-oxo-1-propenyl moiety protons of N2-(3-oxo-1-propenyl)-dG to DNA protons. Molecular dynamics calculations, restrained by 602 distance restraints derived from experimental NOE measurements and 23 empirical distance restraints, converged with pairwise rmsd differences of <0.90 A. The sixth-root residual factor with the NMR data was 9.1 x 10(-2). The cytosine complementary to N2-(3-oxo-1-propenyl)-dG was pushed toward the major groove but maintained partial stacking interactions with its neighboring bases. The modified guanine remained in the anti conformation, while the 3-oxo-1-propenyl moiety was positioned in the minor groove of the duplex. Possible correlations between the relatively small structural perturbations induced in this DNA duplex by N2-(3-oxo-1-propenyl)-dG and the mutagenic spectrum of M(1)G are discussed.