Missing-base and ethylation interference footprinting of P1 plasmid replication initiator.

RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA. Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA. In this study, base elimination was used to identify the contribution of all four bases to the binding reaction. Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding. These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA. We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation. These results indicate that certain bases (e.g. G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.     

DNA and its counterions: a molecular dynamics study

The behaviour of mobile counterions, Na⁺ and K⁺, was analysed around a B-DNA double helix with the sequence CCATGCGCTGAC in aqueous solution during two 50 ns long molecular dynamics trajectories. The movement of both monovalent ions remains diffusive in the presence of DNA. Ions sample the complete space available during the simulation time, although individual ions sample only about one-third of the simulation box. Ions preferentially sample electronegative sites around DNA, but direct binding to DNA bases remains a rather rare event, with highest site occupancy values of <13%. The location of direct binding sites depends greatly on the nature of the counterion. While Na⁺ binding in both grooves is strongly sequence-dependent with the preferred binding site in the minor groove, K⁺ mainly visits the major groove and binds close to the centre of the oligomer. The electrostatic potential of an average DNA structure therefore cannot account for the ability of a site to bind a given cation; other factors must also play a role. An extensive analysis of the influence of counterions on DNA conformation showed no evidence of minor groove narrowing upon ion binding. A significant difference between the conformations of the double helix in the different simulations can be attributed to extensive α/γ transitions in the phosphate backbone during the simulation with Na⁺. These transitions, with lifetimes over tens of nanoseconds, however, appear to be correlated with ion binding to phosphates. The ion-specific conformational properties of DNA, hitherto largely overlooked, may play an important role in DNA recognition and binding.     

Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site

The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.     

DNA ligases ensure fidelity by interrogating minor groove contacts

DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3′-hydroxyl and 5′-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3′ side of the ligase junction, but tolerant of mispairs on the 5′ side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3′ side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson–Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson–Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson–Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3′ side of the ligase junction. The NAD⁺-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.     

Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy

Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine {[H₃N(CH₂)₃NH₂(CH₂)₄NH₃]³⁺} and spermine {[(CH₂)₄(NH₂(CH₂)₃NH₃)₂]⁴⁺}. In solutions containing 60 mM DNA phosphate (~20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H₃) are less affected than phosphates by polyamine–DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.     

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

Interaction of Sulforaphane with DNA and RNA

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO₂), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO₂) interaction. Overall binding constants were estimated to be K_(SFN–DNA)=3.01 (± 0.035)×10⁴ M^-1 and K_(SFN–RNA)= 6.63 (±0.042)×10³ M^-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure.     

Hexahydrated magnesium ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes

Magnesium ions play important roles in the structure and function of nucleic acids. Whereas the tertiary folding of RNA often requires magnesium ions binding to tight places where phosphates are clustered, the molecular basis of the interactions of magnesium ions with RNA helical regions is less well understood. We have refined the crystal structures of four decamer oligonucleotides, d(ACCGGCCGGT), r(GCG)d(TATACGC), r(GC)d(GTATACGC) and r(G)d(GCGTATACGC) with bound hexahydrated magnesium ions at high resolution. The structures reveal that A-form nucleic acid has characteristic [Mg(H₂O)₆]²⁺ binding modes. One mode has the ion binding in the deep major groove of a GpN step at the O6/N7 sites of guanine bases via hydrogen bonds. Our crystallographic observations are consistent with the recent NMR observations that in solution [Co(NH₃)₆]³⁺, a model ion of [Mg(H₂O)₆]²⁺, binds in an identical manner. The other mode involves the binding of the ion to phosphates, bridging across the outer mouth of the narrow major groove. These [Mg(H₂O)₆]²⁺ ions are found at the most negative electrostatic potential regions of A-form duplexes. We propose that these two binding modes are important in the global charge neutralization, and therefore stability, of A-form duplexes.     

Unusual DNA duplex and hairpin motifs

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G·A base pairs are separated by one or two pairs of purine·purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G·C or A·T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G·A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G·A, A·A, A·C or sheared-like G_anti·C_syn base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.     

A molecular dynamics simulation study of oriented DNA with polyamine and sodium counterions: diffusion and averaged binding of water and cations

Four different molecular dynamics (MD) simulations have been performed for ordered DNA decamers, d(5′-ATGCAGTCAG)·d(5′-TGACTGCATC). The counterions were the two natural polyamines spermidine³⁺ (Spd³⁺) and putrescine²⁺ (Put²⁺), the synthetic polyamine diaminopropane²⁺ (DAP²⁺) and Na⁺. The simulation set-up corresponds to an infinite array of parallel DNA mimicking the state in oriented DNA fibers or crystals. This work describes general properties of polyamine and Na⁺ binding to DNA. Simulated diffusion coefficients show satisfactory agreement with experimental NMR diffusion data of comparable systems. The interaction of the polyamines with DNA is dynamic in character and the cations mostly form short-lived contacts with the electronegative binding sites of DNA. Polyamines, Na⁺ and water interact most frequently with the charged phosphate atoms with preference for association from the minor groove side with O1P over O2P. There is a strong anti-correlation in the cation binding to the electronegative groups of DNA, i.e. the presence of a cation near one of the DNA sites repels other cations from binding to this and to the other sites separated by <7.5 Å from each other. In contrast to the other polyamines, DAP²⁺ is able to form ‘bridges’ connecting neighboring phosphate groups along the DNA strand. A small fraction of DAP²⁺ and Put²⁺ can be found in the major grooves, while Spd³⁺ is absent there. The results of the MD simulations reveal principal differences in the polyamine–DNA interactions between the natural (Spd³⁺, Put²⁺ and spermine⁴⁺) and synthetic (DAP²⁺) polyamines.     

Recognition of 5-aminouracil (U(#)) in the central strand of a DNA triplex: orientation selective binding of different third strand bases

A necessary feature of the natural base triads for triplex formation is the requirement of a purine (A or G) in the central position, since only these provide sets of two hydrogen bond donors/acceptors in the major groove of the double helix. Pyrimidine bases devoid of this feature have incompatible complementarity and lead to triplexes with lower stability. This paper demonstrates that 5-aminouracil (U^#) (I), a pyrimidine nucleobase analogue of T in which 5-methyl is replaced by 5-amino group, with hydrogen bonding sites on both sides, is compatible in the central position of triplex triad X*U^#·A, where X = A/G/C/T/2-aminopurine (AP), and * and · represent Hoogsteen and Watson–Crick hydrogen bonding patterns respectively. A novel recognition selectivity based on the orientation (parallel/antiparallel) of the third strand purines A, G or AP with A in the parallel motif (A_p*U^#·A), and G/AP in the antiparallel motif (G_ap/AP_ap*U^#·A) is observed. Similarly for pyrimidines in the third strand, C is accepted only in a parallel mode (C_p*U^#·A). Significantly, T is recognised in both parallel and antiparallel modes (T_p/T_ap*U^#·A), with the antiparallel mode being stable compared to the parallel one. The ‘U^#’ triplexes are also more stable than the corresponding control ‘T’ triplexes. The results expand the lexicon of triplex triads with a recognition motif consisting of pyrimidine in the central strand.     

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]₂, in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]₂ as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]₂. Long-lived water molecules with correlation time τ_c longer than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA–DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA–DNA junction adopts an O4′-endo sugar conformation, while the other DNA residues including 3C in the DNA–RNA junction, adopt C1′-exo or C2′-endo conformations. The exchange rates for RNA C2′-OH were found to be ~5–20 s^–1. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂ is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.     

Similarities and differences in interaction of K+ and Na+ with condensed ordered DNA. A molecular dynamics computer simulation study

Four 20 ns molecular dynamics simulations have been performed with two counterions, K⁺ or Na⁺, at two water contents, 15 or 20 H₂O per nucleotide. A hexagonal simulation cell comprised of three identical DNA decamers [d(5′-ATGCAGTCAG) × d(5′-TGACTGCATC)] with periodic boundary condition along the DNA helix was used. The simulation setup mimics the DNA state in oriented DNA fibers or in crystals of DNA oligomers. Variation of counterion nature and water content do not alter averaged DNA structure. K⁺ and Na⁺ binding to DNA are different. K⁺ binds to the electronegative sites of DNA bases in the major and the minor grooves, while Na⁺ interacts preferentially with the phosphate groups. Increase of water causes a shift of both K⁺ and Na⁺ from the first hydration shell of O1P/O2P and of the DNA bases in the minor groove with lesser influence for the cation binding to the bases in the major groove. Mobility of both water and cations in the K–DNA systems is faster than in the Na–DNA systems: Na⁺ organizes and immobilizes water structure around itself and near DNA while for K⁺ water is less organized and more dynamic.     

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements

The interaction of hexamminecobalt(III), Co(NH₃)₆³⁺, with 160 and 3000–8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH₃)₆³⁺ binding up to the molar ratio [Co(NH₃)₆³⁺]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH₃)₆³⁺]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH₃)₆³⁺]/[P] = 0.3. In the case of 3000–8000 bp DNA only two processes were observed: (i) binding up to [Co(NH₃)₆³⁺]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B→Ψ transition after [Co(NH₃)₆³⁺]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH₃)₆³⁺ binding on volume and compressibility have been obtained. The binding of Co(NH₃)₆³⁺ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH₃)₆³⁺: ΔV = 9 cm³ mol^–1 and Δκ = 33 × 10^–4 cm³ mol^–1 bar^–1. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH₃)₆³⁺ and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.     

Two-dimensional NMR studies of d(GGTTAATGCGGT).d(ACCGCATTAACC) complexed with the minor groove binding drug SN-6999.

The dodecadeoxynucleotide duplex d(GGTTAATGCGGT).d(ACCGCATTAACC) and its 1:1 complex with the minor groove binding drug SN-6999 have been prepared and studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments have been obtained for the free duplex by standard methods. The line widths of the resonances in the complex are greater than those observed for the free duplex, which complicates the assignment process. Extensive use of two-quantum spectroscopy was required to determine the scalar correlations for identifying all of the base proton and most of the 1'H-2'H-2'H spin subsystems for the complex. This permitted unambiguous sequence-specific resonance assignments for the complex, which provides the necessary background for a detailed comparison of the structure of the duplex, with and without bound drug. A series of intermolecular NOEs between drug and DNA were identified, providing sufficient structural constraints to position the drug in the minor groove of the duplex. However, the combination of NOEs observed can only be rationalized by a model wherein the drug binds in the minor groove of the DNA in both orientations relative to the long helix axis and exchanges rapidly between the two orientations. The drug binds primarily in the segment of five consecutive dA-dT base pairs d(T3T4A5A6T7).d(A18T19T20A21A22), but surprisingly strong interactions are found to extend one residue in the 3' direction along each strand to G8 and C23. The observation of intermolecular contacts to residues neighboring the AT-rich region demonstrates that the stabilization of the bis(quaternary ammonium) heterocycle family of AT-specific, minor groove binding drugs is not based exclusively on interactions with dA-dT base pairs.     

High salt solution structure of a left-handed RNA double helix

Right-handed RNA duplexes of (CG)_n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)₂ determined in 6 M NaClO₄. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6_(n)-H5′/H5″_(n)-H8_(n+1)-H1′_(n+1)-H6_(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4–7°), whereas GpC steps have large negative values (−61°). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2′-OH groups, and the major groove are well defined. The 2′-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2′-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.     

Change of RNase P RNA function by single base mutation correlates with perturbation of metal ion binding in P4 as determined by NMR spectroscopy

The solution structures of two 27 nt RNA hairpins and their complexes with cobalt(III)-hexammine [Co(NH₃)₆³⁺] were determined by NMR spectroscopy. The RNA hairpins are variants of the P4 region from Escherichia coli RNase P RNA: a U-to-A mutant changing the identity of the bulged nucleotide, and a U-to-C, C-to-U double mutant changing only the bulge position. Structures calculated from NMR constraints show that the RNA hairpins adopt different conformations. In the U-to-C, C-to-U double mutant, the conserved bulged uridine in the P4 wild-type stem is found to be shifted in the 3′-direction by one nucleotide when compared with the wild-type structure. Co(NH₃)₆³⁺ is used as a spectroscopic probe for Mg(H₂O)₆²⁺ binding sites because both complexes have octahedral symmetry and have similar radii. Intermolecular NOE crosspeaks between Co(NH₃)₆³⁺ and RNA protons were used to locate the site of Co(NH₃)₆³⁺ binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop in both mutants, but is shifted 3′ by about one base pair in the double mutant. The change of the metal ion binding site is compared with results obtained on corresponding mutant RNase P RNA molecules as reported by Harris and co-workers (RNA, 1, 210–218).     

Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR

We have performed solid-state ³¹P-¹⁹F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA^2′FUTGGC)·d(GCCAAT(pS)TT GCG) (A₃T₂) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the ^2′FU7–pS19 inter-label distance, was 9.4 ± 0.7 Å, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 Å decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 Å, consistent with the results of a previous solution NMR study of the 2:1 complex. These ³¹P-¹⁹F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7–14 Å in DNA–drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-stranded Polydeoxyribonucleotides. 3. Binding at the Minor Groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dGdC) · poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism signal of TPT bound to DNA was shown to have positive sign in the range 260–295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT forms an angle less than 54° with the long axis of DNA, and hence the TPT molecule cannot intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose cytosines in the major groove were all glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC) · poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.