Alternative heterocycles for DNA recognition: a 3-pyrazole/pyrrole pair specifies for G.C base pairs

Synthetic ligands comprising three aromatic amino acids, pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp), specifically recognize predetermined sequences as side-by-side pairs in the minor groove of DNA. To expand the repertoire of aromatic rings that may be utilized for minor groove recognition, three five-membered heterocyclic rings, 3-pyrazolecarboxylic acid (3-Pz), 4-pyrazolecarboxylic acid (4-Pz), and furan-2-carboxylic acid (Fr), were examined at the N-terminus of eight-ring hairpin polyamide ligands. The DNA binding properties of 3-Pz, 4-Pz, and Fr each paired with Py were studied by quantitative DNase I footprinting titrations on a 283 bp DNA restriction fragment containing four 6-bp binding sites 5'-ATNCCTAA-3' (N = G, C, A, or T; 6-bp polyamide binding site is underlined). The pair 3-Pz/Py has increased binding affinity and sequence specificity for G.C bp compared with Im/Py.     

Alternative heterocycles for DNA recognition: an N-methylpyrazole/N-methylpyrrole pair specifies for A.T/T.A base pairs

Side-by-side pairs of three five-membered rings, N-methylpyrrole (Py), N-methylimidazole (Im), and N-methylhydroxy-pyrrole (Hp), have been demonstrated to distinguish each of the four Watson Crick base pairs in the minor groove of DNA. However, not all DNA sequences targeted by these pairing rules achieve affinities and specificities comparable to DNA binding proteins. We have initiated a search for new heterocycles which can expand the sequence repetoire currently available. Two heterocyclic aromatic amino acids. N-methylpyrazole (Pz) and 4-methylthiazole (Th), were incorporated into a single position of an eight-ring polyamide of sequence ImImXPy-gamma-lmPyPyPy-beta-Dp to examine the modulation of affinity and specificity for DNA binding by a Pz/Py pair and or a Th/Py pair. The X/Py pairings Pz/Py and Th/Py were evaluated by quantitative DNase I footprint titrations on a DNA fragment with the four sites 5'-TGGNCA-3' (N=T, A, G, C). The Pz/Py pair binds T.A and A.T with similar affinity to a Py/Py pair but with improved specificity. disfavoring both G.C and C.G by about 100-fold. The Th/Py pair binds poorly to all four Watson Crick base pairs. These results demonstrate that in some instances new heterocyclic aromatic amino acid pairs can be incorporated into imidazole-pyrrole polyamides to mimic the DNA specificity of Py/Py pairs which may be relevant as biological criteria in animal studies become important.     

Oligonucleotide-minor groove binder conjugates and their complexes with complementary DNA: effect of conjugate structural factors on the thermal stability of duplexes

Synthetic polycarboxamide minor groove binders (MGB) 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 in side-by-side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence-specific manner, similarly to the corresponding mono-conjugated hairpin structures. The series of conjugates with the general formula Oligo-(L-MGB-R)m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = -(Py)n-, -(Im)n- or -[(Py/Im)n-(CH2)3CONH-(Py/Im)n-] and I < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis-phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence-specific parallel ligand [-L(Py)4R] to monophosphoroamidate conjugate CGTTTATT-L(Py)4R leads to the increase of the duplex Tm, whereas attachment of [-L(Im)4R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

Shape selective recognition of T.A base pairs by hairpin polyamides containing N-terminal 3-methoxy (and 3-chloro) thiophene residues

Hairpin polyamides selectively recognize predetermined DNA sequences with affinities comparable to naturally occurring proteins. Internal side-by-side pairs of unsymmetrical aromatic rings within the minor groove of DNA distinguish each of the four Watson-Crick base pairs. In contrast, N-terminal ring pairs exhibit less specificity, with the exception of Im/Py targeting G.C base pairs. In an effort to explore the sequence specificity of new ring pairs, a series of hairpin polyamides containing 3-substituted-thiophene-2-carboxamide residues at the N-terminus was synthesized. An N-terminal 3-methoxy (or 3-chloro) thiophene residue paired opposite Py displayed 6- (and 3-) fold selectivity for T.A relative to A.T base pair, while disfavoring G,C base pairs by >200-fold. Our data suggests shape selective recognition with projection of the 3-thiophene substituent (methoxy or chloro) to the floor of the minor groove.     

DNA minor-groove recognition by 3-methylthiophene/pyrrole pair

Hairpin polyamides are synthetic oligomers, which fold and bind to specific DNA sequences in a programmable manner. Internal side-by-side pairings of the aromatic amino acid residues 1-methyl-1H-pyrrole (Py), 1-methyl-1H-imidazole (Im), and 3-hydroxy-1-methyl-1H-pyrrole (Hp) confer the ability to distinguish between all four Watson-Crick base pairs in the minor groove of B-form DNA. In a broad search to expand the heterocycle repertoire, we found that when 3-methylthiophene (Tn), which presents a S-atom to the minor groove, is paired with Py, it exhibits a modest threefold specificity for TA>AT presumably by shape-selective recognition. In this study, we explore the scope and limitations of this lead by incorporating multiple Tn residues within a single hairpin polyamide. It was found that hairpin polyamides containing more that one Tn/Py pair exhibit lowered affinities and specificities for their match sites. It appears that little deviation is permissible from the parent five-membered ring 1-methyl-1H-pyrrole-2-carboxamide scaffold for DNA recognition.     

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.     

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.     

Oligonucleotide–Minor Groove Binder Conjugates and Their Complexes with Complementary DNA: Effect of Conjugate Structural Factors on the Thermal Stability of Duplexes

Synthetic polycarboxamide minor groove binders (MGB) 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 in side‐by‐side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence‐specific manner, similarly to the corresponding mono‐conjugated hairpin structures. The series of conjugates with the general formula Oligo‐(L‐MGB‐R)_m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = –(Py)_n–, –(Im)_n– or –[(Py/Im)_n–(CH₂)₃CONH–(Py/Im)_n–] and 1 < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis‐phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence‐specific parallel ligand [–L(Py)₄R] to monophosphoroamidate conjugate CGTTTATT–L(Py)₄R leads to the increase of the duplex Tm, whereas attachment of [–L(Im)₄R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

DNA sequence recognition of thiazole-containing cross-linked polyamides can be favored

The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove ligands

The effect of structural factors on the stability of duplexes formed by DNA minor groove binders conjugated with oligonucleotide mono- or diphosphoramidates of the general formula Oligo-MGBm (where Oligo is an oligonucleotide; m = 1 or 2; MGB is -L(Py)2R, L(Py)4R, -L(Im)4R, or -L(Py)4NH(CH2)3CO(Py)4R; Py is a 4-aminopyrrol-2-carboxylic acid residue, L is a gamma-aminobutyric acid or an epsilon-aminocaproic acid residue, R = OEt, NH(CH2)6NEt2, or NH(CH2)6N+Me3) was studied by the method of thermal denaturation. The mode of binder interaction with minor groove depends on the conjugate structure; it may be of the parallel head to head type for bisphosphoramidates and of the antiparallel head to tail type for monophosphoramidates of a hair-pin structure. The effects of the duplexes with parallel orientation (bisphosphoramidates, MGB is L(Py)4R, m = 2) and those of the hairpin structure with the antiparallel orientation (monophosphoramidates, MGB is L(Py)4(CH2)3CO(Py)4R, m = 1) on Tm values were close. The influence of the linker (L) and substituent (R) structures upon Tm was more pronounced for monophosphoramidate (MGB is L(Py)nR, m = 1) than for bisphosphoramidate (MGB is L(Py)nR, m = 2). No more than two oligopyrrolcarboxamide residues (either in parallel or antiparallel orientations) can be incorporated into the duplex minor groove. Moreover, it was shown by the example of monophosphoramidates (Oligo-L(Py)4R and Oligo-L(Py)4NH(CH2)3CO(Py)4R) that the addition of a second ligand capable of incorporation into the minor groove increased Tm of the corresponding duplex in comparison with the duplex formed by the starting monophosphoramidate. At the same time, the introduction of the ligand incapable of incorporating decreased the Tm value. The mode of interaction of the conjugated ligand with the oligonucleotide duplex is determined by its structure. For example, dipyrrolcarboxamide containing an ethoxy group at the ligand C-end stabilizes the duplex due to the stacking interaction with the terminal A*T pair, whereas tetrapyrrolcarboxamides stabilize the duplex by incorporation into the minor groove.     

Crystallographic study of one turn of G/C-rich B-DNA

The DNA decamer d(CCAGGCCTGG) has been studied by X-ray crystallography. At a nominal resolution of 1.6 A, the structure was refined to R = 16.9% using stereochemical restraints. The oligodeoxyribonucleotide forms a straight B-DNA double helix with crystallographic dyad symmetry and ten base-pairs per turn. In the crystal lattice, DNA fragments stack end-to-end along the c-axis to form continuous double helices. The overall helical structure and, notably, the groove dimensions of the decamer are more similar to standard, fiber diffraction-determined B-DNA than A-tract DNA. A unique stacking geometry is observed at the CA/TG base-pair step, where an increased rotation about the helix axis and a sliding motion of the base-pairs along their long axes leads to a superposition of the base rings with neighboring carbonyl and amino functions. Three-center (bifurcated) hydrogen bonds are possible at the CC/GG base-pair steps of the decamer. In their common sequence elements, d(CCAGGCCTGG) and the related G.A mismatch decamer d(CCAAGATTGG) show very similar three-dimensional structures, except that d(CCAGGCCTGG) appears to have a less regularly hydrated minor groove. The paucity of minor groove hydration in the center of the decamer may be a general feature of G/C-rich DNA and explain its relative instability in the B-form of DNA.     

Molecular topology of polycyclic aromatic carcinogens determines DNA adduct conformation: a link to tumorigenic activity

We report below on the solution structures of stereoisomeric "fjord" region trans-anti-benzo[c]phenanthrene-N2-guanine (designated (BPh)G) adducts positioned opposite cytosine within the (C-(BPh)G-C).(G-C-G) sequence context. We observe intercalation of the phenanthrenyl ring with stereoisomer-dependent directionality, without disruption of the modified (BPh)G.C base-pair. Intercalation occurs to the 5' side of the modified strand for the 1S stereoisomeric adduct and to the 3' side for the 1R stereoisomeric adduct, with the S and R-trans-isomers related to one another by inversion in a mirror plane at all four chiral carbon atoms on the benzylic ring. Intercalation of the fjord region BPh ring into the helix without disruption of the modified base-pair is achieved through buckling of the (BPh)G.C base-pair, displacement of the linkage bond from the plane of the (BPh)G base, adaptation of a chair pucker by the BPh benzylic ring and the propeller-like deviation from planarity of the BPh phenanthrenyl ring. It is noteworthy that intercalation without base-pair disruption occurs from the minor groove side for S and R-trans-anti BPh-N2-guanine adducts opposite C, in contrast to our previous demonstration of intercalation without modified base-pair disruption from the major groove side for S and R-trans-anti BPh-N6-adenine adducts opposite T. Further, these results on fjord region 1S and 1R-trans-anti (BPh)G adducts positioned opposite C are in striking contrast to earlier research with "bay" region benzo[a]pyrene-N2-guanine (designated (BP)G) adducts positioned opposite cytosine, where 10S and 10R-trans-anti stereoisomers were positioned with opposite directionality in the minor groove without modified base-pair disruption. They also are in contrast to the 10S and 10R-cis-anti stereoisomers of (BP)G adducts opposite C, where the pyrenyl ring is intercalated into the helix with directionality, but the modified base and its partner on the opposite strand are displaced out of the helix. These results are especially significant given the known greater tumorigenic potential of fjord region compared to bay region polycyclic aromatic hydrocarbons. The tumorigenic potential has been linked to repair efficiency such that bay region adducts can be readily repaired while their fjord region counterparts are refractory to repair. Our structural results propose a link between DNA adduct conformation and repair-dependent mutagenic activity, which could ultimately translate into structure-dependent differences in tumorigenic activities. We propose that the fjord region minor groove-linked BPh-N2-guanine and major groove-linked BPh-N6-adenine adducts are refractory to repair based on our observations that the phenanthrenyl ring intercalates into the helix without modified base-pair disruption. The helix is therefore minimally perturbed and the phenanthrenyl ring is not available for recognition by the repair machinery. By contrast, the bay region BP-N2-G adducts are susceptible to repair, since the repair machinery can recognize either the pyrenyl ring positioned in the minor groove for the trans-anti groove-aligned stereoisomers, or the disrupted modified base-pair for the cis-anti base-displaced intercalated stereoisomers.     

Minor groove deformability of DNA: a molecular dynamics free energy simulation study

The conformational deformability of nucleic acids can influence their function and recognition by proteins. A class of DNA binding proteins including the TATA box binding protein binds to the DNA minor groove, resulting in an opening of the minor groove and DNA bending toward the major groove. Explicit solvent molecular dynamics simulations in combination with the umbrella sampling approach have been performed to investigate the molecular mechanism of DNA minor groove deformations and the indirect energetic contribution to protein binding. As a reaction coordinate, the distance between backbone segments on opposite strands was used. The resulting deformed structures showed close agreement with experimental DNA structures in complex with minor groove-binding proteins. The calculated free energy of minor groove deformation was approximately 4-6 kcal mol(-1) in the case of a central TATATA sequence. A smaller equilibrium minor groove width and more restricted minor groove mobility was found for the central AAATTT and also a significantly ( approximately 2 times) larger free energy change for opening the minor groove. The helical parameter analysis of trajectories indicates that an easier partial unstacking of a central TA versus AT basepair step is a likely reason for the larger groove flexibility of the central TATATA case.     

Molecular recognition of DNA base pairs by the formamido/pyrrole and formamido/imidazole pairings in stacked polyamides

Polyamides containing an N-terminal formamido (f) group bind to the minor groove of DNA as staggered, antiparallel dimers in a sequence-specific manner. The formamido group increases the affinity and binding site size, and it promotes the molecules to stack in a staggered fashion thereby pairing itself with either a pyrrole (Py) or an imidazole (Im). There has not been a systematic study on the DNA recognition properties of the f/Py and f/Im terminal pairings. These pairings were analyzed here in the context of f-ImPyPy, f-ImPyIm, f-PyPyPy and f-PyPyIm, which contain the central pairing modes, –ImPy– and –PyPy–. The specificity of these triamides towards symmetrical recognition sites allowed for the f/Py and f/Im terminal pairings to be directly compared by SPR, CD and ΔT_M experiments. The f/Py pairing, when placed next to the –ImPy– or –PyPy– central pairings, prefers A/T and T/A base pairs to G/C base pairs, suggesting that f/Py has similar DNA recognition specificity to Py/Py. With –ImPy– central pairings, f/Im prefers C/G base pairs (>10 times) to the other Watson–Crick base pairs; therefore, f/Im behaves like the Py/Im pair. However, the f/Im pairing is not selective for the C/G base pair when placed next to the –PyPy– central pairings.     

Crystallographic insights into DNA minor groove recognition by drugs

This review surveys the crystal structures between minor groove drugs and oligonucleotides, of which over thirty have now been determined. The various factors that are involved in the observed A/T sequence selectivity of these drugs are examined in structural terms. The roles of, in particular, hydrogen-bond recognition and sequence-dependent groove width, are assessed, and as a consequence the minor groove drugs have been classified into two categories, dependent on the relative roles played by these two factors in sequence recognition. Implications for the recognition of non-A/T sequences are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 44: 105–121, 1997     

Structure of a beta-alanine-linked polyamide bound to a full helical turn of purine tract DNA in the 1:1 motif

Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy-beta-Im-beta-ImPy-beta-Dp bound in a single orientation to its match site within the DNA duplex 5'-CCAAAGAGAAGCG-3'.5'-CGCTTCTCTTTGG-3' (match site in bold), as determined by 2D (1)H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 A and a pairwise RMSD over the binding site of 0.80 A. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N-C with the 3'-5' direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 A, twist=38 degrees ) with a narrow minor groove closing down to 3.0-4.5 A in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of beta-alanine and imidazole for 1:1 binding are discussed.     

Minor groove binding DNA ligands with expanded A/T sequence length recognition, selective binding to bent DNA regions and enhanced fluorescent properties

DNA minor groove ligands provide a paradigm for double-stranded DNA recognition, where common structural motifs provide a crescent shape that matches the helix turn. Since minor groove ligands are useful in medicine, new ligands with improved binding properties based on the structural information about DNA-ligand complexes could be useful in developing new drugs. Here, two new synthetic analogues of AT specific Hoechst 33258 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole (DMA) and 5-(4-methylpiperazin-1-yl)-2-[2'[2'-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl] benzimidazole (TBZ) were evaluated for their DNA binding properties. Both analogues are bisubstituted on the phenyl ring. DMA contains two ortho positioned methoxy groups, and TBZ contains a phenolic group at C-4 and a methoxy group at C-3. Fluorescence yield upon DNA binding increased 100-fold for TBZ and 16-fold for DMA. Like the parent compound, the new ligands showed low affinity to GC-rich (K approximately 4 x 10(7) M(-1)) relative to AT-rich sequences (K approximately 5 x 10(8) M(-1)), and fluorescence lifetime and anisotropy studies suggest two distinct DNA-ligand complexes. Binding studies indicate expanded sequence recognition for TBZ (8-10 AT base pairs) and tighter binding (DeltaT(m) of 23 degrees C for d (GA(5)T(5)C). Finally, EMSA and equilibrium binding titration studies indicate that TBZ preferentially binds highly hydrated duplex domains with altered A-tract conformations d (GA(4)T(4)C)(2) (K= 3.55 x 10(9) M(-1)) and alters its structure over d (GT(4)A(4)C)(2) (K = 3.3 x 10(8) M(-1)) sequences. Altered DNA structure and higher fluorescence output for the bound fluorophore are consistent with adaptive binding and a constrained final complex. Therefore, the new ligands provide increased sequence and structure selective recognition and enhanced fluorescence upon minor groove binding, features that can be useful for further development as probes for chromatin structure stability.