Sequence-specific recognition of B-DNA by oligo(N-methylpyrrolecarboxamide)s.

Abstract

Four homologous oligopeptide-EDTA molecules, tri-, tetra, penta-, and hexa(N-methylpyrrolecarboxamide)-EDTA, in the presence of Fe(II), 02, and dithiothreitol, cleave 32P-end-labeled restriction fragments from plasmid pBR322 DNA at common locations rich in A-T base pairs that differ in the size of the binding site. From analysis of the cleavage patterns visualized by high-resolution denaturing gel electrophoresis, the oligopeptides with three, four, five, and six Nmethylpyrrolecarboxamide units, containing four, five, six, and seven amide NHs, bind sites of A-T-rich DNA consisting of five, six, seven, and eight contiguous base pairs, respectively. The general rule of n amides affording binding site sizes of n + 1 base pairs is consistent with the oligopeptides binding in the minor groove of right-handed DNA, with the amide NH groups forming bridges between the adjacent N-3 and 0-2 atoms of adenine or thymine on opposite strands of the DNA helix.Netropsin and distamycin A are antibiotics active against fungi, bacteria, and viruses. These di-and tripeptides show a marked preference for DNA rich in adenine (A) and thymine (T) and have binding sites four to six base pairs in size (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). From a recent x-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G, Dickerson and coworkers provide a molecular basis for the sequence-specific recognition of DNA by netropsin, and by extension, distamycin (12). They find that netropsin sits symmetrically in the center of the minor groove of right-handed DNA and displaces the water molecules of the spine of hydration (12). Each of its three amide NH groups forms a bridge between adjacent adenine N-3 or thymine 0-2 atoms on opposite helix strands (12). This explains recent solution data that distamycin analogues having n amides characteristically bind to n + 1 successive base pairs (13)(14)(15). Dickerson and coworkers suggest that the base specificity of netropsin for contiguous sequences of A-T base pairs in B-DNA is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the oligopeptide molecules (12). Because N-methylpyrrolecarboxamides could be used as recognition elements for the design of synthetic sequence-specific DNA-binding molecules, the question arises whether higher numbers of contiguous Nmethylpyrrolecarboxamides in synthetic oligopeptides would still fit the natural twist of the B-DNA helix (13-18).Affinity Cleaving. Attachment of DNA-cleaving moieties, such as EDTA-Fe(II) (19,20), to DNA-binding molecules followed by DNA cleavage pattern analysis using high-resolution denaturing gel electrophoresis is a useful method for determining the binding locations, site size, and orientation of small molecules on native DNA (14-18). The resulting cleavage patterns are simply the positive image visualized on an autoradiogram with respect to the negative image produced by cleavage inhibition patterns ("footprint...

Origins of netropsin binding affinity and specificity: correlations of thermodynamic and structural data.

“…The relevance of these two biophysical model studies to native DNA was demonstrated by the Dervan group. Their "footprinting" investigations revealed that netropsin binds to similar AT-rich sites of five base pairs in naturally occurring DNA polymers (8)(9)(10).…”

Toward rules for 1:1 polyamide:DNA recognition

“…The 2:1 motif has proven versatile, whereby pairs of amino acids can be assembled side-by-side to distinguish all four Watson⅐Crick base pairs (Im͞Py ϭ G⅐C; Py͞Im ϭ C⅐G; Hp͞Py ϭ T⅐A; and Py͞Hp ϭ A⅐T) (3). Py-Im polyamides are overcurved with respect to the DNA helix (4)(5)(6). By substituting the aliphatic amino acid ␤-alanine (␤) for Py, the ligand curvature is reset, allowing specific recognition of 16 contiguous base pairs of DNA (7)(8)(9).…”

Ultraviolet light-induced cleavage of DNA in presence of iodoHoechst 33258: the sequence specificity of the reaction

“…In some exceptional cases, the ligand in this 'reverse' polarity also results in some (a little) breakage in to labelled (top) strand. The fact that these cleavage sites seem to be almost 'within' the binding sites, is a consequence of the 'staggered' nature of the DNA helix (28), across the axis of the minor groove.…”