Design of Sequence‐specific DNA Cleaving Molecules

Abstract

The design of sequence-specific cleaving molecules for double-helical DNA requires the attachment of DNA cleaving moieties to sequence-specific DNA binding molecules. The site of attachment and choice of cleaving strategy will be dependent on the availability of molecular models for the sequence-specific binding of small molecules (i.e., 1000 MW) to right-handed double-helical DNA. Productive model building is possible because of recent progress in the development of analytical methods, such as footprinting and affinity cleaving, to determine the preferred binding locations, binding site sizes, and orientations of natural or synthetic sequence-specific DNA binding molecules on large DNA such as DNA restriction fragments (100-500 bp). The natural product distamycin is a crescent-shaped tripeptide containing three N-methylpyrrolecarboxamides which binds in the minor groove of right-handed double-helical DNA with a strong perference for A•T rich sequences (FIG. I). The preferred sequences, binding site size, and orientation preference for tris-N-methylpyrrolecarboxamide are known from footprinting and affinity cleaving data. Moreover, from recent X-ray analysis of the complex of the dipeptide, netropsin, with the B-DNA dodecamer, S-CGCGAATTCGCG-3', Dickerson and co-workers have provided a molecular basis for the recognition of DNA by netropsin, and by extension, distamycin. They find that netropsin sits symmetrically in the center of the minor groove of A•T rich right-handed DNA and displaces the water molecules of the spine of hydration. Each of its three amide NH groups forms a bridge between adjacent adenine N3 or thymine O2 atoms on opposite helix strands. 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 C2 hydrogens and CH groups on the pyrrole rings of the oligopeptide molecules.