MODULATION OF RNASE-H ACTIVITY BY MODIFIED DNA PROBES - MAJOR GROOVE VS MINOR-GROOVE EFFECTS

We have previously prepared ribozyme mimics and chemical nucleases-fro m modified DNA containing pendant bipyridine and terpyridine groups. T he ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes were formed using DN A probes designed to deliver metal complexes via either the major groo ve or the minor groove of the duplex. The duplexes were treated with E scherichia coli RNase H. Modifications in the major groove produced th e same RNA cleavage pattern as unmodified DNA probes. However, minor g roove substituents inhibited RNA cleavage over a four-base region. Com parison was made with a DNA probe containing a 2'-OMe modification. Ou r results support enzyme binding in the minor groove of a DNA/RNA dupl ex. We do not observe cleavage directly across from the modified nucle oside. The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA greater than or equal to C-5 mod ified DNA much greater than C2'-modified DNA > Cl'-modified DNA. Resul ts with 28-mer RNA substrates roughly parallel those obtained with a 1 59-mer RNA target. The differences observed between low and high MW RN A substrates can be explained by a much higher enzyme-substrate bindin g constant for the high MW target. (C) 1997 Elsevier Science Ltd.