OLIGONUCLEOTIDE-DIRECTED NUCLEIC-ACID SCISSION BY MICROCOCCAL NUCLEASE

''Sequence-dictated'' scission of DNA can be achieved by tethering a f usion protein composed of glutathione S-transferase and attenuated mic rococcal nuclease (MN) to a targeting oligonucleotide using Cibacron b lue (CB) F3G-A. Deoxyoligonucleotides derivatized with this dye bind t o the fusion protein in gel mobility shift assays. This binding scheme was successfully used to achieve site-specific scission of a single-s tranded DNA substrate after hybridization with a CB-derivatized comple mentary oligonucleotide. Although covalently cross-linked hybrids of M N and oligonucleotides have been successfully used in the past to targ et nucleolytic activity, this novel scheme opens new possibilities for targeting and probing both DNA and RNA sequences by allowing the addi tion of the nuclease subsequent to hybridization.