Oligonucleotide-directed nucleic acid scission by micrococcal nuclease.
"Sequence-dictated" scission of DNA can be achieved by tethering a fusion protein composed of glutathione S-transferase and attenuated micrococcal nuclease (MN) to a targeting oligonucleotide using Cibacron blue (CB) F3G-A. Deoxyoligonucleotides derivatized with this dye bind to the fusion protein in gel mobility shift assays. This binding scheme was successfully used to achieve site-specific scission of a single-stranded DNA substrate after hybridization with a CB-derivatized complementary oligonucleotide. Although covalently cross-linked hybrids of MN and oligonucleotides have been successfully used in the past to target nucleolytic activity, this novel scheme opens new possibilities for targeting and probing both DNA and RNA sequences by allowing the addition of the nuclease subsequent to hybridization.
Landgraf
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R
Department of Biological Chemistry, School of Medicine, University of California at Los Angeles 90024-1570.
Chen
C H
CH
Sigman
D S
DS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase; D008836:Micrococcal Nuclease