Self-quenched covalent fluorescent dye-nucleic acid conjugates as polymeric substrates for enzymatic nuclease assays.
A fluorescent method is described for assessing nuclease activity. The technique is based on the preparation of quenched fluorophore-nucleic acid covalent conjugates and their subsequent dequenching due to degradation by nucleases. The resulting fluorescence increase can be measured by a spectrofluorometer and exhibits subpicogram per milliliter sensitivity level for RNase A and low picogram per milliliter level for DNase I. The method is adaptable for quantitative nuclease inhibitor testing.
Trubetskoy
Vladimir S
VS
Mirus Corporation, 505 South Rosa Road, Madison, Wisconsin 53719, USA. vladimirt@genetransfer.com
Hagstrom
James E
JE
Budker
Vladimir G
VG
D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D012313:RNA; D004247:DNA; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic