Radioactive end labeling to determine hydrolytic rates of nuclease mimics.
Radioactive end labeling can be used to determine the hydrolytic rates of nuclease mimics on moderate to long lengths of RNA or DNA. However, the reliability of end labeling as an assay can vary depending on how well the unincorporated label is removed from the labeled RNA or DNA products. Therefore, gel filtration, acid precipitation, membrane diafiltration, and paper chromatography were tested to determine which technique was the most effective at such separation. The results in order of decreasing contamination by [gamma-32P]ATP were gel filtration (40%), acid precipitation (5%), diafiltration (2%), and paper chromatography (1%); and, in order of decreasing loss of RNA, were acid precipitation (30%), diafiltration (11%), gel filtration (10%), and paper chromatography (1%). In order for the resultant radioactive counts to be linearly proportional to the number of cleavage sites, the total ATP in the end-labeling reaction should be in excess of 5'-hydroxyl ends by a factor of 10 or more. Interference by nuclease mimics in the end-labeling reaction should be accounted for by including the mimics when developing a standard curve based on known concentrations of 5'-hydroxyl ends.
Smith
J
J
Department of Chemistry and Biochemistry, University of Texas at Austin 78712.
Anslyn
E V
EV
D002850:Chromatography, Gel; D003851:Deoxyribonucleases / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry*
D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D003851:Deoxyribonucleases; D012260:Ribonucleases