Ribonuclease H renaturation gel assay using a fluorescent-labeled substrate.
Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids. These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation. One of the most valuable tools for assaying RNase H activity is the renaturation gel assay with which such activities can be detected using purified protein preparations or crude extracts. Radioactive substrates [32P labeled poly(rA)-poly(dT) hybrid] are commonly used with exposure of the gel to X-ray film; this is possible at any time without disturbing the renaturation-degradation process. Here, we describe a method using fluorescent-labeled substrates. RNA-DNA substrates are synthesized by first transcribing DNA with T7 RNA polymerase using Bodipy-TR-14-UTP and the four normal nucleoside triphosphates. The run-off transcript is annealed to a short oligomeric DNA complementary to the 3'-end of the transcript, and the DNA portion of the hybrid is formed by RT. This RNA-DNA is added to the polyacrylamide mixture before polymerization, and SDS-PAGE is performed as usual. After various periods of renaturation, the gel is scanned to detect fluorescent substrate using the red-excited laser of a fluorescence scanner. This fluorescence method has all of the advantages of using radio-labeled substrates and none of its disadvantages, and the sensitivities of the two methods are comparable. In addition, we show that the sensitivity of this procedure can be increased if damaging chemicals remaining in the gel after polymerization are eliminated by simultaneous electrophoresis of the RNase H and a protein with higher mobility.
Han
L Y
LY
National Institute of Child Health and Human Development, Bethesda, MD, USA.
Ma
W P
WP
Crouch
R J
RJ
D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D005782:Gels; D009113:Muramidase; D010957:Plasmids; D011489:Protein Denaturation; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D016914:Ribonuclease H / Q000032:analysis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D005782:Gels; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D009113:Muramidase