RIBONUCLEASE-H RENATURATION GEL ASSAY USING A FLUORESCENT-LABELED SUBSTRATE

Ribonucleases H (RNases H) are enzymes that specifically degrade the R NA of RNA-DNA hybrids. These enzymes are involved in DAN replication, reverse transcription (RT) and antisense oligodexoyribonucleotide-medi ated arrest of translation. One of the most valuable tools for assayin g RNase H activity is the renaturation gel assay with which such activ ities can be detected using purified protein preparations or crude ext racts. Radioactive substrates [P-32-labeled poly(rA)-poly(dT) hybrid] are commonly used with exposure of the gel to X-ray film; this is poss ible at any time without disturbing the renaturation-degradation proce ss. Here, we describe a method using fluorescent-labeled substrates. R NA-DNA substrates are synthesized by first transcribing DNA with T7 RN A polymerase using Bodipy(R)-TR-14-UTP and the four normal nucleoside triphosphates. The run-off transcript is annealed to a short oligomeri c DNA complementary to the 3'-end of the transcript, and the DNA porti on of the hybrid is formed by RT. This RNA-DNA is added to the polyacr ylamide mixture before polymerization, and SDS-PAGE is performed as us ual. After various periods of renaturation, the gel is scanned to dete ct 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 sensit ivities of the two methods are comparable. In addition, we show that t he sensitivity of this procedure can be increased if damaging chemical s remaining in the gel after polymerization are eliminated by simultan eous electrophoresis of the RNase H and a protein with higher mobility .