CYCLING PROBE TECHNOLOGY WITH RNASE-H ATTACHED TO AN OLIGONUCLEOTIDE

A streptavidin-RNase H gene fusion was constructed by cloning the Ther mus thermophilus RNase H coding sequence in the streptavidin expressio n vector pTSA18F. The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity. The fus ion protein was shown to have a molecular weight of 128 kDa and to con sist of four subunits. Furthermore, hear treatment of the fusion enzym e showed that it was stable as a tetramer at 65 degrees C. The fusion enzyme was shown to have both biotin binding and RNase H catalytic pro perties. Using cycling probe technology (CPT), the fusion enzyme was c ompared to the native RNase H with a biotinylated probe at different r atios of probe:enzyme and varying amounts of synthetic target DNA. At a ratio of 1:1, the fusion enzyme was active in CPT, but the native en zyme was not; both enzymes were active at a 1:5000 ratio of probe:enzy me. The fusion enzyme was further tested using biotinylated and non-bi otinylated probes and was shown to be active at a 1:1 ratio with the b iotinylated probe but not with the non-biotinylated probe. These exper iments show that through binding of the streptavidin-RNase H fusion en zyme to the biotinylated probe, the efficiency of the cycling probe re action is enhanced.