Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H

To construct a DNA-linked RNase H, which cleaves RNA site-specificallyat high temperatures, the 15-mer DNA, which is complementaryto the polypurine-tract sequence of human immunodeficiency virus-1RNA (PPT-RNA), was cross-linked to the unique thiol group ofCys135 in the Thermus thermophilus RNase HI variant. The resultantDNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH,in which the RNase H portion of the d15-C135/TRNH is replacedby the Escherichia coli RNase HI variant, cleaved the 15-merPPT-RNA site-specifically. The mixture of the unmodified enzymeand the unlinked 15-mer DNA also cleaved the PPT-RNA but ina less strict manner. In addition, this mixture cleaved thePPT-RNA much less effectively than the DNA-linked enzyme. Theseresults indicate that the cross-linking limits but acceleratesthe interaction between the enzyme and the DNA/RNA substrate.The d15-C135/TRNH cleaved the PPT-RNA more effectively thanthe d15-C135/ERNH at temperatures higher than 50°C. Thed15-C135/TRNH showed the highest activity at 65°C, at whichthe d15-C135/ERNH showed little activity. Such a thermostableDNA-linked RNase H may be useful to cleave RNA molecules withhighly ordered structures in a sequence-specific manner.