RNase H1 Can Catalyze RNA/DNA Hybrid Formation and Cleavage with Stable Hairpin or Duplex DNA Oligomers

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Abstract
Cleavage of a RNA target site by RNase H1 from Escherichia coli was examined in the presence of complementary DNA sequences in the form of single-stranded, duplex, and hairpin structures. The target site was a 15 nt sequence in the middle of a 79 nt RNA transcript. DNA molecules employed included seven single-stranded oligodeoxynucleotides 10 or 15 nt long, and five hairpin DNAs each with a 10 bp stem and 5 nt loop. The loop and 3‘ side of the stem of two of the hairpin DNAs were fully complementary to the target site, while the other hairpin DNAs had sequence changes. A 10 bp duplex DNA with one strand complementary to the target site was also employed. A gel electrophoresis mobility shift assay examined hybrid formation between the RNA and the single-stranded 15 nt DNA and two hairpin DNAs that contained 15 complementary bases. RNA titration of the 32P-labeled single-stranded DNA produced a shifted band indicative of RNA/DNA complex formation. No RNA/DNA complex was detected when the more stable (Tm = 71 °C) hairpin DNA was combined with excess RNA. The less stable hairpin DNA (Tm = 62 °C) showed a small amount (∼8%) of hybrid formation. Thermodynamic analysis of RNA binding to the DNAs was in qualitative agreement with the results. Although no RNA/DNA hybrid was expected from thermodynamic calculations, a RNase H assay at 25 °C showed that hairpin or duplex DNAs with a 10 nt complementary sequence catalyzed RNA degradation. A complementary loop sequence in the hairpin DNA was not required. Cleavage of the RNA did not occur with hairpin DNAs containing three or four noncomplementary bases in the stem. The results show that RNase H can promote the formation and cleavage of a RNA/DNA hybrid between an RNA site and a base paired strand of a stable hairpin or duplex DNA at temperatures below their Tm.