RNase H1 can catalyze RNA/DNA hybrid formation and cleavage with stable hairpin or duplex DNA oligomers

Biochemistry
J Li, R M Wartell

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 degrees C) hairpin DNA was combined with excess RNA. The less stable hairpin DNA (Tm = 62 degrees C) showed a small amount ( approximately 8%) of hybrid formation. Thermodynamic analysis of RNA binding to the DNAs was in qual...Continue Reading

Citations

Jun 1, 2001·Journal of Biochemical and Biophysical Methods·E ZamaratskiJ Chattopadhyaya
May 22, 2008·Molecular BioSystems·Julian L Huppert
Apr 7, 1999·Antisense & Nucleic Acid Drug Development·V K Rait, B R Shaw
Dec 7, 2007·Nucleic Acids Research·XinJing TangIvan J Dmochowski
Oct 2, 2007·Nucleic Acids Research·Igor D VilfanNynke H Dekker
Sep 11, 2010·Biosensors & Bioelectronics·Hana SípováJiří Homola
Nov 1, 2011·Journal of Molecular Recognition : JMR·Bo ZhengGu Yuan
Dec 1, 2004·EcoSal Plus·Zhongwei Li, Murray P Deutscher

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