title: Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate.
abstract: In a previous study, we demonstrated that the sugar conformation and helical geometry of the heteroduplex substrate at the catalytic site of human RNase H1 directs the selective recognition of the substrate by the enzyme (J Biol Chem 279: 36317-36326, 2004). In this study, we systematically introduced 2'-methoxyethoxy (MOE) nucleotides into the antisense oligodeoxyribonucleotide (ASO) of the heteroduplex to alter the helical geometry of the substrate. The MOE substitutions at the 3' and 5' poles of the ASO resulted in fewer cleavage sites and slower cleavage rates compared with the unmodified substrates. Furthermore, a greater reduction in cleavage activity was observed for MOE substitutions at the 5' pole of the ASO. The 3'- and 5'-most cleavage sites were positioned two and four to five base pairs, respectively, from the nearest MOE residues, suggesting a conformational transmission of the MOE/RNA helical geometry into the DNA/RNA portion of the heteroduplex. Similar conformational transmission was observed for Okazaki-like substrates containing deoxyribonucleotide substitutions at the 3' pole of the oligoribonucleotide. Finally, the heteroduplex substrates exhibited preferred cleavage sites that were cleaved 2- to 3-fold faster than other sites in the substrate, and these sites exhibited the greatest influence on the initial cleavage rates. The data presented here offer further insights into the role substrate structure plays in directing human RNase H1 activity as well as the design of effective ASOs.
authors.affiliation: Department of Molecular and Structural Biology, Isis Pharmaceuticals, Carlsbad, California, USA. wlima@isisph.com
authors.forename: Walt F
authors.initials: WF
authors.lastname: Lima
authors.forename: John B
authors.initials: JB
authors.lastname: Rose
authors.forename: Josh G
authors.initials: JG
authors.lastname: Nichols
authors.forename: Hongjiang
authors.initials: H
authors.lastname: Wu
authors.forename: Michael T
authors.initials: MT
authors.lastname: Migawa
authors.forename: Tadeusz K
authors.initials: TK
authors.lastname: Wyrzykiewicz
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Siwkowski
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009692:Nucleic Acid Heteroduplexes; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D020319:Oligodeoxyribonucleotides, Antisense; D011994:Recombinant Proteins; D016914:Ribonuclease H; C078856:ribonuclease HI