QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.106.025429v1 71/1/73    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lima, W. F. Articles by Crooke, S. T. Search for Related Content PubMed PubMed Citation Articles by Lima, W. F. Articles by Crooke, S. T.

The Positional Influence of the Helical Geometry of the Heteroduplex Substrate on Human RNase H1 Catalysis

Department of Molecular and Structural Biology, Isis Pharmaceuticals, Carlsbad, California

In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2'-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA. Conformationally biased and flexible modified nucleotides were positioned at the junctions between the DNA and MOE residues of the chimeric substrates to modulate the effects of the MOE residues on human RNase H1 activity. The strong northern-biased locked-nucleic acid modification exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates. Enhanced cleavage rates were observed for the eastern-biased 2'-ara-fluorothymidine and bulge inducing N-methylthymidine modifications positioned at the 5'-DNA/3'-MOE junction as well as the southern-biased 2'-methylthiothymidine and conformationally flexible tetrafluoroindole (TFI) modifications positioned at the 5'-MOE/3'-DNA junction. The heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity, whereas nucleotide substitutions adjacent the TFI significantly affected the cleavage rate. Mismatch base pair(s) exhibited similar effects on human RNase H1 activity as the TFI modifications. The effects of the TFI modification and mismatch base pair(s) on human RNase H1 activity were influenced by the position of the modification relative to the nucleotides interacting with the catalytic site of the enzyme rather than the juxtaposition of the modification to the MOE residues. Finally, these results provide a method for enhancing the human RNase H1 activity of chimeric antisense oligonucleotides (ASO) as well as the design of more potent ASO drugs.

Address correspondence to: Walt Lima, 2292 Faraday Ave. Carlsbad, CA 92008. E-mail: wlima{at}isisph.com