Format

Send to

Choose Destination
See comment in PubMed Commons below
FASEB J. 2013 Oct;27(10):4017-26. doi: 10.1096/fj.13-228668. Epub 2013 Jun 14.

Single modification at position 14 of siRNA strand abolishes its gene-silencing activity by decreasing both RISC loading and target degradation.

Author information

1
1Z.L., Institute of Molecular Medicine, Peking University, Beijing, 100871, China. liangz@pku.edu.cn.

Abstract

Normally siRNA has to be chemically stabilized in therapeutic applications. It is a challenge to obtain optimal stabilizing effects while maintaining full silencing activity due to a lack of understanding of how different chemical modifications would influence the efficacy of siRNA. In the current study, the effect of single 2'-sugar modifications was profiled across the length of the siRNA guide strand. This led to the surprising finding that a single 2'-OMe modification at position 14 of the siRNA guide strand substantially compromised its gene-silencing activity in a manner that was independent of the nucleotide identity at this site or the sequence context around it. We found that modification at position 14 of the siRNA guide strand reduced its RNA-induced silencing complex (RISC) loading tremendously, whereas the loading of the siRNA sense strand was only marginally affected. When comparing the silencing potency of 14th position-modified siRNA (transfected at 16.7 nM) and native control (transfected at 1 nM) at equivalent Ago2 loading levels, the silencing potency of modified siRNA was much lower, even lower than the level of native siRNA transfected at 0.1 nM. These data indicated that modification at position 14 of the siRNA guide strand abolishes its gene-silencing activity by decreasing both RISC loading and target degradation. Using a computational modeling approach, we demonstrated an intimate interaction between the 14th nucleotide of guide strand and the amino acid Q675 in the AGO protein, which is located in a highly conserved loop of PIWI domain. In addition to gaining insights into siRNA-AGO interactions, this study of structure-activity relationship further established a general principle for siRNA modification in siRNA drug development.

KEYWORDS:

AGO; RNA immunoprecipitation; computational modeling

PMID:
23771927
DOI:
10.1096/fj.13-228668
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire
    Loading ...
    Support Center