RNA major groove modifications improve siRNA stability and biological activity - PubMed (original) (raw)
RNA major groove modifications improve siRNA stability and biological activity
Montserrat Terrazas et al. Nucleic Acids Res. 2009 Feb.
Abstract
RNA 5-methyl and 5-propynyl pyrimidine analogs were substituted into short interfering RNAs (siRNAs) to probe major groove steric effects in the active RNA-induced silencing complex (RISC). Synthetic RNA guide strands containing varied combinations of propynyl and methyl substitution revealed that all C-5 substitutions increased the thermal stability of siRNA duplexes containing them. Cellular gene suppression experiments using luciferase targets in HeLa cells showed that the bulky 5-propynyl modification was detrimental to RNA interference activity, despite its stabilization of the helix. Detrimental effects of this substitution were greatest at the 5'-half of the guide strand, suggesting close steric approach of proteins in the RISC complex with that end of the siRNA/mRNA duplex. However, substitutions with the smaller 5-methyl group resulted in gene silencing activities comparable to or better than that of wild-type siRNA. The major groove modifications also increased the serum stability of siRNAs.
Figures
Figure 1.
The 5-substituted nucleosides employed in this study.
Figure 2.
Sequences of modified guide-RNAs (1–9) and of passenger RNA used in the RNAi experiments and _T_m data. Sites of propynylU (‘pU’), methylU (‘mU’) and methylC (‘mC’) substitutions are indicated in bold. _T_ms were measured in 15 mM HEPES–KOH (pH 7.4), 1 mM MgCl2 and 50 mM KOAc. _T_m data from lower ionic strength solution are included in the
Supplementary Table S2
.
Figure 3.
Plot of gene-specific RNAi activity for 5-propynyl and 5-methyl substituted siRNAs at different sites in the Renilla luciferase mRNA expressed in HeLa cells. Varied amounts of RNA were added as shown.
Figure 4.
Plot comparing RNAi activity of methylated siRNAs (2, 7, 9) to unmodified siRNA (1). Data are from Figure 3, plotted on a scale that allows for ready comparison of highest activity RNAs. Note the consistently greater gene supression by 7 and 9 relative to 1.
Figure 5.
Dual plot comparing helix stability (_T_m) and RNA gene knockdown activity (percent suppression) with 5-propynyl and 5-methyl substitutions along the guide strand of siRNA duplexes at different positions. Suppression data are for siRNA concentrations of 0.3 nM.
Figure 6.
Models of interactions between the PIWI domain and the 5′-nucleobases of a siRNA-like guide strand. Adapted from the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a siRNA-like duplex (pdb code 2bgg). The figures were prepared with PyMOL (
).
Figure 7.
Oligoribonucleotide [modified (guide) and unmodified (passenger) strands] half lives in 90% human serum, from stained gel data. Half-life is defined as the time at which full-length RNA staining density is half that of the density at time zero. Error is estimated at ± 30%.
References
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. - PubMed
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494–498. - PubMed
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