Modulation of Splicing by Single-Stranded Silencing RNAs.

Nucleic acid therapeutics (2015-03-11)
Jing Liu, Jiaxin Hu, Jessica A Hicks, Thazha P Prakash, David R Corey
ABSTRACT

Single-stranded silencing RNAs (ss-siRNAs) are chemically modified single-stranded oligonucleotides that can function through the cellular RNA interference (RNAi) machinery to modulate gene expression. Because their invention is recent, few studies have appeared describing their use and the potential of ss-siRNAs as a platform for controlling gene expression remains largely unknown. Using oligonucleotides to modulate splicing is an important area for therapeutic development and we tested the hypothesis that ss-siRNAs targeting splice sites might also be capable of directing increased production of therapeutically promising protein isoforms. Here we observe that ss-siRNAs alter splicing of dystrophin. Altered splicing requires a seed sequence complementarity to the target and expression of the RNAi factor argonaute 2. These results demonstrate that ss-siRNAs can be used to modulate splicing, providing another option for therapeutic development programs that aim to increase production of key protein isoforms. Splicing is a classical nuclear process and our data showing that it can be modulated through the action of RNA and RNAi factors offers further evidence that RNAi can take place in mammalian cell nuclei.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Minimum Essential Medium Eagle, With Earle′s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
SAFC
MEM Non-essential Amino Acid Solution (100×), without L-glutamine, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
DL-Glyceraldehyde 3-phosphate solution, 45-55 mg/mL in H2O
Sigma-Aldrich
Anti-RNA polymerase II Antibody, clone CTD4H8, clone CTD4H8, Upstate®, from mouse