A simple and cost-effective method for producing small interfering RNAs with high efficacy

Abstract

Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity. This approach is expensive due to the cost of chemical synthesis of RNAs. We report a technically simple, quick and cost-effective method for the production of siRNAs that makes use of in vitro transcription and deoxyribozyme digestion of the transcripts to produce the desired sequence and length. The method allows for several siRNAs to be produced in parallel at much reduced costs. The siRNAs produced with this method were tested in MDA-MB-231 human breast cancer cells for efficacy against the type 1 insulin-like growth factor receptor (IGF1R) mRNA and they caused dose-dependent inhibition of IGF1R expression comparable to that induced by chemically synthesised siRNAs of the same sequence. This method is also useful for producing long RNA fragments of defined length and sequence that may be difficult to synthesise chemically, and also for producing large quantities of RNAs for applications including structural studies and the study of interactions between RNA and other molecules, such as proteins, other nucleic acids and drugs.

Gene silencing of HPV16 E6/E7 induced by promoter-targeting siRNA in SiHa cells

“…The criteria described by Sohail et al (2003) and the computer programme available at http://www.promega.com/siRNADesigner/ program/ were used to design siRNA. The T7 RiboMAX Express RNAi System Kit (Promega, Madison, WI, USA) was used to synthesise siRNA.…”

RNA Interference with Special Reference to Combating Viruses of Crustacea

“…The polymerase produces individual sense and antisense siRNA strands that form siRNAs when annealed [34,173,206]. However, the purity and specificity using enzymatic synthesis is variable [36].…”

“…Transcription is generated from short double stranded oligo cassettes containing the promoter sequence upstream of the siRNA template sequence that is to be transcribed [34,173]. Transcription begins and terminates at specific initiation and termination sequences, determined by the promoter [40].…”

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting

“…These investigators further demonstrated that the presence of triphosphates on the 59 end of T7 transcripts is required for interferon induction, since the treatment of T7 transcripts with phosphatase could abrogate this effect. Sohail et al (2003) reported an alternative approach for producing a desired siRNA sequence using T7 polymerase in vitro transcription followed by transcript digestion by deoxyribozymes, which are known as ''DNAzymes'' or catalytic DNA. The cleavage of RNA by DNAzymes occurs between two specific nucleotides, and the requirement for this dinucleotide sequence is different for different DNAzyme groups, making them flexible tools for digesting a variety of sequences (Breaker and Joyce 1994;Feldman and Sen 2001).…”