RNA Interference

RNA has been used for almost 15 years to reduce (or interfere with) expression of targeted genes in a variety of systems. Historically, these methods have been called post transcriptional gene silencing (PTGS) in plants, quelling in fungi and RNA interference (RNAi) in higher organisms.¹ Although originally thought to require use of long double-stranded (DS) RNA molecules², the active mediators are now known to be short DS RNAs³. Short single-stranded antisense RNA molecules were demonstrated to be effective inhibitors of gene expression over a decade ago⁴ but are susceptible to degradation by a variety of nucleases and are therefore of limited utility without chemical modification. DS RNAs are surprisingly stable and, unlike single-stranded DNA or RNA antisense oligos, do not need extensive modification to survive in tissue culture media or even within living cells.

Short interfering RNAs are naturally produced by degradation of long DS RNAs by Dicer, an RNase III class enzyme. While these fragments are usually around 21 bases long, synthetic DS RNAs of a variety of lengths (ranging from 18 bases to 30 bases)⁸ can be used to suppress gene expression depending on the system studied. These short DS RNAs are bound by the RNA Induced Silencing Complex (RISC), which contains several protein components including a ribonuclease which degrades the targeted mRNA. The antisense strand of the DS RNA directs target specificity of the RISC RNase activity while the sense strand of an RNAi duplex appears to function mainly to stabilize the RNA prior to entry into RISC and is degraded or discarded after entering RISC.

Based on studies done using Drosophila extracts, chemically synthesized RNAi duplexes have historically been made as two 21-mer oligos that form a 19-base RNA duplex with two deoxythymidine bases added as 3'-overhangs.⁵ Blunt 19-mer duplexes can also be used to trigger RNAi in mammalian systems ⁶, however these duplexes are generally less potent. Blunt duplexes can be effectively used for longer RNAs that are Dicer-Substrates⁸. In this case, the duplex is processed by Dicer to 21-mer length with 2-base, 3'-overhangs before entry into RISC.

Similar to experience with selecting antisense oligos, it is now clear that certain sites in a target mRNA sequence perform better than others for RNAi. To find an effective RNAi (with >75% knockdown), a good approach is to synthesize several duplexes and compare results. While pools can be employed to speed up high-throughput screening projects, it is better to employ well characterized single duplexes for in-depth study of a target gene of interest. Use and cross-comparison of two distinct RNAi duplexes against the same target, or an RNAi duplex and an antisense oligo, can provide support that conclusions are valid and do not arise from some artifact or off-target effect. An RNAi duplex design tool is available on the web at http://scitools.idtdna.com/RNAi/.

Chemical Modifications

While chemical modification of RNAi oligos is not required, certain modifications can sometimes be useful.⁷ The antisense strand must either have a free 5'-OH or 5'-phosphate terminus ^6,7. 5'-end modification of the sense strand RNA does not alter the efficacy of silencing and IDT recommends that, if addition of a fluorescent dye is desired to track transfection efficiency, Cy^TM be coupled to the 5'-end of the sense strand oligo. Modifications, such as 2'-O-methyl RNA, phosphorothioate bonds, inverted-dT and a variety of other options are also available. Please contact Technical Support at 800-328-2661 for consultation.

Purity and QC

RNAi pricing includes RNase-Free HPLC purification with a >80% duplex purity, verified by HPLC QC. In addition, each oligo is examined by mass spectrometry. HPLC mass spectrometry data are provided free on IDT's website. With each 10 nmole or 40 nmole RNAi duplex order, Nuclease-Free Duplex Buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.5) is included at no additional charge. Duplex Buffer is certified nuclease-free through the use of IDT's RNaseAlert^TM and DNaseAlert^TM products. RNAi sequences must be 100% complementary with up to a 3 base overhang.

Length Restrictions: RNAi oligos are available from 18-23 RNA and up to 3 DNA bases per strand.

RNAi Modifications

5' Phosphorylation may be incorporated into either the sense or antisense strand of an RNAi duplex.
5' 6-FAM^TM and 5' Biotin may only be incorporated on the sense strand.