DNA-Directed RNA Interference (ddRNAi, shRNA)

siRNA or shRNA made in cells from DNA templates in plasmid or viral vectors.

RNA interference can be achieved using RNA made enzymatically by transcription from DNA templates. This method has been called DNA-Directed RNAi, or ddRNAi. In its simplest form, ddRNAi can be done in vitro where a T7 or any other convenient promoter/polymerase combination is used to make RNA from template DNA oligos¹. These products are heterogeneous and can sometimes result in interferon induction or other related problems². However, this approach does offer an inexpensive way to make RNA.

DNA oligos can be cloned into a variety of vectors, ranging from expression plasmids to lentiviral vectors. These constructs can be introduced into cells or animals. The cloned vectors will express the dsRNA molecules and initiate an RNAi response in the transfected or infected cells. This method can give long term instead of transient RNAi. DNA oligos can be cloned with the sense and antisense stands in tandem³ or in hairpin configuration, which mimics naturally occurring miRNAs. Expressed hairpins, or short-hairpin RNAs (shRNAs) are the current favored design for the ddRNAi approach and the construction and systematic use of large-scale libraries of this kind for functional genomics screens are showing great promise^{4, 5, 6}

To make an shRNA expression plasmid, two DNA oligos need to be ordered (sense and antisense strands). Normally the strands are designed to leave sticky ends when annealed which enable cloning into a specific expression vector. Each sequence will contain the target sequence in forward and reverse orientation separated by a hairpin loop. A variety of loop sequences have been used with good results, including certain sequences derived from natural miRNA hairpins, which can sometimes result in enhanced functional expression. For use with some vectors, inclusion of promoter and/or termination signals in the cloning oligos may also be necessary. Depending on the length of the duplex (stem) region, the size of the loop, and need for regulatory domains, the DNA oligos needed for cloning can range in size from around 50 to 100 bases, but are more typically in the 57-80 base range.

Normally IDT recommends the use of purified oligos when cloning DNA sequences over 40 bases long. However, the hairpin present in the shRNA cloning oligos makes purification difficult. Further, purification is not feasible for large volume shRNA cloning projects, where many thousands of oligos are needed quickly at low cost. IDT has developed a new synthesis scale that results in an ultra-high quality product. This process has been optimized for manufacturing oligos in the 60-200 base range. Product identity is confirmed by LC MS spectrometry. These oligos are intended for use without additional purification.

IDT is licensed under patents owned by Benitec Ltd., to sell oligos for use in ddRNAi applications.

References

1. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Donze, O., and Picard, D. Nucleic Acids Res., 30:e46 (2002).

2. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Kim, D.-H., Longo, M., Han, Y., Lundberg, P., Cantin, E., and Rossi, J.J. Nature Biotechnology, 22:321-325 (2003).

3. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P., and Rossi, J. Nature Biotechnology, 19:500-505 (2002)

4. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., and Schultz, P.G. Proc. Natl. Acad. Sci., USA, 101:135-140(2004).

5. A resource for large-scale RNA-interference-based screen in mammals. Paddison, P.J., Silva, J.M., Conklin, D.S., Schlabach, M., Li, M., Aruleba, S., Balija, V., O'Shaughnessy, A., Gnoj, L., Scobie, K., Chang, K., Westbrook, T., Cleary, M., Sachidanandam, R., McCombie, W.R., Elledge, S.J., and Hannon, G.J. Nature, 428:427-431(2004).

6. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M., Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P.S., Beigersbergen, R.L., and Bernards, R. Nature, 428:431-7(2004).