A team led by Harvard University researchers this week published data on a new method of generating effective siRNAs in bacteria.
To date, the majority of siRNAs are chemically synthesized. They can also be made from transcribed longer dsRNAs that are processed in vitro by RNase III family enzymes.
“In the latter case, the resulting siRNAs contain many sequences against one target,” wrote the investigators in a paper published in Nature Biotechnology. “A pool of several siRNAs can sometimes be more effective and have fewer off-target effects than any one single siRNA. However, thus far, functional siRNAs have not been produced in living cells.”
To address this, the team ectopically expressed p19, an siRNA-binding protein found in a plant RNA virus, in Escherichia coli, and found that the protein stabilized a roughly 21 nucleotide siRNA-like species produced by bacterial RNase III.
“When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by [around 90 percent] without immunogenicity or off-target effects,” according to the paper.
These so-called pro-siRNAs, the investigators concluded, could prove to be a useful addition to existing RNAi molecules for both research and therapeutic applications.
“Use of pro-siRNAs would eliminate the need to purchase and test multiple individual chemically synthesized siRNAs,” they wrote. “When generated from longer hairpins, pro-siRNA preparations containing multiple sequences might trigger fewer off-target effects than individual siRNAs and, in the cases of virus infection or cancer, might more effectively prevent target gene escape by mutation.”
The technique for the molecules’ generation was adapted from “well-established techniques for making recombinant proteins from E. coli and could easily be adapted and scaled up in an industrial setting,” they added. “In addition, mammalian cDNA libraries might be used to generate pro-siRNA libraries for siRNA screening purposes.”
Still, chemical synthesis allows for the introduction of chemical modifications that enhance stability and potency, and additional work needs to be conducted to see if such modifications can be made to pro-siRNAs, they noted.