If you are a scientist and you want to turn off a gene, one option that’s been gaining traction is RNA interference (or RNAi). In this molecular process—first discovered in plants and only 12 years ago detected in mammals—bits of RNA called small interfering RNAs (siRNAs) cancel out a gene’s messenger RNA, effectively muffling that gene.
Labs can order custom-made, chemically synthesized siRNAs for just about any DNA sequence they want to silence. The tricky part is deciding what the right sequence is—especially when that gene is part of a virus, where genes can mutate pretty quickly.
However, a biotechnology approach to producing siRNAs could make it relatively easy for just about any lab that can master recombinant DNA technologies to make a number of siRNAs against multiple sequences within the same target gene: a potential bonus for companies seeking to make drugs that rely on RNAi.
“To turn off one gene, you have to order multiple siRNAs, each complementary to a different portion of the gene, and they don’t always work well,” says Judy Lieberman, MD, PhD, of Boston Children’s Program in Cellular and Molecular Medicine. “This can be especially problematic with genes from viruses like HIV, where one can find a great deal of sequence diversity for any one gene.”
Lieberman has been working for years on RNAi-based therapeutic approaches against HIV, herpes, breast cancer and more. Now, she and postdoctoral fellow Linfeng Huang, PhD, with collaborators at New England Biolabs, have developed a new way of generating siRNAs that gets bacteria to do the work—using a recombinant DNA approach similar to that used in making many biological or protein-based drugs.
Turning bacteria into siRNA factories
“Until recently it was thought that only plant and animal cells could carry out RNA interference,” Lieberman explains. “But now we are finding that bacteria make lots of small RNAs that play a role in regulating bacterial gene expression.”
In their method, Lieberman and Huang start with the sequence of the gene to be silenced, and insert it and its complementary reverse sequence into the DNA of E. coli bacteria, one of the workhorses of recombinant DNA technology. The bacteria transcribe this foreign DNA to produce a double stranded RNA, which gets chopped into smaller pieces that resemble siRNAs. They call them pro-siRNAs—short for prokaryotic siRNAs (prokaryote is the scientific name for the group of organisms like bacteria that do not have a cell nucleus).
“This approach opens the door for any lab that can make recombinant proteins to make their own siRNAs.”
To get the pro-siRNAs out, Lieberman and Huang also insert into the bacteria the gene for a protein called p19. Originally discovered in a plant virus, p19 binds to and stabilizes small RNA pieces, including siRNAs. In essence, it acts like a hook, fishing pro-siRNAs out of the bacteria.
The end product of this process is a pool of ready-to-use siRNAs targeting multiple sequences within a single gene of interest. As Lieberman writes in the paper, this pooling approach can sometimes be more effective and precise than any single siRNA.
“With this approach, we get multiple siRNAs against multiple portions of a gene’s sequence, and can reproducibly shut off the expression of a gene using only very low concentrations of siRNAs,” Lieberman says. “This could especially be important when developing siRNAs against viruses and also cancers, where genes mutate readily.”
RNA interference for the masses?
The process still needs work, including optimizing the method to increase the yield of siRNAs and also to enrich the pool of siRNAs for those for the target gene. But as a proof of concept, it holds a lot of promise for better RNAi screening techniques and development of drugs based on RNA interference.
It could also democratize RNA interference technology by making it more accessible.
“This approach opens the door for any lab that can make recombinant proteins to make their own siRNAs,” Lieberman notes. “And these days, almost any laboratory can do that.”