A team of scientists led by the Cold Spring Harbor Laboratory (CSHL) researchers have developed a system that allows them to sift through thousands of candidate hairpin-shaped Ribonucleic acid (RNA) molecules at one time and pull out only those RNAs that potently shut down the movement of a target gene. This achievement of the researchers will now allow biologists to fully utilize RNA interference (RNAi), a natural cellular mechanism that has already been chosen by researchers for a myriad of purposes such as hunting for cancer genes, stopping viral infections and more recently, treating diseases in clinical trials.

Professor of CSHL and investigator of Howard Hughes Medical Institute (HHMI), Scott Lowe, along with another lead author of the study, Gregory Hannon said that RNAi is a great tool that in theory can be used to knock down any gene of interest, but has been difficult to apply in practice. One main challenge has been the difficulty of finding the right molecular trigger for RNAi. He said that the trigger is a small piece of RNA, which, by connecting to a matching piece of the target gene’s RNA, spurs its damage, so shutting down the production of protein from that gene.

Hannon explains that for every gene, depending on the size of its protein-coding RNA, there are potentially 500 to 5000 different small RNAs that can trigger RNAi and most of these are weak triggers that do not close down gene action completely or end up targeting a different gene resulting in so-called “off-target” effects. Lowe said that the scenario can damage experiments that use RNAi to examine diseases in the lab, lead to useless or even toxic treatments in the clinic and picking the right trigger has been like trying to pull a needle out of a haystack.

The Scientists’ team, in an online journal Molecular Cell, explain how they succeeded in solving this problem. Gregory Hannon is a pioneer in RNAi technology, and using molecular tools developed in the Lowe laboratory, the research team designed assesses that tests thousands of short hairpin RNA (shRNA) molecules at a time for their capability to shut down genes of interest in cells and identifies the most potent RNAi triggers.

In an experiment to find the most powerful RNAi triggers for nine target genes including a few hard-to-suppress cancer genes, researchers generated genetic codes for about 20,000 shRNAs, each with the potential to shut down any one of the nine target genes. Each and every genetic code was then added into a retrovirus that was also engineered to carry the target gene or the sensor and a gene for a fluorescent protein, called a marker. This design made sure that cells growing in dishes were tainted with the genetically engineered viruses, both the sensor and the marker would always be co-produced within each cell along with one shRNA.

Hannon said that there is still very little that is known about small RNA biogenesis, but this evaluation has now allowed researchers to explore this process at a scale that wasn’t feasible before. Researchers’ study of the 20,000 small hairpin RNAs, and particularly those recovered from cells in which the target gene had been efficiently shut off, has discovered new insights into this process and considerably improved the recipe for creating a potent RNAi trigger. Lowe said that their assay has given them a strong framework for rational design of small hairpin RNAs.
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