To a non-biologist, it’s a little known and somewhat surprising side of genetics: In the deepest innards of our cells, tiny bits of RNA regulate the expression of myriad genes. Their activities are governed by an enzyme called Dicer, which snips the tiny workhorses called miRNA molecules from larger RNA molecules; the exact activity of the molecules depends on their sequence.
Scientists are interested in the process because of its potential for therapeutic manipulation: If they can harness the technique, they could flip gene expression on and off at will to learn more about the internal workings of the cell, to replace damaged or missing molecules, or even to block viral infections such as hepatitis and HIV. Dicer and the RNAs it processes play important roles in the pathogenesis of cancer as well as many other acquired diseases.
There’s one problem, though: Like a kindergartner wielding his first pair of scissors, Dicer is not all that picky as to exactly where it cleaves the larger RNA molecules, called pre-miRNAs and shRNAs. As a result, the smaller, inhibiting molecules can vary in their exact sequence and biological activity. Such unpredictable ‘off-target’ effects are a death knell for therapeutic uses, which naturally demand a high level of precision and reproducibility.
Now Stanford geneticists Shuo Gu, PhD, and Mark Kay, MD, PhD, have hit upon a way to specifically target Dicer’s activity by positioning the desired cleavage point a precise distance away from a naturally occurring loop or bulge in the RNA molecule. The research was published today in Cell (subscription required). According to Kay:
We discovered a new loop-counting rule that Dicer employs to process pre-miRNAs. Using this rule, we were able to design shRNAs that produced a homogenous population of siRNAs capable of repressing targeted gene expression in cells while restricting the generation of fragments with off-target activities.
The researchers showed that they could use the technique to block the expression of hepatitis C viral proteins in an infected cell with fewer unwanted side-effects. "Our findings will impact the future design of more-efficient and safer shRNAs for therapeutic uses and increase the specificity of RNAi-based genetic screens used in biological discovery," Kay told me.