Small pieces of RNA in our cells can act like molecular switches that turn genes off by binding to them. These pieces, called small interfering RNAs (siRNAs) are also used by researchers to design experiments to understand what certain genes do.
Scientists can design siRNA molecules aimed at turning off specific genes they are trying to study. Though such siRNA ‘switches’ can be very useful, they are often non-specific, turning off hundreds of genes that they should not have an effect on. As a result, it is difficult for a biologist to conclude whether an experimentally observed effect is due to turning off the gene they meant to turn off or the hundreds that they didn’t (called “off-target effects”). Eugen Buehler of the National Center for Advancing Translational Sciences (NCATS), a new center at the NIH, describes an alternate approach to dealing with these off-target effects of siRNAs in his recent PLOS ONE paper. Read on to find out more about this research:
How did you become interested in improving siRNA experiments?
I’ve been working on siRNAs for about the last five years. When I would talk to my wife (a cell biologist) about my research, I would go on and on about all the problems created by these non-specific effects. Since she uses siRNAs in her research, she would ask, “Well, what should I do to avoid it?”
I didn’t have an answer. All the methods I had involved a statistical analysis of a large number of results from high-throughput screens, which look at several thousand genes at once. They couldn’t be applied to experiments that only involved one or a few genes, which is what many researchers do. It frustrated me not being able to help her, and so this question of what to do about off-target effects in small-scale experiments kept nagging me, until I found an answer.
And what was that answer?
As is often the case, the answer involved looking at the problem a different way. For years, people have been trying to solve the problem by getting rid of the non-specific effects. There are many ways to do this, but they still have a high incidence of these effects.
So, rather than trying to eliminate the off-target activity, we took the opposite approach. We changed three points (bases 9-11) where an siRNA makes contact with its target, so it couldn’t have the effect it was designed for. In this way, we created the C911 version, an anti-siRNA of sorts, which had all the off-target effects but none of the on-target effects.
So if an siRNA has an effect in a cell that is different from what the C911 version of the same siRNA has, we can conclude that the effect is because it silenced the intended target.
Which figure in the manuscript do you think best summarizes your results?
Definitely Figure 3B. Here, we compare siRNAs that appear to have specific effects but don’t (false positives), with ones that do have a specific action on a target gene. We took ten of each kind and created C911 versions for all twenty.
When we compared the two we found that for the false positives, the siRNA and the anti-siRNA had the same effects (left hand panel). But for the siRNAs which really did have an effect on their target, there was a big difference between the siRNA and its C911 version. As it happened, the C911 controls worked perfectly for all twenty siRNAs we had selected for the experiment.
Where do you hope to go from here?
For a tool as well established as siRNA, it will take a while to change the way we design our experiments. The first step is for there to be a reasonable alternative, and that is what this paper is meant to supply. The next is to make that alternative easy to choose. Part of that will involve getting companies that manufacture siRNAs to eventually make negative controls like this, so that negative controls like C911 can be easily and affordably obtained for any siRNA.
My hope is that someday when a researcher orders an siRNA, they won’t even have to ask; they’ll get a tube with their siRNA and a tube with the appropriate negative control by default.
Citation: Buehler E, Chen Y-C, Martin S (2012) C911: A Bench-Level Control for Sequence Specific siRNA Off-Target Effects. PLoS ONE 7(12): e51942. doi:10.1371/journal.pone.0051942
Image: Target by Ivan McClellan on flickr