Researchers have long considered off-target effects to be one of the most significant hurdles to RNAi-based screening experiments. Although they assumed that siRNA/mRNA complementarity triggers these unintended gene-silencing events, new research from Dharmacon suggests that 3' untranslated region seed matches are the true culprit in the vast majority of cases.
The findings call into question the effectiveness of current protocols used to minimize off-target effects via homology matching, such as BLAST and the Smith-Waterman local alignment algorithm.
"For the last year and a half … off-target [effects] have been abuzz in RNAi research," William Marshall, vice president of technology and business development at Dharmacon parent firm Fisher Biosciences, told RNAi News this week. "We … have a very large amount of data around real off-target events through global expression profiling, and we decided … to really dig into this and think mechanistically about what could be going on [to] start looking for the root cause for off-target events."
Marshall noted that while much importance has been placed on BLAST and Smith-Waterman to find the long stretches of sequence homology thought to be causing off-target events, "much to our surprise, we found that there wasn't a very good correlation between long stretches of exact complementarity determined by [these tools] and true experimentally validated off-target events."
"It had become somewhat of a dogma that the long stretches of homology and … doing things like BLAST or Smith-Waterman … were the important factors in minimizing off-target events."
Dharmacon's research, which appears in the March issue of Nature Methods, found that, except in cases where there is a near-perfect complementarity, "the level of overall complementarity between an siRNA and any given mRNA is not associated with off-target identity."
"We really thought that this may be a microRNA-based phenomenon," Marshall, one of the Nature Methods co-authors, said. As such, the researchers then posed the question: "If this actually is a microRNA-based phenomenon, what do we know about microRNA biology?
"We know that the microRNAs use the seed region to bind to a host of different RNAs in their untranslated regions," he said. "So our next thinking was [to] … start searching for seed regions and complementarity in that short 6- to 7-nucleotide region between the siRNA that's causing the off-target [event] and the gene that's being affected by it."
Ultimately, he said, the Dharmacon group uncovered "a very compelling story:" that off-targeting is associated with the presence of one or more perfect 3' untranslated region matches with the hexamer or heptamer seed region of the antisense strand of the siRNA.
"It had become somewhat of a dogma that the long stretches of homology and … doing things like BLAST or Smith-Waterman … were the important factors in minimizing off-target events," Marshall said. "We would assume from first principle that these long stretches of homology would be an important factor. What was surprising was that when we did the true analysis and really dug into this, we couldn't see this correlation that we were hoping to see."
According to the authors of the Nature Methods paper, "current protocols used to minimize off-target effects," such as BLAST and Smith-Waterman, "have little merit aside from eliminating the most obvious off-targets … and likely discard substantial numbers of functional siRNAs owing to unfounded specificity concerns."
"What was surprising was that when we did the true analysis and really dug into this, we couldn't see this correlation [between long stretches of homology and off-target events] that we were hoping to see."
However, they caution that "the siRNA seed-3' UTR match is only one parameter in what is assumed to be an extremely complex phenomenon. Because the sheer number of genes that contain matches with any given siRNA seed region is very large in comparison to the number of actual off-targets for that siRNA, the value of the identified parameter by itself is limited.
"The identification of additional factors that have roles in off-targeting will likely lead to development of predictive algorithms that minimize off-target effects and enhance siRNA design," they add.
For Dharmacon, the findings are expected to be incorporated into future products and services, Marshall said. However, it is still "very early. We did the science and moved quickly to publish because we thought it was so important," and it will take time to actually apply the research, he noted.
In the end, Marshall expects the findings to be most beneficial to those conducting large-scale RNAi screens. "If you're doing one-off gene-silencing experiments, or smaller sets of gene-silencing experiments, you can go through and correct for these things to get some pretty good ideas of off-target versus on-target gene silencing," he said.
"But [with] things like genome-wide screens [that generate] massive amounts of data … [the findings will provide] you a tool to go in and help guide whether or not you have on-target gene silencing versus the potential for off-target gene silencing."
Doug Macron ([email protected])