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The researchers found that slicing rate increased when the base at position 7 was an A or a U. The bases A and U pair more weakly than C and G. The researchers found that having a weak A-U pair at that position, or a fully mismatched pair at position 6 or 7, may allow a kink to form in the double helix shape that actually makes the target easier to slice. Wang also found that slicing rate increases with certain substitutions at the 10th and the 17th base positions, although the researchers could not yet determine possible underlying mechanisms.<\/p>\n
These observations correspond to existing recommendations for RNAi design, such as not using a G at position 7. The new work demonstrates that the reason these recommendations work is because they affect the slicing rate, and, in the case of position 7, the new work further identifies the specific mechanism at play.<\/p>\n
Interplay between regions matters<\/h6>\n People designing synthetic guide RNAs thought that the bases at the tail end, past the 16th position, were not very important. This is because in the case of the most commonly used guide RNAs, the target will be rapidly cleaved even if all of the tail end positions are mismatches that cannot pair.<\/p>\n
However, Wang and Bartel found that the identity of the tail end bases are only irrelevant in a specific scenario that happens to be true of the most commonly used guide RNAs: when the bases in the center of the guide RNA (positions 9-12) are strong-pairing Cs and Gs. When the center pairings are weak, then the tail end bases need to be perfect matches to the target RNA. The researchers found that guide RNAs could have up to a 600-fold difference in tolerance for tail end mismatches based on the strength of their central pairings.<\/p>\n
The reason for this difference has to do with the final set of motions that the two RNAs must perform in order to assume their final double helix shape. A perfectly paired tail end makes it easier for the RNAs to complete these motions. However, a strong enough center can pull the RNAs into the double helix even if the tail ends are not ideally suited for doing so.<\/p>\n
The observation that weak central pairing requires perfect or near perfect tail end matches could provide a useful new guideline for designing synthetic RNAs. Any guide RNA runs the risk of sometimes binding other messenger RNAs that are similar enough to the intended target RNA. In the case of a therapy, this off-target binding can lead to negative side effects. Bartel and Wang suggest that researchers could design guide RNAs with weak centers, which would require more perfect pairing in the tail end, so that the guide RNA will be less likely to bind non-target RNAs; only the perfect pairing of the target\u2019s RNA sequence would suffice.<\/p>\n
Altogether, Wang and Bartel\u2019s findings explain how small differences between guide RNAs can make such large differences in the efficacy of RNAi, providing a rationale for the long-standing RNAi design guidelines. Some of the findings even suggest new guidelines that could help with future synthetic guide RNA designs.<\/p>\n
\u201cDiscovering the interplay between the center and tail end of the guide RNA was unexpected and satisfying,\u201d says Bartel, who is also a professor at the Massachusetts Institute of Technology and a Howard Hughes Medical Investigator. \u201cIt explains why, even though the guidelines suggested that tail-end sequence doesn\u2019t matter, the target RNAs that are sliced in our cells do have pairing to the tail end. This observation could prove useful to reduce off-target effects in RNAi therapeutics.\u201d<\/p>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"
RNA interference (RNAi) is a process that many organisms, including humans, use to decrease the activity of target RNAs in cells by triggering their degradation or slicing them in half. If the target is a messenger RNA, the intermediary between gene and protein, then RNAi can decrease or completely silence expression of the gene. Researchers […]<\/p>\n","protected":false},"author":1783,"featured_media":29527,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[6212,6221,6240],"tags":[],"class_list":["post-29526","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-news-briefs","category-whitehead-institute","category-whitehead-institute-faculty-locations","placement-placement-homepage","research-area-biochemistry-biophysics-and-structural-biology","research-area-computational-biology","research-area-genetics"],"acf":[],"yoast_head":"\n
Gene silencing tool has a need for speed - MIT Department of Biology<\/title>\n \n \n \n \n \n \n \n \n \n \n \n\t \n\t \n\t \n \n \n \n\t \n\t \n\t \n