| RNA interference,also known as RNA silencing,is a highly universal,eukaryotic biological process in which RNA regulate gene expression inpost-transcriptional level.Classical RNA silencing describes the process that 21?23bp RNA duplex activate endogenous RNAi pathway,siRNA is used to lead RISC to recognize target RNA in base-pairing manner,then degrades target RNA or inhibits translation process,and finally leads to a significant decline in both RNA level and protein level.The discovery of asymmtric RNAi refreshes our understanding of RNAi:the 19+2 structure of classical siRNA is not necessary.Two strands of siRNA that do not totally match,nor have same length are still capable of triggering RNAi.Compared with classical siRNA,asymmetric RNA can efficiently reduce off-target effect.This provide new ways to enhance accuracy of RNAi.We believe furthering the study on aiRNA will help understand the roles played by the two strands.As is known to all,siRNA mediated mRNA degradation was driven by asRNA,ssRNA's function is not clear,while single-stranded asRNA is not enough to trigger RNAi.David Corey from Southwestern Medical Center in US suggests ssRNA is capable of protecting asRNA from endougenous ribozymes' destruction,thus keeping asRNA intact.Based on this suggestion,we promote the RNA trimming process:We believe,ssRNA is capable of protecting asRNA from exo-ribonuclease;in asymmetric RNA structure,asRNA gets trimmed at the ends because of losing part of the protection from ssRNA;Short ssRNA makes asRNA not long enough to trigger RNAi.In order to prove this hypothesis,we focused on detecting RNAi of different kinds of aiRNA.We found that,the decrease of RNAi caused by overexposure of asRNA's 3' ends can be rescued by adding phosphate at 5' ends of asRNA.The decrease of RNAi caused by overexposure of asRNA's 5'ends can be rescued by adding two nucleotids at the 3'end of asRNA.asRNA whose ends match ssRNA but the middle don't triggers RNAi,while asRNA whose middle matche ssRNA but the ends don't trigger RNAi.We use NGS to sequence small RNA library originated from aiRNA transfecting cell lines in order to see if ssRNA and asRNA show any difference in their dosage.We found that amount of ssRNA were far less than asRNA even though they were equally transfected.aiRNA with 3'ends of asRNA overhanging(Type A aiRNA)shows lower amount in both ssRNA and asRNA;while aiRNA with 5' ends of asRNA overhanging(Type B aiRNA)shows lower amount only in ssRNA.We next confirm dosage asymmetric siRNA can mediate very strong RNAi with less off-target effect.And we found that by increasing the dosage of aiRNA,we can rescue weakened RNAi ofType A aiRNA.By adding the amount of ssRNA,we can save the reduced RNAi of Type B aiRNA.We construct aiRNA expressing plasmids with shRNA expressing patterns and single-stranded RNA expressing patterns and checked their RNAi and off-target effect using LAR assay.Results show both aiRNA patterns can mediate RNAi with less off-target effect.Compared with classical RNA transfecting effect,aiRNA with 5' ends of asRNA overhanging does not show RNAi attenuation.In sum,we find the difference between phosphorylated and non-phosphorylated RNA in RNAi activity,the interplay of ssRNA and asRNA in RNAi activity.We find the asymmetric dosage of siRNA by NGS.We construct aiRNA expressing plasmids and evaluate their RNAi activity and off-target effect. |
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