Functional polarity is introduced by Dicer processing of short substrate RNAs.
Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.
Rose
Scott D
SD
Integrated DNA Technologies, Inc. 1710 Commercial Park, Coralville, IA 52241, USA.
Kim
Dong-Ho
DH
Amarzguioui
Mohammed
M
Heidel
Jeremy D
JD
Collingwood
Michael A
MA
Davis
Mark E
ME
Rossi
John J
JJ
Behlke
Mark A
MA
D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D043244:Ribonuclease III