Defining the optimal parameters for hairpin-based knockdown constructs.
Induction of gene silencing using intracellularly expressed silencing triggers has been explored for large-scale loss-of-function screening, creation of knockdown cell lines or knockdown animals, and disease intervention. In all of these applications, the use of highly potent silencing constructs can maximize the possibility of obtaining target knockdown and thereby is intrinsically important for the chance of success. Several attempts have been made to improve the potency of a silencing construct. Results published in high profile journals such as Nature Biotechnology and Nature Genetics suggest that shRNAs with a 29-nucleotide (nt) stem is much more potent than shRNAs with a 19-nt stem, and miR30-based silencing constructs are much more potent than shRNA-based constructs. In this study, we systematically investigated several parameters, including the use of shRNA- or miR30-based scaffolds, the length of shRNA, and the selection of shRNA sequences for their impact on the knockdown efficiency of a silencing construct. Our studies revealed that the optimal configurations for a potent silencing trigger could be an shRNA with a 19-nt stem and a 9-nt loop. By comparing properties that favor the functional shRNAs and siRNAs using a set of 190 shRNAs against 19 targets and 360 siRNAs against four targets, we found that the functional shRNAs and siRNAs displayed similar but not identical nucleotide preferences. Based on the characteristic nucleotide preferences in the functional versus the nonfunctional shRNAs, we developed a computer program that outperforms an advanced siRNA selection algorithm for the enrichment of highly functional shRNAs.
Li
Leiming
L
Cancer Research, Global Pharmaceutical Research and Development, AP10, Abbott Laboratories, Abbott Park, Illinois 60064, USA.
Lin
Xiaoyu
X
Khvorova
Anastasia
A
Fesik
Stephen W
SW
Shen
Yu
Y
D001483:Base Sequence; D045744:Cell Line, Tumor; D006801:Humans; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D022661:RNA, Untranslated / Q000737:chemistry* / Q000378:metabolism*
D022661:RNA, Untranslated
EMBO J. 2005 Jan 12;24(1):138-48
15565168
Nature. 2006 May 25;441(7092):537-41
16724069
Nat Biotechnol. 2005 Aug;23(8):995-1001
16025102
Genes Dev. 2001 Jan 15;15(2):188-200
11157775
Nature. 2001 May 24;411(6836):494-8
11373684
Genes Dev. 2002 Apr 15;16(8):948-58
11959843
Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5515-20
11960009
Science. 2002 Apr 19;296(5567):550-3
11910072
Nat Biotechnol. 2002 May;20(5):497-500
11981564
Nat Biotechnol. 2002 Oct;20(10):1006-10
12244328
Nat Struct Biol. 2003 Feb;10(2):91-2
12536207
Nat Genet. 2003 Mar;33(3):401-6
12590264
Nature. 2003 Aug 14;424(6950):797-801
12917690
Cell. 2003 Oct 17;115(2):209-16
14567918
Nat Biotechnol. 2004 Mar;22(3):326-30
14758366
Nature. 2004 Mar 25;428(6981):427-31
15042091
Nature. 2004 Mar 25;428(6981):431-7
15042092
Nat Rev Drug Discov. 2004 Apr;3(4):318-29
15060527
Nat Med. 2004 Aug;10(8):816-20
15235598
Nature. 2004 Sep 16;431(7006):371-8
15372045
Nature. 1998 Feb 19;391(6669):806-11
9486653
Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15502-7
9860998
FEBS Lett. 2004 Nov 19;577(3):376-80
15556613
Mol Cell. 2004 Dec 22;16(6):861-5
15610730
Methods Enzymol. 2005;392:371-80
15644193
Cancer Res. 2005 Aug 15;65(16):7249-58
16103076
Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13212-7
16141338
Nat Genet. 2005 Nov;37(11):1289-95
16200064
Nat Genet. 2005 Nov;37(11):1281-8
16200065
Mol Ther. 2005 Nov;12(5):900-9
16115802
Nat Biotechnol. 2005 Feb;23(2):227-31
15619616