Tài liệu Báo cáo khoa học: Effect of siRNA terminal mismatches on TRBP and Dicer binding and

Effect of siRNA terminal mismatches on TRBP and Dicer

binding and silencing efficacy

Hemant K. Kini and S. P. Walton

Applied Biomolecular Engineering Laboratory ⁄ Cellular and Biomolecular Laboratory, Department of Chemical Engineering and Materials

Science, Michigan State University, East Lansing, MI, USA

Introduction

Short interfering RNAs (siRNAs) can be designed to

target and regulate the expression of any gene of

interest. Gene silencing by RNA interference (RNAi)

is mediated by endogenous proteins, resulting in tar￾get mRNA cleavage or translational inhibition [1]. In

the cytoplasm of human cells, the dsRNA binding

proteins HIV transactivating response RNA-binding

protein (TRBP) and Dicer recognize and bind the

siRNA and form RNA-induced silencing complex

(RISC) loading complexes (RLCs) [2–4]. Argonaute 2

(Ago2), the catalytic core of the RISC [5,6], is then

recruited by the RLC to form a holo-RISC [7].

Although other proteins such as protein activator of

protein kinase R (PACT) might also be associated

with the formation of holo-RISCs [8–12], in vitro

experiments have shown that TRBP, Dicer and

Ago2 alone are capable of forming an active mini￾mal RLC [13].

Being double-stranded, either strand of the siRNA

can be used as the guide strand of an active RISC.

Keywords

Dicer; mismatches; RNA interference; short

interfering RNA; TRBP

Correspondence

S. P Walton, Applied Biomolecular

Engineering Laboratory ⁄ Cellular and

Biomolecular Laboratory, Department of

Chemical Engineering and Materials

Science, Michigan State University, 3249

Engineering Building, East Lansing, MI

48824-1226, USA

Fax: +1 517 432 1105

Tel: +1 517 432 8733

E-mail: [email protected]

Website: http://www.egr.msu.edu/abel/

(Received 2 July 2009, revised 28 August

2009, accepted 7 September 2009)

doi:10.1111/j.1742-4658.2009.07364.x

To enhance silencing and avoid off-target effects, siRNAs are often

designed with an intentional bias to ensure that the end of the siRNA that

contains the guide strand 5¢ end is less stably hybridized relative to the end

containing the passenger strand 5¢ end. One means by which this is accom￾plished is to introduce a terminal mismatch, typically by changing the

passenger strand sequence to impair its hybridization with the guide strand

5¢ end. However, there are conflicting reports about the influence of termi￾nal mismatches on the silencing efficacy of siRNAs. Here, the silencing effi￾ciency of siRNAs with a terminal mismatch generated either by altering

the guide strand (at the 5¢ end, nucleotide 1) or the passenger strand

(nucleotide 19 from the 5¢ end) was examined. Subsequently, we studied

the relationship between the silencing efficiency of the siRNAs and their

binding to the RNA-induced silencing complex loading complex proteins

HIV transactivating response RNA-binding protein and Dicer in H1299

cytoplasmic extracts. Binding of siRNA and the transactivating response

RNA-binding protein was significantly reduced by terminal mismatches,

which largely agrees with the reduction in eventual silencing efficacy of the

siRNAs. Single terminal mismatches led to a small increase in Dicer

binding, as expected, but this did not lead to an improvement in silencing

activity. These results demonstrate that introduction of mismatches to

control siRNA asymmetry may not always improve target silencing, and

that care should be taken when designing siRNAs using this technique.

Abbreviations

Ago2, Argonaute 2; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; RISC, RNA-induced silencing

complex; RLC, RISC loading complex; siRNA, short interfering RNA; TRBP, HIV transactivating response RNA-binding protein.

6576 FEBS Journal 276 (2009) 6576–6585 ª 2009 The Authors Journal compilation ª 2009 FEBS