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How to design siRNA

Tom Tuschl's Method:

1. Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start codon
2. Look for 23-nt sequence motif AA(N19) first. If there is no suitable sequence look for 23-nt sequence motif NA(N21) and convert the 3' end of the sense siRNA to TT
3. Alternatively look for NAR(N17)YNN
4. Target sequence should have a GC content of around 50%

A = Adenine
T = Thymine
R = Adenine or Guanine (Purines)
Y = Thymine or Cytosine (Pyrimidines)
N = Any.

Rational siRNA design

By experimentally analyzing the silencing efficiency of 180 siRNAs targeting the mRNA of two genes and correlating it with various sequence features of individual siRNAs, Reynolds et al at Dharmacon, Inc identified eight characteristics associated with siRNA functionality. These characteristics are used by rational siRNA design algorithm to evaluate potential targeted sequences and assign scores to them. See company's disign website siDESIGN below. The sequences with higher scores will have higher chance of success in RNAi.

Design with Softwares

siRNA Target Finder of Ambion. The background materials can be seen here.

siDESIGN Center at Dharmacon is an advanced, user-friendly siRNA design tool, which significantly improves the likelihood of identifying functional siRNA when compared to other publicly available design tools that rely on on early design guidelines. The algorthem is based on the publication of A. Reynolds and colleagues.

Construct Builder and Target Finder of siRNA from GenScript. The cited reference for the website can be found here.

Designer - Invitrogen Block-iT RNAi.

Deqor of Scionics Computer Innovation according to their publication.

Proligo by Sigma - siRNA design tool created based on Tuschl's rules.

siDirect University of Tokyo is particularly targeted to mammalian and chick RNAi interference.

Sfold software at Wadsworth Center, New York State Department of Health.

siRNA Design at Whitehead Institute based on two high profile papers - Asymmetry in the assembly of the RNAi enzyme complex and Functional siRNAs and miRNAs exhibit strand bias.

siRNA Selector at Wistar Institute based on publication called Gene specific siRNA selector

E-RNAi: a web application to design optimized RNAi constructs published paper can be found here.

RNAi Caculator

TROD - T7 RNAi Oligo design

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