Rules of siRNA design for RNA interference (RNAi) | |
Source: Protocol Online | |
Date Added: Sat May 29 2004 | |
Date Modified: Mon Feb 02 2009 | |
Abstract: Summarize both Tom Tuschl's motif siRNA design rules and siRNA rational design. | |
General Guidelines
Tom Tuschl's rules
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. Sequences with higher scores will have higher chance of success in RNAi. The table below lists the 8 criteria and the methods of score assignment.
Criteria | Description | Score | |
Yes | No | ||
1 | Moderate to low (30%-52%) GC Content | 1 point |
|
2 | At least 3 A/Us at positions 15-19 (sense) | 1 point /per A or U |
|
3 | Lack of internal repeats (Tm*<20ˇăC) | 1 point |
|
4 | A at position 19 (sense) | 1 point |
|
5 | A at position 3 (sense) | 1 point |
|
6 | U at position 10 (sense) | 1 point |
|
7 | No G/C at position 19 (sense) |
| -1 point |
8 | No G at position 13 (sense) |
| -1 point |
A sum score of 6 defines the cutoff for selecting siRNAs. All siRNAs scoring higher than 6 are acceptable candidates.
*Tm = 79.8 + 18.5*log<sub>10([Na+]) + (58.4 * GC%/100) + (11.8 * (GC%/100)2) - (820/Length)
For example, the Tm can be calculated as follows for the siRNA UUCUCCAGCUUCUAAAAUA
Tm = 79.8 + 18.5*log<sub>10(0.05) + (58.4 * 31.6/100) + (11.8 * (31.6/100)2) - (820/19)
Tm = 32.19
There are two siRNA design tools which implement this siRNA design
algorithm: one is offered by Dharmacon, Inc; the other is a downloadable Excel
template, written by Maurice Ho at http://boz094.ust.hk/RNAi/siRNA.
References