Current Considerations for siRNA Design

1.Choose a 21 or a 23 nt sequence in the coding region of the mRNA with a GC ratio as close to 50% as possible. Ideally the GC ratio will be between 45% and 55%. An siRNA with 60% GC content has worked in many cases, however when an siRNA with 70% GC content is used for RNAi typically a sharp decrease in the level of silencing is observed. Avoid regions within 50-100 nt of the AUG start codon or 50-100 nt of the termination codon.

2.Avoid more than three guanosines in a row. Poly G sequences can hyperstack and therefore form agglomerates that potentially interfere in the siRNA silencing mechanism.

3.Preferentially choose target sequences that start with two adenosines . This will make synthesis easier, more economical, and create siRNA that is potentially more resistant to nucleases. When a sequence that starts with AA is used, siRNA with dTdT overhangs can be produced.
      If it is not possible to find a sequence that starts with AA and matches rules 1and 2, choose any 23 nt region of the coding sequence with a GC content between 45 and 55% that does not have more than three guanosines in a row.

4.Ensure that your target sequence is not homologous to any other genes. It is strongly recommended that a BLAST search of the target sequence be performed to prevent the silencing of unwanted genes with a similar sequence.

5.Based on feedback from various customers, labelling the 3'-end of the sense strand gives the best results with respect to not interfering with the gene silencing mechanism of siRNA. Consult our custom siRNA page for available modifications.

       When these rules are used for siRNA target sequence design RNAi effectively silences genes in more than 80% of cases. Current data indicate that there are regions of some mRNAs where gene silencing does not work. To help ensure that a given target gene is silenced, it is advised that at least two target sequences as far apart on the gene as possible be chosen.
       This method does not take into account mRNA secondary structure. At present it does not appear that mRNA secondary structure has a significant impact on gene silencing.