siRNA design-RNAi

How to design effective siRNA has always been a hot topic in current RNAi research The effectiveness of gene suppression largely depends on the selection of the target gene sequence. The target sequence can be randomly selected or tested on different regions of the target gene to determine which sequence is most effective. The following points are extremely important for the success of siRNA design.

1. Starting from the AUG start codon of the transcript (mRNA), search for the "AA" double stranded sequence and record its 19 base sequence at the 3 'end as a potential siRNA target site. Both the sense chain and the antisense chain are designed using these 19 bases (excluding AA repeats).

2. Avoid selecting the destination sequence near the starting codon or meaningless area.

3.The GC content of SiRNA sequence should be around 30% -50%.

4. Tuschl et al. suggest not targeting the 5 'and 3' untranslated regions (UTRs) when designing siRNA, as these regions have abundant regulatory protein binding regions, and these UTR binding proteins or translation initiation complexes may affect the binding of siRNP endonuclease complexes to mRNA, thereby affecting the effectiveness of siRNA.

5. Compare the selected sequence in a public database to ensure that the target sequence has no homology with other genes.

6. Compare potential sequences with corresponding genomic databases (human, mouse, rat, etc.) to exclude sequences that are homologous to other coding sequences/ESTs. For example, using BLAST (www.ncbi. nlm. nih. gov/BLAST/)

7. Select the appropriate target sequence for synthesis. Usually, a gene requires the design of multiple siRNA target sequences to find the most effective siRNA sequence.

8. Negative control: A complete siRNA experiment should have a negative control, and the siRNA used as the negative control should have the same composition as the selected siRNA sequence, but without obvious homology with mRNA. The usual practice is to scramble the selected siRNA sequence and also check the results to ensure that it has no homology with other genes. If we plan to synthesize siRNA, we can directly provide a 21 base sequence starting with AA, and the manufacturer will synthesize a complementary pair of sequences. It should be noted that siRNA synthesized usually ends with 3'dTdT, and if UU termination is required, special instructions should be provided. There are results showing that there is no difference in the effectiveness between UU terminated and dTdT terminated siRNA. Because this protruding end does not need to be complementary to the target sequence.

1.The shRNA cloned into the shRNA expression vector consists of two short reverse repeat sequences separated by a loop sequence, forming a hairpin structure controlled by the pol III promoter. Subsequently, 5-6 T were connected as transcription terminators for RNA polymerase III.

2. Two complementary oligonucleotides must have restriction enzyme cleavage sites at both ends.

3.Stratagene found that 29 oligonucleotides were more effective in inhibiting the target gene compared to the originally recommended 23 oligonucleotides.

4. Attach a C tightly below the enzyme cleavage site downstream of the promoter, creating a certain spatial gap between the insertion fragment and the promoter to ensure transcription occurs.

5.The first base of the ShRNA target sequence must be G to ensure transcription by RNA polymerase. If the selected destination sequence does not start with G, a G must be added upstream of the closely connected justice chain.

6.The stem loop in the ShRNA insertion fragment should be located near the center of the oligonucleotide. Stem loops of different sizes and nucleotide sequences have been successfully utilized. The stem loop containing a unique restriction enzyme cleavage site facilitates the detection of clones with shRNA insertion fragments. After comparing stem loops of various lengths and sequences, the 5 'TCAAGAG3' sequence was found to be the most effective.

7.5-6 T must be placed at the tail of the shRNA insertion fragment to ensure RNA polymerase III terminates transcription.

8. There cannot be three or more consecutive T's on the sequence of the justice chain and the antonym chain. This may lead to premature termination of shRNA transcription.