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Several guidelines have been proposed to design effective siRNA (3-5). The strategy for siRNA design is based on our present understanding of the biochemical mechanisms involved in RNA interference and, in particular, structural features that allow the antisense-strand of the siRNA duplex to be more efficiently incorporated into the RNA-induced silencing complex (RISC). The main characteristics are listed below (3).
Default parameters: (N19)TT
1. Low to medium GC content (30-50%).
2. Absence of internal repeats or palindromes.
3. Presence of an A at position 3 of the sense strand.
4. Presence of A at position 19 of the sense strand.
5. Absence of G or C at position 19 of the sense strand.
6. Presence of U at position 10 of the sense strand.
7. Absence of a G at position 13 of the sense strand.
8. At least 3 A/Us at positions 15-19 of the sense strand.
There are several other factors that influence effective functional performance of the designed siRNA; half-life of the siRNA is crucial for sustained activity and in many cases to increase effect for a longer time the dosage (concentration) is increased that leads to toxicity and off target effects. Similarly serum or cellular delivery methods can be improved. A common list of features for improvement is given below and possible sites in the nucleobase that can be modified to impart specific customized properties.
Common Features for Improvement
1. Increased nuclease resistance.
2. Increased duplex stability and manipulation of duplex stability.
3. Cellular delivery.
4. Surface attachment.
Common Modification Sites
1. Phosphodiester linkages.
2. Nucleic acid bases.
3. Sugar moieties.
4. Functional group addition.
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