Going Beyond Terminal Tm
In addition to G/C content at the termini of the siRNAs, traits that were found to be influential in defining optimal siRNA sequences include the Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region) and nucleotide content of the 3' overhangs. These findings are incorporated into the Cenix algorithm used to design Ambion's Silencer Pre-designed siRNAs and SilencerValidated siRNAs as well as the Silencer siRNA Libraries. The design process also includes a specificity check whereby both siRNA strands are subjected to customized homology searches to minimize risks of generating off-target effects, and putative siRNA target sequences are screened against the most updated single-nucleotide polymorphism (SNP) databases to avoid variability in siRNA efficacy. Target mRNA sequences known to mediate regulatory processes through binding to protein factors are similarly avoided, as are CpG motifs.
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