Well designed siRNAs are potent and specific modulators of gene expression and have immense potential in therapeutic gene silencing. Very successful design rules and algorithms have been established based on performance of hundreds of siRNA and miRNAs (miRNAs are naturally occurring and structurally resemble siRNA and use a similar silencing complex). The most important determinant of siRNA function is sequence specific recognition and Watson-Crick base pairing of the target single stranded mRNA sequence facilitated by the RISC complex followed by cleavage of the target mRNA leading to gene silencing. Robust algorithms are available for predictive selection of sequences that are patterned on established design rules (1-4).
Other than specific sequence requirements, effective siRNA functionality requires the following attributes.
1. Effective cell membrane permeation.
2. Targeted delivery.
3. Nuclease resistance confers increased half-life.
4. Sustained gene silencing with increased duplex stability.
5. Minimal off-target seeding and silencing
6. Low dosage. Minimal Toxicity
siRNAs are conveniently synthesized chemically similar to common primers; synthetic oligos are used ubiquitously for molecular applications from the simplest as amplification primers to the more complex as siRNA and aptamers. In general a well designed oligo to serve as a primer, probe or siRNA will perform using standard bases for hybridization to its cognate sequence(s); but we can make it perform better using modified bases that are specifically developed by nucleic acid chemists to enhance exacting characteristics.
As with most natural molecules oligos are prone to degradation under normal conditions, specifically once introduced in body fluids. Ubiquitous nucleases as well as chemical instability lead to fast degradation with a finite half life.
Gene Link presents various design options for synthesizing effective siRNAs, probes and oligos based on the application. SmartBase™ siRNA can be synthesized in a predetermined way to exhibit the features that is desired; for instance to increase duplex stability 2’-fluoro C and U bases and 5-methyl dC and 2-Amino dA can be substituted, for nuclease resistance the phosphodiester linkages can be selectively substituted with phosphorothioate and for cellular delivery we may add cholesterol to the synthetic oligonucleotide sequence or modify with thiol or amine for post synthesis conjugation with Cell Penetrating Peptides (CPP’s) that are known to aid transport and facilitate cellular uptake.
The premise of this product guide is to introduce the use of SmartBase™ modifications and go beyond the traditional use of standard DNA and RNA bases for constructing synthetic siRNAs in particular and as well emphasize their use in primers, oligos and probes. SmartBase™ modifications also introduces the molecular biologists to develop a cross-disciplinary synergy of molecular applications to a wealth of nucleic acid chemistry tools available as modified bases to impart specific properties compatible with biological applications and gene expression pathways.
