46 BIOTECHNOLOGY
Fig. 2. IDT synthesises and ships an average of 36,000 custom oligos per day to more than 86,000 customers worldwide.
product. DsiRNAs duplexes have a single 2-base, 3’ overhang on the antisense strand and are blunt on the other end. Te blunt end is further modified with DNA bases. Tis design provides Dicer with a single favorable binding site that helps direct the cleavage reaction.
Te functional polarity introduced by this processing event favours antisense strand loading into RISC. It is thought that the increased potency of DsiRNAs is related to this linkage between Dicer processing and RISC loading7
¾ that is,
increased antisense loading will result in increased target mRNA cleavage.
Designing siRNAs Te ability of a particular siRNA to silence gene expression is predominantly determined by its sequence, and not all target sites are equal6, 9
. In addition to the
sequence, other considerations, such as cross-hybridization and
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Additionally, modification can be used to increase cellular uptake and
chemical modifications, can alter the effectiveness of an siRNA 5
.
Location: Te localization of an siRNA within a target gene, either internal or at 5’ or 3’ ends, is not a major factor for silencing efficacy. However, complete knowledge of specific splice variants is important for targeting of the desired isoform(s) of the gene, as is locations of common polymorphisms8
.
Modifications: Chemical modification is not required for siRNA function, but certain modifications are sometimes useful. Chemical modifications can decrease the susceptibility of synthetic nucleic acids to nuclease degradation, thus increasing siRNA stability10
. Tey
can also reduce siRNA activation of an innate immune response during in vivo applications9
.
to prevent unwanted participation in miRNA pathways that create off-target effects9
. However, chemical
modification can also alter the potency of an siRNA and modified siRNAs should be empirically tested to ensure that they are effective.
siRNAs must have phosphate groups at the 5’ end in order to have activity so it is important to not block the 5’ end of the antisense strand with modification. Tat said, 5’-OH ends are rapidly phosphorylated by cellular kinases, in vitro or in vivo, so it is not necessary to phosphorylate synthetic siRNAs5
.
Termodynamic stability: Te most effective siRNAs have a relatively low melting temperature (Tm
more A/U rich) toward the 5’-end of the guide strand and a relatively high Tm
(more stable, more G/C
rich) toward the 5’-end of passenger strand5
. When options for a target
sequence are limited, it may not be possible to select thermodynamically favorable regions. For these situations, it is possible to introduce mismatches (to lower Tm
) or to
add modified bases (to increase Tm
) to the siRNA duplex to create thermodynamic asymmetry. If non-complementary bases are introduced, it is important that they are on the 3’-end of the passenger strand rather than the 5’-end of the guide strand to avoid impairing the ability of the guide strand to anneal to the target.
) and duplex stability (less stable,
■ Sequence characteristics and specificity: To maintain specificity, the guide strand should not contain sequence characteristics such as homopolymeric runs (those with 4 or more identical nucleotides) or 9-base or greater segments of G/C bases5
. In addition, target
accessibility ¾ eg, secondary structure, is an important factor affecting siRNA efficacy5
. A
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