Chemical Modifications
While chemical modification of RNAi oligos is not required for siRNA function, certain modifications are sometimes useful. The antisense strand must have either a free 5′-OH or 5′-phosphate terminus [6-7]. A 5′-phosphate results in natural Dicer processing and this is the active form of the molecule. For 21-mer siRNAs, 5′-end modification of the sense strand RNA does not alter the efficacy of silencing and addition of a fluorescent dye, biotin, or other similar modifier can safely be done at this position. IDT recommends use of TYE™563 or Cy3™ when a fluorescent RNA is desired. Cellular autofluorescence in the spectrum of fluorescein limits its utility to track siRNAs in cell culture; TYE™563 and Cy3™ are brighter and spectrally does not suffer from the autofluorescence problem.
Chemical modification of DsiRNAs is slightly different due to the need to interact with Dicer. Modifications placed at the 3′-end of the sense strand have minimal impact on function or potency (IDT recommends this position for routine modification needs). Note that modifications placed at the 3′-S will be cleaved off the functional siRNA by Dicer processing; if a modification (such as biotin) is desired to be retained in the siRNA after cleavage, then 5′-S modification is preferred.
All RNAs have the potential to trigger IFN responses in cells [8-9]. Certain sequences and cell types are more at risk. Incorporation of 2′-O-methyl RNA residues can prevent activation of IFN responses [10] and should be considered for all in vivo applications. IDT has developed modified DsiRNAs that evade immune detection and have improved nuclease stability in serum.
In vivo use of siRNAs
Use of siRNAs in vivo is showing great potential as both research tools and as therapeutic agents [11]. DsiRNAs have been successfully used in vivo [12] and are available from milligram to gram scale both as chemically stabilized and as unmodified RNA. Large-scale sterile, endotoxin-free preparations are available specifically for in vivo research needs.