Modified 27-nt dsRNAs with Dramatically Enhanced Stability in Serum and Long-Term RNAi Activity

Abstract

The present study describes improved properties of 27-nt dsRNAs over 21-nt siRNAs, and accents on the possibility to use their modifications and conjugates for direct long-term gene silencing in viable cells and animals, avoiding conventional transfectants. Using a Renilla Luciferase gene-silencing system and cultured cell lines, we established that 27-nt dsRNAs possessed about three to five times higher "long-term" RNAi activity than 21-nt siRNAs and 21-nt dsRNAs. Moreover, if RNA duplexes were preincubated with cell-cultured medium for several hours before their transfection in cells, 21-mer completely lost its RNAi effect, while 27-mer, its amino modifications, thiol modifications, and cholesterol conjugates manifested a strong gene silencing. In attempts to clarify the reason(s) for the higher RNAi activity of 27-nt dsRNAs, we found that they were approximately 100 times more stable than 21-nt siRNA and 21-nt dsRNA in cell-cultured medium supplemented with 10% inactivated serum, approximately 50 times more stable in 90% inactivated serum, and approximately six times more stable in active serum. The 5' sense modification was selected as the most stable, accessible to Dicer, and with highest RNAi potential. The RNAi activity of 5' sense modifications was higher even than the activity of nonmodified 27-nt dsRNA. The 5' sense amino modification also did not influence the activity of 21-nt siRNA, right overhang 25/27-nt (R25D/27), and 25D/27-nt RNAs. The stability of 5' sense modified R25D/27-nt and 25D/27-nt RNAs in serum was lower than that of blunt 27-nt dsRNA. However, these asymmetric RNAs were more active than modified and nonmodified blunt 27-nt dsRNAs, which demonstrates the superiority of the asymmetric design. The 5' sense modifications were considered as most appropriate for conjugation with small signal molecules to facilitate the intracellular delivery of RNA duplex, to preserve its RNAi capacity, and to ensure a possibility for rapid long-term gene silencing in viable cells and animals. The 5' sense conjugation with cholesterol approved this assumption.

Chemical Modification Patterns Compatible with High Potency Dicer-Substrate Small Interfering RNAs

“…No intact 21-mer siRNA is seen after 6 hours incubation in fetal calf serum. Consistent with earlier reports, the unmodified 27-mer duplexes were somewhat more nuclease resistant than 21-mer duplexes at the same site (Kubo et al, 2007), and small amounts of the unmodified 27-mer DsiRNA were still present at 6 hours. In contrast, an appreciable amount of the 2′OMe ASm modified 27-mer DsiRNA remained intact after 24 hours incubation in serum.…”

Noncanonical Synthetic RNAi Inducers

“…It was found that bonds with 3′ pyrimidine nucleotides are cleaved faster than bonds with 3′ purines. Kubo and his colleagues demonstrated that degradation rate of dsiRNAs correlated with the amount of pyrimidines at the 3′ end [31]. At the same time, degradation rate of dsiRNAs also correlates with the presence of AU-rich domains that might be related to low thermal stability, easy dissociation, and faster cleavage by both endo-and exonucleases.…”

“…On the other hand, dsiRNAs with chemical modifications of the 5′ end possess high nuclease stability and RNAi activity in the cell-cultured medium. Thus, 5′-end amino-modified dsiRNA demonstrated improved RNAi activity and stability in the cell-cultured medium [31].…”

“…The stability of dsiRNA in physiological fluids is extremely an important factor for its applications in vivo [31]. Although dsRNAs are more stable in comparison with singlestranded RNAs and 21-bp siRNA, they still rapidly degrade in the serum [32].…”

Structure - Functions Relations in Small Interfering RNAs

“…The other approach utilize the inactivation of the sense strand of siRNA (for example, segmentation) to guarantee the incorporation of the antisense strand into activated RISC* independent from the thermodynamic properties of the duplex [99]. The silencing activity of siRNA can be increased by the lengthening of the duplex and converting siRNA into Dicer substrate DsiRNA [160,161] or by the modifications of the overhands affecting duplex thermodynamic asymmetry [162]. Single stranded analogs of siRNA can be also used as inducers of RNAi, in this case the problem of the strand selection does not exist [40,77].…”