Elimination pathways of systemically delivered siRNA.

The elimination process of systemically administered small interfering RNA (siRNA) was investigated by using siRNA labeled with an infrared fluorescent dye. A novel siRNA elimination pathway was identified. In this pathway, liver-enriched siRNA is secreted into the gallbladder and then emptied into the intestine. Blocking this pathway resulted in the absence of siRNA fluorescence within the intestine, with greatly enhanced siRNA accumulation in liver and gallbladder at the same time. Furthermore, we demonstrated that delivery carriers play an essential role in siRNA distribution and elimination, highlighting their importance in siRNA therapeutics.

Comprehensive analysis of sequence-specific stability of siRNA.

Double-stranded small interfering RNAs (siRNAs) are important modulators of biological processes and hold great promise for therapeutic applications. However, serum processing of synthetic siRNAs is still largely unknown. To address this issue, serum degradation assays of 125 siRNAs were first performed in this study. Four siRNA categories of distinct serum stability were identified, including a group of siRNAs that were stable in their native form for both in vitro and in vivo assays. Fine mapping of the cleavage events occurring in serum treatment demonstrated that most occurred at two vulnerable sites, leading to a speculation that rational modification of these sites might protect most siRNAs from serum degradation. For proof of concept, an exhaustive siRNA modification study was performed. In addition to the consistent stabilization pattern revealed at these sites, our study further showed that a single modification made at the cleavage site stabilized the siRNAs to a large extent, highlighting the importance of these sites in siRNA degradation. In summary, the present study provided a comprehensive picture of serum processing of siRNA as well as a starting point for a rational siRNA modification strategy, both of which are of great importance to in vivo and therapeutic applications of siRNA.

Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs.

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.

Focusing on RISC assembly in mammalian cells.

RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.