Research

 The main interest of our group is to elucidate the molecular basis of gene regulation by tiny protein-noncoding RNAs including short interfering RNAs (siRNAs) and microRNAs (miRNAs). Our major activities may be summarized as follows.

 

1) Establishment of guidelines for the selection of highly effective siRNA sequences for RNA interference.

High effectvive & ineffective siRNA sequences

RNAi is the process of double-stranded RNA (dsRNA) dependent, post-transcriptional gene silencing. In mammalian cells, siRNA is a promising reagent for RNAi; however, since only a limited number of siRNAs appear to be capable of producing highly effective RNAi in mammalian cells, the relationship between an siRNA sequence and its ability to induce RNAi was examined in detail. On the basis of our results, it was found that siRNAs simultaneously satisfying the four following sequence conditions at the same time is functional (Figure 1): A/U at the 5’ end of the antisense strand; G/C at the 5’ end of the sense strand; AU-richness in the 5’ terminal, 7 bp-long region of the antisense strand and the absence of any long GC stretch of more than 9 bp in length.

 

Until now,several different algorithms for selecting functional siRNA sequences have been published. To find a robust siRNA selection procedure by integrating these algorithms, we performed validation studies on some published rational siRNA design algorithms. In this study, we used a single reporter assay with the firefly luciferase gene, since it is likely that the efficiencies of siRNA sequences vary between target genes, partially depending on the detection sensitivity of the expression of each gene. The results suggest that it is best to select siRNAs that generally satisfy a combination of some reliable integrated algorithms (Figure 2). Our results should be considered when conducting RNAi experiments.

Comparison of siRNA desing algorithms for functional RNAi

 

2) Functional dissection of siRNA sequences by systematic DNA substitution.

siRNA with a DNA seed arm

To clarify the position-dependent functions of the ribonucleotides in siRNAs, we examined the effects of systematic deoxyribonucleotide substitutions in siRNAs on gene silencing. The 5’ proximal “seed” sequence (positions 1-8 from the 5’ end of the siRNA guide strand) could be completely replaced with cognate deoxyribonucleotides with little or no loss of gene silencing activity (Figure 3). In contrast, the remaining region could not be replaced with DNA, probably because of binding by RNA-binding proteins such as Argonaute and TRBP2.The passenger strand with DNA in the 3’end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA-RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA-RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.

 

3) Thermodynamic stability in the seed duplex is a primary determinant of the efficiency of the siRNA-based off-target effect.

Seed-dependent off-target effect is correlated with Tm of seed/target duplex

Seed-dependent off-target effect

A growing body of evidence from large-scale knockdown experiments suggests that siRNAs generate off-target effects through a mechanism similar to that of target silencing by miRNAs, which influence the expression of many transcripts in a tissue-specific manner. However, little was known about the molecular basis for the efficiency of seed-dependent off-target gene silencing. We found that the seed-dependent off-target effect is highly correlated with the thermodynamic stability in the duplex formed between the seed region of the siRNA guide strand and its target mRNA (Figure 4 & 5). ΔG and Tm for the formation of the seed duplex, respectively, are positively and negatively correlated with seed-dependent gene silencing activity. The seed-dependent off-target gene silencing effect is almost completely eliminated with G:U pairing in the seed duplex. In this case, off-target effect is avoided probably because of the structural perturbation of seed duplex but not seed duplex stability.