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Researchers Publish Data on Role of Target Accessibility in RNAi

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While RNA interference is widely considered one of the most important tools available to researchers studying gene function, the technology does have its limitations.

But recent data published by University of Massachusetts Medical School researcher Tariq Rana in Nature Structural & Molecular Biology has some suggestions on how to possibly overcome one of those limitations.

In the paper, published online this week, Rana and his colleagues note that while researchers' overall understanding of RISC catalysis and function has increased in recent years, and while "many steps of the RNAi pathway have been elucidated, the effect of target accessibility on [activated] RISC catalysis remains unclear."

"We had hypothesized that the accessibility of the target played a role in the efficiency of RNAi, whereas before the general assumption in the field was that it didn't matter," Rana said statement.

According to the paper, the UMMS researchers used HIV-1 transactivation responsive element, which has a stable stem-loop structure, as a target for RNAi in order to assess the role of target site accessibility on RISC catalysis. "We hypothesized that the well-defined secondary structure of TAR RNA would not be accessible for cleavage by RISC programmed with a TAR siRNA, limiting the efficacy of target cleavage," they wrote in the paper.

"We designed an siRNA that was complementary to the stem region of TAR and predicted that the double-stranded stem region of TAR would hamper [siRNA-programmed RISC (siRISC)] binding to the target site," they noted. "As expected, cytoplasmic extracts prepared from HeLa cells programmed with TAR siRNA exhibited minimal target cleavage activity when monitored in vitro using a capped TAR target RNA substrate."

Rana and his colleagues then synthesized 20-nucleotide-long 2'-O-methyl oligonucleotides complementary to the 5' and 3' regions of the TAR siRNA-binding site in order to disrupt TAR's secondary structure and enhance target site accessibility.

"TAR siRISC cleavage efficiency was measured in vitro in the presence of one or both 2'-O-methyl oligonucleotide clamps pre-annealed to the target RNA," the scientists stated in the paper. "Notably, the cleavage of clamped TAR target RNA was substantially increased to [roughly] 65 percent in comparison to [about 2 percent with] free TAR target RNA."

The UMMS team wrote in the paper that the increase in TAR RNA cleavage suggested that the kinetic properties of TAR siRISC would increase when directed against a clamped TAR RNA. However, measuring the increase in RISC catalysis proved difficult when clamp oligos were used due to the low efficiency of free TAR RNA cleavage.

Therefore, "we tested whether the cleavage efficiency and kinetics of siRISC catalysis of other target mRNAs encoding green fluorescent protein or transcription elongation factor Cdk9 could be influenced by the presence of clamps bound [about] 6-10 nt 5' and/or 3' of their respective siRNA-binding sites." The result was that in the presence of one or both clamps, the in vitro efficiency of clamped GFP target RNA was substantially increased in comparison to free GFP target RNA.

Additionally, a comparison of the kinetics of Cdk9 siRISC and GFP siRISC targeting of free and clamped target RNA indicated that increased target access boosted the rate of target cleavage. "At constant concentrations of Cdk9 siRISC, the efficiency of target cleavage increased with increasing substrate concentration," the team wrote in the paper.

"The kinetics of siRISC directed against a clamped target RNA were also determined for free target RNA except that clamps were pre-annealed to the target RNA before in vitro cleavage reactions," they added. "At constant concentrations of siRISC, the efficiency of target cleavage increased with increasing substrate concentration."

According to Rana and his colleagues, "target site recognition by RISC during RNAi is complicated by the vast number of sequences RISC encounters. The propensity of RNA to form secondary structures may also hamper physical access to the target site sequence, explaining the low cleavage efficiency of highly structured TAR. We propose that the 5' and/or 3' clamps disrupt the secondary structure of the RNA immediately surrounding the target site and thereby increase RISC access to the target site," the paper states.

The UMMS researchers also note in their paper that their findings argue against a mechanism of target site recognition, like the one used by robosomes to located translation initiation sites, in which siRISC scans the length of an mRNA.

"If siRISC adheres to the directional scanning mechanism, at least one or both of the clamps would have been expected to cause a decrease in cleavage efficiency," they stated. "Hence, the observed increase of target site recognition … favors a diffusion-controlled mechanism in which RISC binds many sites non-specifically until the correct target site is found.

"In summary, our results highlight the importance of target site access during RISC catalysis," the researchers concluded.

— DM

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