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Washington University Team Develops Molecules Combining siRNA, microRNA Effects

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Looking to harness the gene-silencing efficiency of siRNAs to enhance the potency of disease-targeting microRNAs, a team of researchers from Washington University has developed new molecules that incorporate features of both classes of small RNAs.

Dubbed artificial interference RNAs, or aiRNAs, these chimeras combine the natural effects of specific miRNAs with the functions of synthetic siRNAs, reducing the potential for off-target effects while enabling silencing of multiple genes, according to a paper appearing in RNA.

Xiaowei Wang, senior author of the study, said that the findings establish a proof of concept for aiRNAs and, while still preliminary, suggest a new area of therapeutic RNAi research where siRNA- and miRNA-based approaches overlap.

At Washington University, Wang and his colleagues are focused on the roles of miRNAs in human disease, particularly cancer. Through the course of this work, they found that, in many cases, the unintended targets of many siRNAs have seed-pairing sites in their 3' UTRs, mirroring the seed-pairing requirement in miRNA-mediated gene silencing, he told Gene Silencing News.

Additionally, bioinformatics studies suggest that multiple targeting determinants outside of the seed match, such as local AU content and base composition at certain sequence positions, are also shared by both miRNAs and siRNAs.

Taken together, these factors show siRNA off-targeting to be highly similar to miRNA gene suppression, he said. As such, he speculated that miRNA-target prediction tools his lab has developed could also be used to predict genes targeted by siRNAs.

As described in RNA, the scientists analyzed the off-target signature of an siRNA targeting GAPDH and found that it matched up to the off-targets computationally predicted by one of their miRNA algorithms.

"To experimentally validate … [the] prediction results, the siRNA was overexpressed in HeLa cells, and microarrays were performed to identify siRNA off-targets globally at the transcriptome level," they wrote in their paper. "As expected, the single intended target, GAPDH, was silenced by the siRNA, with only 4 percent remaining mRNA level."

Of all the siRNA off-targets predicted by the algorithm, the majority were downregulated by at least 10 percent, they added, while more than one-fifth of all predicted off-targets were downregulated by at least 30 percent — a finding consistent with the observation of other groups that siRNA off-targets are generally moderately silenced.

Having established the similarity between the targeting rules of siRNAs and miRNAs, the investigators began exploring whether siRNAs could be designed to mimic the activity of natural miRNAs, while including enhanced functionality.

Given the importance of seed sequence match to miRNA target specificity, the Washington University group hypothesized that an siRNA designed against a specific target but carrying the seed sequence against a particular miRNA could silence the targets of both.

"In this way, we can design a single RNA to combine the functions of both the original siRNA and the newly introduced microRNA," Wang said.

In line with its overarching interest in cancer, Wang's lab focused on miR-200a — a member of a miRNA family known to suppress cancer cell motility by targeting multiple genes involved in epithelial-mesenchymal transition — and the well-established oncogene AKT1, which is involved in cell proliferation.

"Our design goal was to engineer novel aiRNAs that not only share miR-200a functions for suppressing tumor cell motility, but also bear new functions for suppressing tumor cell proliferation" by also targeting AKT1, the researchers wrote. "By simultaneously suppressing both the motility and proliferation of tumor cells, [our gene-silencing molecule] was expected to have enhanced functions for tumor suppression as compared with naturally processed miR-200a."

Although the selection of a potent AKT1 siRNA that shares the same seed sequence with miR-200a would have been the most "straightforward" design for such a molecule, this was not possible as the AKT1 transcript sequence does not contain any miR-200a seed-binding site, Wang and his team wrote.

In light of studies from other groups showing that siRNAs with a few mismatched bases to the target binding sites could still potentially knock down their intended gene targets, they determined that their AKT1-targeting aiR-200a could be designed by introducing a few mismatched bases into an AKT1 siRNA.

"In this way, these aiRNAs may still silence AKT1 via RNAi-mediated transcript degradation despite the presence of base mismatches," they noted.

The researchers selected a series of AKT1-targeting siRNAs based on their predicted knockdown efficiency, then replaced their seed regions with miR-200a's seed sequence. They winnowed this collection down to three aiRNAs — one had two interspersed mismatches, and the others had two and three contiguous mismatches, respectively.

A bioinformatics analysis showed that the "vast majority" of miR-200a's targets were shared by the three aiRNAs, despite the fact the 3' portions of the miRNA and aiRNAs were completely different.

To experimentally validate the aiRNAs, Wang and his colleagues analyzed the suppression of three of the molecules' potential targets: AKT1, as well as ZEB1 and ZEB2, which are both known to be miR-200a targets and have been linked to cancer metastasis.

Real-time PCR showed that all three aiRNAs were able to suppress the mRNA expression of both ZEB1 and ZEB2 by around 50 percent — a level comparable to that observed in miR-200a-mediated target suppression. Meanwhile, one aiRNA was able to silence AKT1 by about 60 percent, although the other two had minimal impact on the gene.

Two AKT1 siRNAs used as controls showed no ability to silence these genes, while miR-200a had no effect on AKT1.

The aiRNA that proved effective against AKT1, ZEB1, and ZEB2 was selected for further evaluation, and RNA-seq analysis showed that it targeted a similar set of genes as miRNA-200a.

In HeLa studies, the aiRNA and miR-200a, but not AKT1 siRNAs, were also able to suppress cell migration by about 60 percent, indicating that the aiRNA inherited the functions of the miRNA. Meanwhile, in cell proliferation assays, overexpression of the aiRNA led to a reduction in cell proliferation on par with AKT1 siRNAs. Overexpression of miR-200a, however, had no impact.

In order to show that their aiRNA approach can be used to mimic other miRNAs and siRNAs, Wang's lab designed ones based on miR-9, which can promote cancer cell motility, and siRNAs against TP53, a well-characterized tumor suppressor.

As expected, the resultant aiRNAs acted as kind of "hyperfunctional oncogene," suppressing cell death in response to chemotherapy and promoting cancer cell motility.

Overall, the findings indicate that modifying the seed region of an siRNA can help convert unintended off-target effects into intended miRNA-like on-target suppression, the scientists concluded in their paper. "Although miRNAs and siRNAs are still two distinct therapeutic strategies at present, we envision that the boundary defining the two would be blurred in future because it is possible to design aiRNAs that combine the advantages of both miRNAs and siRNAs for RNAi-based therapeutics.

Wang cautioned, however, that the work described in RNA is still early stage, and additional work is underway to validate the findings in vivo.

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