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Archive for the ‘RNAi’ Category

RNAi is fast emerging as a wonderful tool for inhibiting gene expression in a sequence specific manner. The applications of this technology are two-fold: to study gene function, and use as a therapeutic agent in treating many diseases. As a therapeutic agent it finds applications in antiviral treatments because RNAi has been shown to successfully inhibit virus replication. In a report published on 8 November, 2007, in the Retrovirology, Naito et al., describe the design of antiviral siRNA targeted against HIV-I. To create effective antiviral siRNAs against HIV is a daunting task as the virus mutates at a very high frequency. The researchers first analyzed the HIV-I group M sequences available in the Los Alamos HIV Sequence Database and then found those regions which are highly conserved. Using these conserved regions as target sites, they designed optimal antiviral siRNAs. 21-mer siRNA sequences were generated for all the possible HIV-I sequences. The conserved sequences identified in HIV-I genome included the TATA sequence, polyadenylation signal (AAUAAA), regions essential for viral replication regulation, the primer activation signal (PAS), primer binding site (PBS), packaging signal (ψ), central polypurine tract (cPPT), central termination sequence (CTS), and 3 polypurine tract (3 PPT). A total of 216 highly conserved (>70%) siRNA targets were identified. 41 siRNAs (23 siRNAs out of 216 mentioned above and 18 more siRNAs targeted against moderately conserved regions) were subjected to target mRNA cleavage assay for functional validation using real-time RT-PCR. HeLa cells were cotransfected with vector expressing reporter mRNA that contains the siRNA target site and the corresponding siRNA. Then the potency of siRNAs was monitored by real-time RT-PCR. siRNAs were evaluated for their antiviral efficacy against HIV subtypes B, B’, C and CRF01_AE.
The study clearly demonstrated that 39 out of the 41 siRNAs gave more than >60% silencing; and 26 of the 41 siRNAs effectively inhibited viral replication of all four strains by >80%. The results of the study are quite remarkable and point towards the efficient use of siRNA for inhibiting viral gene expression and replication. The study also paved the way for using siRNAs against divergent HIV-I strains. However, the extreme genetic diversity and high mutation rate of the virus has hindered the creation of a single siRNA effective against all HIV-I strains found in the world. The use of this technology for as a highly effective treatment might be possible in the future and this study could be applied to other pathogens like SARS, influenza virus, etc.

Read the article here.


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Transgenic plants expressing Bt gene have been widely used for pest control for the past several years. However, due to certain limitations associated with using this technology scientists have been looking for alternatives, and they found one in RNAi. The researchers at the Chinese Academy of Sciences, Shanghai, and at Monsanto and Devgen, a Belgian company have shown for the first time that RNAi could be used as an efficient means of pest control. In two independent studies, when scientists fed the insect larvae with the plant material expressing dsRNA for the insect genes, it was found that it triggered the RNAi pathway in insect larvae and blocked the expression those genes.

In the first study, cotton bollworm (Helicoverpa armigera) was the target. dsRNA for a cotton bollworm cytochrome P450 gene, CYP6AE14, was made to express in the plants. This plant material was then fed to the insect larvae, and it was found that the levels of the cytochrome P450 transcript in the larval midgut decreased and larval growth retarded. This cytochrome P450 gene permits Helicoverpa armigera to tolerate inhibitory concentrations of the cotton metabolite, gossypol, and survive. In the absence of this gene, the insect showed decline in its growth. The abstract is here.

In another study, scientists made corn plants that silenced a gene essential for energy production in corn rootworms. Read the abstract here.

Many research workers are optimistic about this technology. Read the full story here.

via: Technology Review


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piRNAs

Piwi-interacting RNAs (piRNA) are a class small of RNA molecules that are expressed uniquely in mammalian spermatogenic cell lines. These are 26–31 nucleotides long and bigger that miRNAs and siRNAs. They are so named because of their capability of forming RNA-protein complexes with Piwi proteins. Piwi proteins are part of the family of Argonaute proteins, which are defined by the PAZ (Piwi Argonaut and Zwille) domain and the PIWI domain. Argonaute proteins interact with small RNAs through PAZ and PIWI domains. A small RNA guides the Argonaute protein to its target molecule, which leads to gene silencing. MIWI, MIWI2 and MILI, three Piwi subfamily proteins, are essential for spermatogenesis in mice. piRNAs are involved in RNA silencing via the formation of RISC. The biogenesis pathway of piRNAs has not been clearly elucidated yet, but they are generated from junk DNA. A review on piRNA can be found here.

In a recent report scientists at Yale University have shown that piRNAs play pivotal role in regulating gene function. They discovered more than 13,000 Piwi-associated piRNAs in fruit flies. Out of these one was found to interact with Piwi, which finally binds to the chromatin and regulates the activity of the gene. Read the full story here.

via: Physorg

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