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Tools & Techniques: Allele-specific siRNAs, RNA Delivery to Tumors, and More


NEW YORK (GenomeWeb) – Despite the specificity offered by RNAi, designing siRNAs capable of silencing specific alleles remains a challenge. As part of an effort to overcome this issue, researchers from Japan's National Institute of Neuroscience have developed two formulas for determining allele-specific siRNAs.

Allele-specific RNAi, or ASP-RNAi, represents a new application of the gene-silencing technology that holds great potential in conditions such as Huntington's disease, where knocking down only mutant alleles can provide a therapeutic benefit without the potential risks associated with inhibiting a wild-type gene.

"To induce ASP-RNAi, the design and selection of siRNAs that confer ASP-RNAi is vital, but quite difficult," the study's authors wrote in Molecular Biology Reports. Typically, in vitro assessment of siRNAs is performed using reporter genes carrying target and non-target allelic sequences, with the effects of the siRNAs against each separately examined. The, careful normalization must be carried out to precisely compare the independent data sets.

There are, however, few appropriate measures in determining optimal allele-specific siRNAs, the researchers noted.

To address this, the scientists created two formulas for calculating a new measure of an siRNA's allele discrimination, or ASP-score, which they say allows for "an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs."

With delivery still a major challenge for RNAi therapeutics, a multi-institute team leas by scientists from the National Institutes of Health has reported a new protocol for producing tumor cell-targeted nanoparticles for siRNA or microRNA delivery in culture and in vivo.

The delivery system is based on the use of a Zn(II)-dipicolylamine (Zn­DPA) analog, an artificial receptor for phosphate anion derivatives," the investigators wrote in Nature Protocols.

A number of groups have reported fluorescent chemosensors based on Zn­DPA analogs owing to the selective Zn­DPA­ coordinated interaction associated with phosphate groups, according to the paper. Meanwhile, similar analogs have been used as probes for necrotic and apoptotic cells by targeting anionic phosphatidylserine on cell surfaces.

"Recently, we applied Zn­DPA analogs as carriers for the delivery of therapeutic molecules, such as siRNA, using the selective and strong binding of Zn­DPA with the phosphodiester groups on the siRNA backbone," they added. "In particular, this Zn­DPA platform for RNA delivery is highly flexible and can be used in conjunction with existing delivery systems" including nanoparticles, polymers, proteins and antibodies by labeling the small-molecule Zn­DPA by established bioconjugation chemistry.

In their paper, the researchers presented the protocol for producing the RNA carriers. They also described the functionalization of the Zn-DPA analog to hyaluronic acid-based self-assembled nanoparticles by conjugating amine-functionalized Zn-DPA molecules onto the nanoparticles through amide formation. This results in efficient tumor-targeted delivery of both RNAs and small-molecule drugs.

Notably, the functional group of Zn-DPA can be converted into other groups such as a carboxylic or thiol group, and the DPA analog can be covalently attached to a variety of platforms or formulations for the development of multifunctional materials via standard bioconjugation techniques.

Functionally redundant gene family members are a common occurrence in plant genomes and can hamper efforts to study individual gene function. To overcome this issue, a Spanish research group used a collection of artificial miRNAs (amiRNAs) to construct Arabidopsis thaliana lines wherein up to six paralogous genes encoding members of transcription factor families can be silenced at once.

They constructed amiRNA-producing transgenes in the backbone of the gene encoding the endogenous Arabidopsis miRNA miR-319a, and generated each by replacing the sequence of the mature miR-319a with that of a given amiRNA.

In a paper appearing in The Plant Journal, the researchers described testing their approach by targeting 576 genes, only 122 of which had been functionally studied before. To confirm the inhibitory effects of the transgenes, they examined the transgenic lines for morphological phenotypes at the rosette stage.

Of 338 tested transgenes, 21 caused a visible morphological phenotype in leaves, a proportion much higher than that expected from insertional mutagenesis, they wrote in their report.

"This robust, versatile method enables functional examination of redundant transcription factor paralogs and is particularly useful for genes that occur in tandem."