Web Date: December 11, 2013
Unlocked Nucleic Acids Help Silence Genes Selectively
Some biologists have long hoped that small pieces of RNA that silence genes could help wipe out genetic diseases. But developing nucleic acid medications with good drug properties, such as selectivity, has proven difficult. Researchers have now added a new, improved weapon to the silencing arsenal: RNAs containing flexible synthetic nucleic acids. These RNAs selectively and potently inhibit the expression of the mutant genes associated with Huntington’s disease and Machado-Joseph disease in cells (Biochemistry 2013, DOI: 10.1021/bi4014209).
Small interfering RNAs (siRNAs) are 20- to 25-nucleotide-long RNA duplexes with one strand that complements a target sequence of messenger RNA. Cellular machinery identifies the duplex RNA and removes the non-complementary strand, which allows the siRNA to bind its target mRNA and inhibit the expression of that gene.
Researchers have looked to these siRNAs as a way to treat Huntington’s disease and other related disorders, because small-molecule drugs are not a viable option, says David R. Corey of the University of Texas Southwestern Medical Center. For example, he says, the protein coded for by the Huntington’s gene is a scaffolding protein that interacts with many partners. A small molecule could inhibit one of those interactions, but would probably leave the others intact. So isolating the defective protein from the rest of the cell would be nearly impossible. “It’s at the undruggable end of undruggable targets,” Corey says.
In a gene silencing approach, patients would receive siRNAs that knock out the mutant gene before cells could produce the defective protein. People with Huntington’s or other trinucleotide disorders typically have one healthy copy of the affected gene and one mutant allele with extra repeats of a three base sequence. Unfortunately, fully complementary sequences of siRNA will bind to wild type and mutant genes, inhibiting both. Corey has been working to develop a selective silencing solution to prevent possible adverse side effects caused by inhibiting the wild type gene.
Unexpectedly, previous research showed that incorporating a mismatch between the siRNA sequence and the repeat target sequence in the mutant gene improves selectivity (Chem. Biol. 2010, DOI: 10.1016/j.chembiol.2010.10.013). However, it also lowers binding affinity. Because an RNA complex with a mismatch has more flexibility than a fully complementary one, Corey thought about other ways to add in flexibility without affecting binding affinity. His solution was to use unlocked nucleic acids, which have the same carbon skeleton as a normal ribose sugar but lack a bond in the sugar ring. With the ring opened, they are extra flexible.
To test the approach, the researchers synthesized several siRNAs targeting CAG repeats, with and without a mismatch. They altered the position and number of unlocked nucleic acids within each sequence. The team delivered the RNA constructs into fibroblast cells derived from a patient with Huntington’s disease and a patient with Machado-Joseph disease, which is another trinucleotide repeat disorder. After incubating the cells for four days, the researchers used Western blots to measure how much mutant and wild type protein the cells produced. The best siRNAs—with one mismatch and one unlocked nucleic acid—were 40-fold stronger inhibitors of the mutant genes than the wild-type ones. An siRNA with the mismatch alone was a 31-fold stronger inhibitor of the mutant allele. Both siRNAs had affinities in the nanomolar range.
This study is “the first convincing evidence that unlocked nucleic acids may be very useful in being able to tune double-stranded RNA constructs,” says Jesper Wengel of the University of Southern Denmark. He says the researchers next should test their siRNAs in animal models of trinucleotide repeat disorders as soon as possible. Indeed, this is Corey’s next step.
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