Getting a Grip on Gene Silencing

Courtesy of Active Motif One challenge in working with a family of homologous genes is finding a reagent that can silence one gene without acting on its brethren. This is a question of specificity, and Active Motif of Carlsbad, Calif., claims its gripNA™ probes are more specific than conventional antisense oligonucleotide reagents and more effective than the peptide nucleic acids (PNAs) from which gripNAs derive. Although PNAs exhibit strong hybridization and specificity properties, the

Apr 7, 2003
Jane Salodof Macneil
Courtesy of Active Motif

One challenge in working with a family of homologous genes is finding a reagent that can silence one gene without acting on its brethren. This is a question of specificity, and Active Motif of Carlsbad, Calif., claims its gripNA™ probes are more specific than conventional antisense oligonucleotide reagents and more effective than the peptide nucleic acids (PNAs) from which gripNAs derive.

Although PNAs exhibit strong hybridization and specificity properties, they tend to aggregate, limiting their use in vivo. The gripNAs posses a negatively charged backbone that increases solubility, yet they retain PNAs benefits, including nuclease resistance and strong sequence specificity. "They don't tolerate mismatches well," says John Archdeacon, research and development manager, citing experiments that compare the binding strengths of gripNA and DNA probes to complementary sequences. The presence of a single-base mismatch in the target sequence either prevents or destabilizes duplex formation by the gripNA probe.

Biochemist Gilles Divita, of the Research Center of Macromolecular Biochemistry (CRBM) in Montpelier, France, is investigating an antisense cancer drug that targets cyclin B1. He used gripNAs to turn off the gene in mammalian cells, to determine whether the cyclin is a good candidate for drug development. "If you downregulate to zero, and you have no effect on the cell cycle, it is the wrong target," he explains.

Gene silencing with gripNAs has been demonstrated in mammalian cells, zebrafish, and in Xenopus. Laboratories can deliver gripNA into mammalian cells with lipid transfection formulations, but Active Motif recommends using its Chariot II reagent, which was developed specifically for gripNA. Chariot II reportedly increases efficiency--Divita estimates it to be as high as 90%--and lowers toxicity.

The company provides gripNAs through a customized synthesis service. Customers identify the sequences they are targeting, and the company provides an 18-mer probe. Amine, biotin, and fluorescein modifications are available, and Archdeacon adds that every probe is verified by mass spec and supplied with a gripNA human CREB-positive control. "It's a comfort factor, so they see it works," he says. The gripNA service costs $395 (US) per probe.

--Jane Salodof MacNeil