Antagomirs Join Chemists' Toolkit

Researchers have invented a new class of RNA oligonucleotides that can silence the short noncoding RNA molecules known as microRNAs in much the same way that short interfering RNAs silence messenger RNA in thephenomenon known as RNA interference. Called antagomirs, these new compounds may help researchers figure out the function of microRNAs and may themselves be potential therapeutics.

MicroRNAs are believed to play a role in regulating gene expression. In recent years, more than 250 microRNAs have been found in the mouse, human, and other genomes. Silencing microRNAs can increase or decrease the expression of other genes, depending on the relationship between the microRNA and the particular gene. Although their function is not fully understood, microRNAs have been shown to be involved in diseases such as hepatitis C, cancer, and diabetes.

Short interfering RNAs (siRNAs), on the other hand, silence gene expression by targeting specific messenger RNA for cleavage, a process called RNA interference. In RNAi, double-stranded RNA composed of 19 to 21 nucleotides targets and guides specific mRNA to a protein assembly known as the RNA-induced silencing complex. The complex cleaves the mRNA and prevents its translation into protein.

Now, researchers at Rockefeller University and Alnylam Pharmaceuticals, Cambridge, Mass., have shown that antagomirs can do to microRNAs what siRNAs do to mRNA. Using antagomirs, which are short single strands of modified RNA conjugated to cholesterol, the researchers have silenced microRNA in mice (Nature, published online Oct. 30, dx.doi.org/ 10.1038/nature04303). This is the first demonstration of microRNA silencing in live animals, although similar effects have previously been seen in cell culture.

Team leader Markus Stoffel of Rockefeller is interested in studying the function of microRNAs. The genetic approach—breeding mice that lack the microRNA genes—is a laborious, expensive, and long process, he says. It's not going to work for many microRNAs because they are often clustered and encoded from different genes. Many microRNAs are stitched together from the introns, or noncoding regions, that are cut out of other genes, with parts of a single microRNA coming from multiple genes, he explains.

His search for a nongenetic way to reduce the expression of microRNAs led to antagomirs, which were developed at Alnylam. The results were even better than the researchers had expected. We thought antagomirs would bind to the microRNA and prevent it from binding to the messenger RNA, Stoffel says. The antagomirs go a step further, however, and actually degrade the microRNA.

We expected [antagomirs] to inhibit the microRNA, not get rid of it, Stoffel says. The scientists don't currently understand how the degradation of the microRNA is occurring.

The chemical modifications to the RNA structure are crucial for the antagomirs to work effectively, says coauthor Muthiah Manoharan, vice president for drug discovery at Alnylam. The company previously used similar cholesterol-conjugated modified siRNAs for RNAi (C&EN, Nov. 15, 2004, page 9).

On each nucleotide, the 2'-hydroxyl on the ribose is replaced with a methoxy group. The backbone is modified such that some of the normal phosphodiester linkages at each end of the strand are replaced with phosphorothioate linkages. These modifications improve the biostability of the antagomirs.

The cholesterol conjugation is important for biodistribution and cell permeation. Normal RNA has no effect on microRNA expression. Chemically modified but unconjugated RNA only partially reduces microRNA expression, whereas the conjugated modified RNA drastically reduces even the most highly expressed microRNAs.

The effects of a single course of treatment consisting of three injections at one-day intervals lasted as long as 23 days, the longest time the researchers tested. By targeting a microRNA expressed in many different tissues, the researchers found that systemically injected antagomirs silenced microRNAs in all tissues except the brain.

Stoffel intends to use antagomirs to study microRNA function. Once we know what microRNAs do and if they play a role in disease processes, we hope to use these compounds to treat diseases, he says.