Aptamers Identify Protein Signatures

Graphic: Leza BerardoneA technique not deemed "sexy" enough for commercialization a decade ago may finally find its niche in proteomics. SomaLogic Inc. of Boulder, Colo. is pioneering aptamers, which are modified DNA molecules that bind specific proteins in body fluid samples. Aptamers are initially selected for specific binding activities from a huge starting pool, then variants are obtained during subsequent rounds of amplification. The technology builds on the ability of aptamers to bind tenaciously to proteins. "Single-stranded oligonucleotides can fold into any shape, and behave like antibodies," explains Larry Gold, SomaLogic founder. To develop an aptamer screen, the researchers begin with a small amount of a purified protein from a particular cell type under particular conditions. That protein is used to select an aptamer from a pool, and the aptamer is eventually incorporated into an array with others that bind different proteins in that cell type, forming a unique protein signature. "The idea is that the pathology of tumors and other conditions will show changes in proteins in the blood, so that simple blood tests can be done routinely, for early warnings," says Gold, who with Craig Tuerk developed aptamer technology at the University of Colorado, Boulder, in 1989.1 Gold and Tuerk used an in vitro selection and evolution method called SELEX (systematic evolution of ligands by exponential enrichment) to investigate RNAs that bind bacteriophage T4 DNA polymerase. From there, the budding technology wound its way through a trajectory of biotech companies--to NeXagen in 1991, which soon merged with another firm to spawn NeXstar Pharmaceuticals. Despite an impressive patent estate, long-standing prejudices delayed the launch of the technology: specifically, the lag in commercialization of antibody-based technologies, and Big Pharma's thirst for orally active compounds. "Aptamers would always have to be injected and aimed extracellularly, because they do not cross cell membranes. So it was a limited niche, but one through which many diseases could be approached," Gold recalls. Circa 1997, however, his view broadened. "Four years ago, I finally understood the potential for research and diagnostics. I took out the Affymetrix chip and added something to make the reporting more accurate, and wondered if we could provide high-density arrays of aptamers. If we could make, quickly enough, aptamers with specificities, we could make proteomics cheap." Then in 1999, Gilead Sciences of Foster City, Calif., bought NeXstar, and soon after sold an exclusive license for in vitro diagnostics using aptamer technology back to Gold, who started SomaLogic. Aptamers are not just oligos--they are modified to resist nuclease digestion, and to remain in the bloodstream longer, or to be sufficiently stable in serum for diagnostics. They differ by a variable 30 to 40 base sequence that is nestled between sequences complementary to PCR primers, which provides the amplification mechanism for the cycles of evolution and selection. Used as a diagnostic--or prognostic, since it will yield information before symptoms arise--arrays of aptamers are bound to a solid support, and a body fluid sample applied. Unbound protein is washed off, and stains that tag proteins but not nucleic acids used to generate protein profiles. "At every address in the array, there will be a billion molecules of each aptamer. Blood or urine is applied, and we collect some number of proteins at each address," says Gold. Aptamers have therapeutic potential too because they block receptors and other protein activities.2 Gilead Sciences has developed an aptamer called NX1838, for example, that inhibits vascular endothelial growth factor (VEGF). "NX1838 functions as an antagonist in an angiogenic disease model--excess blood vessels. It inhibits VEGF activity, binding it tightly, so that the protein cannot bind its receptor," Gold says. It is currently in clinical trials to treat age-related macular degeneration. Ricki Lewis (rickilewis@nasw.org) is a contributing editor for The Scientist. References 1. C. Tuerk and L. Gold. "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase," Science, 249:505-10, 1990. 2. E. N. Brody et al., "The use of aptamers in large arrays for molecular diagnostics," Molecular Diagnostics, 4:381-8, 1999.

Aptamers Identify Protein Signatures

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