Book of Abstracts: Albany 2007
June 19-23 2007
Aptabodies: RNA Aptamer-derived Constructs that Mimic Antibodies in Immunochemical Assays
Aptamers are generated through applied molecular evolution experiments (aka SELEX, or systematic evolution of ligands by exponential enrichment) to have appropriate binding affinities and specificities towards certain target molecules. They are usually short strands of RNA or DNA that can adopt highly specific three-dimensional conformations. Compared to small organic compounds, a unique feature of RNA aptamers is that they can be stitched together with other RNA structural or functional elements to form molecules with multiple functional sites, which resembles a protein. This allows RNA-aptamer-based molecular constructs to function not only as inhibitors by blocking binding sites on proteins, but also as novel connectors.
Inside cells, mechanisms bringing two protein molecules together play an important role in cellular regulatory networks. The same principle can be employed for therapeutic or experimental purposes. As a preliminary study to explore the utility of aptamers along this line, here I demonstrate in vitro that two unrelated proteins from two different organisms, the splicing factor B52 of Drosophila melanogaster and streptavidin from Streptomyces avidinii, can be bridged by a constructed RNA molecule derived from aptamers against these proteins. These constructs with multivalency and multi-specificity recapitulate features of antibodies in RNA molecules, and thus were termed "aptabodies." They functioned in three standard immuno-chemical assay formats defined by antibodies. First, immunofluorescence staining of polytene chromosomes showed that the B52 aptabody stains polytenes in a distinct pattern characteristic of antibody against B52 protein. Second, a Western blot showed that the aptabody can detect B52 protein with similar sensitivity to that seen with a monoclonal antibody. Third, dot blots showed that the aptabody easily detects 1 ng of protein.
In addition, I have developed a general method for rational modular design of composite aptamer molecules with multiple valencies and specificities. These composite molecules are termed ?apta-dendrimers? because they comprise primarily aptamers presented on a dendritic scaffold. By splicing together more than one type of RNA aptamers, this method promises to create combinations of binding sites that do not correspond to that of any existing protein, and can be used to modify the connectivity of protein networks both in vitro and in vivo. Non-biological utility of these molecules includes making nano-grids for supramolecular engineering.
Department of Biological Sciences