We describe an integrated approach for High Throughput Screening of AptamersTM (HTSATM, patent pending) to discover small, structurally defined nucleic acid aptamer sequences that bind with high affinity and selectivity to a target of interest. Such targets are proteins, bio-terror agents, pathogenic organisms and viruses. These aptamers will be engineered into the """"""""probe"""""""" sequence of our patented AlloSwitchTM indicators that re- spond rapidly to the presence of the target, giving rise to a fluorescent or luminescent signal. In phase I we demonstrated that it is feasible to rapidly screen and isolate high-affinity HT-aptamers. We have used HTSA to isolate previously known aptamers with nanomolar affinity for human athrombin (from DNA hairpin loops) and for human coagulation factor IXa (from RNA with concatenated hairpin and internal loops), and an unprece- dented aptamer with apparent specificity for hexose sugars (from DNA hairpins) with affinity in the top third of known aptamers for small molecules. Thus, the feasibility of HTSA has been demonstrated. In phase II, the approach will be expanded to include a wider variety of structural motifs and new targets. The targets for discovery of probes and creation of AlloSwitches will include proteins involved in clotting and thrombosis, and growth factors involved in wound healing, angiogenesis, and proliferative diseases. AlloSwitch based assays will assist in the discovery of small molecules that can be used for therapies to regulate the interactions of these proteins in blood. This stands in contrast to therapies based directly on aptamers or antibodies, which are difficult to protect from hydrolysis by enzymes in plasma and to formulate as pharmaceuticals in pills. This project will also accelerate the commercialization of our biosensor technology in other fields.
We present an integrated approach for rapid discovery of small, structurally defined nucleic acid """"""""HT- aptamer"""""""" sequences that bind with high affinity and selectivity to proteins and other targets. The approach will be used to create sensors to discover small molecules as candidates for drugs against selected protein targets involved in wound healing and thrombosis. Beyond this project, discovery of high affinity HT-aptamers will be used in detecting waterborne pathogens, biological warfare agents, and diagnostic applications with costs ex- pected to be reduced by a factor of ten or more compared to antibody-based reagents.
|Kupakuwana, Gillian V; Crill 2nd, James E; McPike, Mark P et al. (2011) Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing. PLoS One 6:e19395|