. There is a need for the high throughput generation of aptamers for diagnostic and basic research. Current technology is based on SELEX, which is very difficult to automate and is not suitable for high-throughput aptamer generation. Recently, Bowser at the University of Minnesota and Krylov at York University developed a high-speed method for aptamer generation based on capillary electrophoresis. Their technology, which Bowser calls capillary electrophoresis-SELEX (CE-SELEX), is based on the observation that the mobility of single stranded DNA in free solution is independent of the oligonucleotide's sequence. In CE-SELEX, a target molecule is incubated with an oligonucleotide library. The uncomplexed DNA library migrates as a tight band during capillary electrophoresis. In contrast, DNA-target complexes migrate with a different mobility. By discarding the uncomplexed DNA and amplifying the DNA that is complexed with the target, high affinity aptamers can be generated in one to four selection cycles. We propose to develop an instrument that will increase the speed of CE- SELEX by two orders of magnitude. Our system is based on two-dimensional capillary electrophoresis, where a protein sample is separated in the first capillary. Up to 100 components can be separated in the first capillary. Fractions are automatically transferred to an incubator, where they are mixed with and can complex with an oligonucleotide library. Each fraction is then transferred to a second capillary, where the contents undergo CE-SELEX. Uncomplexed DNA is discarded and the DNA in the protein-complexes is amplified. The amplified DNA from each cycle of the two-dimensional system is pooled and used as the DNA library for subsequent SELEX steps. After SELEX is complete, the product DNA is cloned and sequenced, and the corresponding target protein is digested and identified by mass spectrometry.
Narrative. Aptamers are synthetic pieces of DNA that tightly bind to a specific target. They are useful in the analysis of the target and have found application as therapeutics. Using current technology, their generation is slow. This proposal will speed aptamer generation by two orders of magnitude.
|Huge, Bonnie Jaskowski; Flaherty, Ryan J; Dada, Oluwatosin O et al. (2014) Capillary electrophoresis coupled with automated fraction collection. Talanta 130:288-93|
|Flaherty, Ryan J; Huge, Bonnie J; Bruce, Spencer M et al. (2013) Nicked-sleeve interface for two-dimensional capillary electrophoresis. Analyst 138:3621-5|
|Zhu, Guijie; Sun, Liangliang; Yang, Ping et al. (2012) On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis. Anal Chim Acta 750:207-11|
|Dada, Oluwatosin O; Huge, Bonnie J; Dovichi, Norman J (2012) Simplified sheath flow cuvette design for ultrasensitive laser induced fluorescence detection in capillary electrophoresis. Analyst 137:3099-101|
|Sun, Liangliang; Li, Yihan; Yang, Ping et al. (2012) High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres. J Chromatogr A 1220:68-74|