In Vitro Evolution of Functional Biomolecules Using CE

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DESCRIPTION (provided by applicant): SELEX is an evolutionary approach to combinatorial chemistry that uses in vitro selection to identify RNA or DNA sequences with affinity for a particular target. These functional sequences, also referred to as aptamers, have found use as drugs that act on specific biological receptors or as diagnostic agents that can be used in biomedical analyses or imaging.This proposal outlines a unique method (CE-SELEX) for selecting functional DNA molecules in free solution for the first time. Selection will be made on the basis of an electrophoretic mobility shift induced by interactions between active DNA sequences and the target performing the selection in free solution will eliminate many of the evolutionary biases introduced by the chromatographic selection in conventional SELEX. The most obvious bias introduced by chromatographic separation is that selection is not performed against the actual target. Instead a sequence is selected to have affinity for the target attached to a stationary support. Another concern is kinetic bias where it is almost impossible to elute very strongly interacting sequences from a chromatography column. Electrophoretic selection in free solution will eliminate these biases, providing aptamers with improved binding efficiency and selectivity. The improved flexibility of CE-SELEX will also allow the fundamentals of the selection process to be studied intensively for the first time, further improving the quality of the selected aptamers.Initially a control experiment using a 20-base ssDNA as a target will be performed to ensure that CE-SELEX does select for the optimum binding sequence. The affinities of aptamers selected using CE-SELEX will then be compared to those obtained using conventional SELEX using both a large (lgE) and small (ATP) target The selectivity of aptamers selected using CE-SELEX will be tested by searching for sequences that specifically bind N-methylmesoporphyrin over mesoporphyrin, D-serine over L-serine and g-ABA over a-ABA and b-ABA. Lastly, selection conditions expected to affect the binding efficiency and selectivity of the resulting aptamers, including target concentration, size of the initial DNA pool and negative selections, will be optimized. It is anticipated that by removing biases introduced by the stationary phase in conventional SELEX, CE-SELEX will provide aptamers with improved binding efficiency and selectivity.Improving the quality of aptamers selected using SELEX will have direct benefits to health research. Increased binding efficiency and selectivity will be beneficial in developing aptamer drugs that act on specific biological receptors. Aptamers with improved binding efficiency and selectivity may show increased pharmacological activity with fewer side effects. Improved aptamers will also find use in many areas as diagnostic markers including medical analyses, in vivo imaging and biosensors.