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Photoaptamer Technology: Development Of Multiplexed Microarray Protein Assays

D. Zichi, T. Koga, C. Greef, R. Ostroff, Helen Petach
Published 2002 · Chemistry

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To study and identify the complex protein expression patterns associated with a disease, efficient methods are necessary to detect and quantify hundreds of proteins simultaneously, many of which are present in exceedingly low concentrations. Photoaptamers are intriguing capture agents for multiplexed proteomics assays because they demonstrate extraordinary specificity and sensitivity toward protein analytes and can be used in the multiplexed array format. Photoaptamers are single-stranded DNA molecules that have the ability to form covalent bonds with their cognate proteins when they are electronically excited. Photoaptamers have been discovered for proteins with a wide range of characteristics, including acidic, basic, large, small, glycosylated, chemically modified, and hydrophobic, and these photoaptamers may be used in a wide variety of formats, including most formats available to antibodies (1)(2)(3)(4)(5). The photoSELEX process has been successfully automated as a high-throughput process so that a wide range of proteins have yielded active photoaptamers that exhibit nanomolar or better affinities (6)(7)(8)(9). The photoaptamers described below were selected for the proteins thrombin and basic fibroblast growth factor (bFGF) (10)(11)(12)(13). The sensitivities, specificities, and cross-linking efficiencies of the photoaptamers suggest that they will be suitable capture agents for microarrays. For example, the bFGF photoaptamer has a K d of ∼16 pmol/L, a photo cross-linking yield of 50%, and specific binding to bFGF that is more than three orders of magnitude higher than binding to other heparin-binding proteins, such as vascular endothelial growth factor and platelet-derived growth factor (13). The bFGF photoaptamer characteristics and sequence have been described previously (13). We identified photoaptamers in vitro by use of the photoSELEX process (12)(14) to select high-affinity sequences from a random pool of oligonucleotides. Photoaptamers were synthesized at SomaLogic, …
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