The use of aptamers in large arrays for molecular diagnostics.
BACKGROUND: Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM. METHODS AND RESULTS: The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. CONCLUSIONS: Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.