Multiplexed Assay\nfor Small-Molecule Quantification via Photo-cross-linking of Structure\nSwitching Aptamers

There is an unmet need for molecular detection assays\nthat enable the multiplexed quantification of small-molecule analytes.\nWe present xPlex, an assay that combines aptamer switches with ultraviolet-cross-linkable\ncomplementary strands to record target-binding events. When the aptamer’s\nsmall-molecule target is present, the cross-linkable strand is displaced,\nenabling PCR amplification and detection of the relevant aptamer.\nIn the absence of that target, the aptamer is readily cross-linked\nto the strand, preventing amplification from happening. The resulting\naptamer-specific amplicons can be detected and quantified in a multiplexed\nfashion using high-throughput sequencing. We demonstrate quantitative\nperformance for a pair of small-molecule analytes, dopamine and glucose,\nand show that this assay retains good specificity with mixtures of\nthe two molecules at various concentrations. We further show that\nxPlex can effectively evaluate the specificity of cross-reactive aptamers\nto a range of different small-molecule analytes. We believe that the\nxPlex assay format could offer a useful strategy for achieving multiplexed\nanalysis of small-molecule targets in a variety of scenarios.