Multiplexed Assay for Small-Molecule Quantification via Photo-cross-linking of Structure Switching Aptamers

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