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

Abstract

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.

Figure 1

Overview of xPlex. (a) xPlex produces a DNA reporter of small molecule-aptamer binding events, which can subsequently be sequenced and quantified. (b) In the absence of target, structure-switching aptamers (blue) are bound to a competitor strand (red) modified with a ^CNVK moiety (top left). Aptamer binding to its target (tan) displaces the competitor strand (top right). UV exposure covalently cross-links nontarget-bound aptamers to the competitor strand (bottom left). Only target-bound, non-cross-linked aptamers can be amplified with PCR (bottom right). (c) Details of the cross-linking reaction; top sequence is the aptamer, and bottom sequence is the competitor strand. Created with BioRender.

Figure 2

xPlex enables quantification of dopamine and glucose. (a) Cross-linked aptamer concentrations decrease with increasing dopamine concentration, as seen on denaturing urea gels. The full gel is shown in Figure S1. NoX denotes the uncross-linked control. (b) qPCR Cq values (n = 3) decrease with increasing dopamine concentrations. Dopamine-free controls with and without cross-linking are indicated in yellow (Buffer) and orange (NoX), respectively. (c, d) Binding curve fits for dopamine and glucose aptamers in response to (c) dopamine and (d) glucose. (e, f) Multiplexed quantification of (e) dopamine and (f) glucose in mixtures of these analytes at varying concentrations, with respective mean squared log errors (MSLE) based on 4PL fits for each aptamer. (g, h) Plot of true versus calculated concentrations for (g) dopamine and (h) glucose. The dashed diagonal line shows 1:1 correlation. Vertical purple lines indicate the lower limit of quantification (LLOQ). Color scale indicates analyte concentration of the nontarget analyte (glucose in mM, dopamine in μM). Dots are individual replicates (n = 3).

Figure 3

Characterization of kynurenine pathway aptamers with xPlex. HTS-derived binding curves for 3HK-1, XA-1, KA-1, and SK-1 with 3hk, xa, ka, and kyn. Each analyte concentration produced four separate reads for each aptamer, and all samples were processed in the same sequencing run. Target-free and no-cross-linking controls are shown in Figure S9. Non-normalized reads are shown in Figure S10. Curve fits are shown in Figure S11.