Nonamplification Multiplexed Assay of Endonucleases and DNA Methyltransferases by Colocalized Particle Counting

Restriction endonucleases (ENases) and DNA methyltransferases (MTases) are important enzymes in biological processes, and detection of ENases/MTases activity is significant for biological and pharmaceutical studies. However, available nonamplification methods with a versatile design, desirable sensitivity, and signal production mode of unbiased quantification toward multiple nucleases are rare. By combining deliberately designed hairpin DNA probes with the colocalized particle counting technique, we present a nonamplification, separation-free method for multiplexed detection of ENases and MTases. In the presence of target ENases, the hairpin DNA is cleaved and the resulting DNA sequence forms a sandwich structure to tie two different-colored fluorescent microbeads together to generate a colocalization signal that can be easily detected using a standard fluorescence microscope. The multiplexed assay is realized via different color combinations. For the assay of methyltransferase, methylation by MTases prevents cleavage of the hairpin by the corresponding ENase, leading to decreased colocalization events. Three ENases can be simultaneously detected with high selectivity, minimal cross-talk, and detection limits of (4.1-6.4) × 10^-4 U/mL, and the corresponding MTase activity can be measured without a change of the probe design. The potential for practical application is evaluated with human serum samples and different ENase and MTase inhibitors with satisfactory results. The proposed method is separation-free, unbiased toward multiple targets, and easy to implement, and the strategy has the potential to be extended to other targets.