Protein-Templated\nClick Ligation Reaction Triggered\nby Protein-Split Aptamer Interactions

DNA-templated reactions have found wide applications\nin sensing\nand drug discovery. However, this strategy has been limited to the\nuse of nucleic acids as templating elements to direct the proximity\neffect. Herein, we describe a versatile <u>p</u>rotein-<u>t</u>emplated <u>sp</u>lit <u>a</u>ptamer <u>c</u>lick <u>l</u>igation <u>r</u>eaction (PT-SpA-CLR) in which the protein template-induced\ncovalent proximity ligation of split aptamer elements enables translating\nprotein/aptamer binding events into the output of ligated DNA products.\nA ligation yield of >80% is observed for three model protein templates,\nincluding VEGF<sub>165</sub>, PDGF-BB, and SARS-CoV-2 S1. The ligation\nreaction compensates for the weakness of reduced binding affinity\nresulting from splitting the aptamer, as evidenced by an approximately\n2-fold lower dissociation constant than the non-ligated system. This\nnewly developed PT-SpA-CLR strategy is further integrated with colorimetric\nor fluorescent reporting mechanisms to achieve easy-to-use and low-cost\nbiosensors utilizing ligation to produce a fully active G-quadruplex\nor an RNA-cleaving DNAzyme to report protein binding. Both assays\ncan achieve specific detection of an intended protein target with\na limit of detection at the picomolar level even when challenged in\nbiological samples. The combined PT-SpA-CLR and versatile sensing\nstrategies offer attractive universal platforms for efficient detection\nof protein biomarkers.