Abstract
Template driven chemical ligation of fluorogenic probes represents a powerful method for DNA and RNA detection and imaging. Unfortunately, previous techniques have been hampered by requiring chemistry with sluggish kinetics and background side reactions. We have developed fluorescent DNA probes containing quenched fluorophore-tetrazine and methyl-cyclopropene groups that rapidly react by bioorthogonal cycloaddition in the presence of complementary DNA or RNA templates. Ligation increases fluorescence with negligible background signal in the absence of hybridization template. Reaction kinetics depend heavily on template length and linker structure. Using this technique, we demonstrate rapid discrimination between single template mismatches both in buffer and cell media. Fluorogenic bioorthogonal ligations offer a promising route towards the fast and robust fluorescent detection of specific DNA or RNA sequences.