Ultramild Protein-Mediated Click Chemistry Creates Efficient Oligonucleotide Probes for Targeting and Detecting Nucleic Acids

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)‐binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine‐based fluorescent probes in vitro and rationalize our results by electronic structure calculations. Functionalized oligonucleotides were prepared in good yields by protein‐mediated CuAAC click reactions for the first time with a human copper‐binding chaperon. The carbohydrate, peptide, and fluorescent derivatives display high binding affinity and selectivity for DNA/RNA targets, as well as pH‐sensitive fluorescence when 5‐JOE dye is incorporated.