Site-Selective and Re-Writable Labelling of DNA Through Enzymatic, Dynamic and Click Chemistries

Current methods for bioconjugation rely on the introduction of stable linkers that lack the required versatility to perform sequential functionalizations. However, sequential manipulations are an increasing requirement in chemical biology because they can underpin multiple analyses of the same sample to provide a wider understanding of cell behavior. Here, we present a new method to site-selec- tive write, erase and re-write chemical functionality to a biomolecule, DNA in this case. Our method com- bines the robustness of methyltransferase-directed labeling with the versatility of dynamic covalent chemistry. Underpinning the method is a new S-adenosyl-L-methionine derivative to site-selectively la- bel DNA with a bifunctional chemical handle containing an acyl hydrazone-linker and a terminal azide. Functional tags are conjugated via the azide, and can be erased when needed at the acyl hydrazone via dynamic exchange. The formed hydrazide-labeled DNA is a versatile intermediate that can be either re- written to reset the original chemical handle, or covalently reacted with a permanent tag. This ability to write, tag, erase and permanently tag DNA is exploited to sequentially introduce two fluorescent dyes on DNA. Finally, we demonstrate the potential of the method by developing a protocol to sort labeled DNA using magnetic beads, with subsequent amplification of the sorted DNA sample for further analysis. The presented method opens new avenues for site-selective bioconjugation, and should underpin integrative approaches in chemical biology where sequential functionalizations of the same sample are required.