An Enzymatic Platform for Late‐Stage (Radio)isotope Labelling of Oligonucleotides With Methyltransferases

The development of therapeutic nucleic acids has become a cornerstone of modern drug discovery. To support the clinical translation of these modalities, radiolabelled analogues are vital for absorption, distribution, metabolism and excretion (ADME) studies, particularly for quantitative whole‐body autoradiography (QWBA). Access to such radio probes is limited because established methods for the preparation of 3 H‐labelled oligonucleotides are characterised by high costs, extensive lead times, low specific activity and the generation of substantial radioactive waste. To address these limitations, we developed a biocatalytic platform for late‐stage 3 H‐radiolabelling of oligonucleotides using methyltransferases, reducing precursor costs and radioactive waste by orders of magnitude. We demonstrate that DNA‐cytosine‐5‐methyltransferases can methylate oligonucleotides with high regioselectivity at a preparative scale, yielding stable‐ and radioisotope‐labelled ( 2 H and 3 H) samples with an unprecedented molar activity up to 3 TBq mmol −1 (81 Ci mmol −1 ). In view of the precision and efficiency of methyltransferase‐mediated radiolabelling, we anticipate that similar approaches will facilitate QWBA studies with other drug modalities.