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 ³H-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 ³H-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 (²H and ³H) 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.