Toward Multi(radio)metalated DNA: Enzymatic Polymerization of Metal‐Chelate‐Modified Deoxyribonucleoside Triphosphates

ABSTRACT We report the first enzymatic synthesis of precisely defined metal‐oligonucleotide conjugates through template‐directed polymerase‐mediated primer extension with two different (radio)metal‐chelator‐modified deoxyribonucleoside triphosphates. We successfully incorporated five different metals (Ga, In, Tb, Lu, and Y) along designed oligonucleotide templates, including the simultaneous incorporation of two different metal chelates within a given strand. To provide bio‐orthogonality, this approach exploits the well‐known enzymatic fidelity of DNA polymerase to generate uniformly metal‐loaded DNA constructs with well‐defined and programmable compositions. The platform is validated using chelators DOTA and DTPA and confirmed by both inductively coupled plasma mass spectrometry (ICP‐MS) quantification and autoradiographic analysis following gel electrophoresis to demonstrate radioisotope incorporation with clinically relevant metals ( 68 Ga for PET, 111 In for SPECT, 161 Tb and 177 Lu for β − therapy), establishing the potential for using the information content of DNA to create multi‐metalated radiopharmaceuticals. This programmable platform establishes a new paradigm for metalated molecular probe development with versatility for potential therapeutic, theranostic, and mass spectrometry‐based applications, and potentially generalizable to other metal isotopes based on diagnostic or therapeutic requirements.