A pre-synthetic conjugation methodology of DNA and RNA: combining on-column conjugation and tandem oligonucleotide...

Amino modifiers are valuable chemical tools for nucleic acid bioconjugation applications and for probing potential mechanisms relevant to the origins of life. Herein, we describe a streamlined and cost-effective strategy for the incorporating amine functionalities at the 3'-end of DNA/RNA constructs through a commercially available diethyl 2,2-bis(hydroxymethyl)malonate-derived controlled pore glass (a so-called version of chemical phosphorylation reagent II CPG, or CPR II CPG). This platform mitigates certain limitations associated with previously explored sulfonyl-based supports, thereby improving the robustness of amine handle conjugation. CPR II CPG is first detritylated, converted to a mixed N-hydroxysuccinimide carbonate followed by efficient derivatization with the amine of interest. Oligonucleotides are then assembled using standard solid-phase synthesis and isolated by standard purification workflows. Using 1,3-diamino-2-propanol as a multivalent conjugation handle allows for the installation of ligands prior to strand elongation, which may be further derivatized post-synthetically. Finally, the integration this advancement, and others, with tandem oligonucleotide synthesis (TOS) enables a versatile conjugation TOS methodology, thereby advancing nucleic acid engineering and broader biotechnology applications.