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

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.