A Facile Strategy for 3'-Terminal Functionalization of DNA and RNA via On-Column Conjugation with Integration into Tandem Oligonucleotide Synthesis.

Amino-functionalized oligonucleotides are valuable tools in therapeutics, biosensing, and nanotechnology. Although 5'-terminal modifications are readily accessible, incorporation at the 3'-terminus remains more challenging and often requires pre-functionalized solid supports or specialized reagents. To address these limitations, we developed a bis(hydroxymethyl)malonyl CPG (CPR II CPG) solid support that can be activated with N,N'-disuccinimidyl carbonate (DSC) to generate a carbamate-forming platform. Using this support, we established an on-support bis-carbamate construction strategy that enables efficient incorporation of amino modifiers in high yields and crude purities. Furthermore, by leveraging a trifunctional linker (1-amino-3-(((4-methoxyphenyl)diphenylmethyl)amino)propan-2-ol), we developed a modular approach in which one amine is tethered to the solid support while the second amine can be selectively detritylated and conjugated to diverse ligands. The free hydroxyl group can then be used to extend an oligonucleotide through automated solid-phase synthesis. This strategy accommodates a broad range of ligands through multiple conjugation chemistries, including NHS esters/carbonates and EDC-mediated amide bond formation, with good conversions. Finally, we describe a tandem oligonucleotide synthesis (TOS) protocol for generating strands of DNA bearing ligands at defined terminal positions, enabling rapid access to highly modified oligonucleotides. All oligonucleotide conjugates were purified using standard workflows, including strong anion exchange high-performance liquid chromatography (SAX-HPLC), and characterized by mass spectrometry (MS). © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of R-CPG and incorporation of bis-amino modifiers such as putrescine Basic Protocol 2: Synthesis of MMTr-1,3-diamino-2-propanol (compound 2) and R-CPG functionalization Alternate Protocol 1: EDC-mediated ligand-amine coupling (4-bromobenzoic acid) and DNA synthesis Alternate Protocol 2: NHS-carbonate-mediated ligand coupling (Boc-OSu) and RNA synthesis Basic Protocol 3: Conjugational tandem oligonucleotide synthesis (cTOS) of highly modified DNA duplexes.