A Versatile Disulfide‐Containing Solid‐Support Strategy for 3′‐Modifiers in Oligonucleotides: Introducing Modular Tandem Oligonucleotide Synthesis

We report a versatile disulfide-containing linkage to solid support that enables on-column synthesis of DNA and RNA containing a 3′-amino or 3′-phosphate group. This orthogonal solid-support linker enables a protecting group strategy for the synthesis of oligonucleotides containing 3′-amino-2′,3′-dideoxyribosides directly from commercially available unprotected mononucleosides. We present an on-column deprotection application to improve oligonucleotide recovery by eliminating the precipitation step typically involved in the RNA workflow. Building on prior work, we introduce a novel modular tandem oligonucleotide synthesis (mTOS) method for selective strand release of downstream strands for the one tethered to the solid support directly.

Abstract

Chemical modifications of oligonucleotides are routinely employed to enhance their functional properties. Amino-modifiers serve as versatile chemical handles for postsynthetic (bio)conjugation, nucleic acid immobilization on solid supports, and investigations into nonenzymatic genome replication relevant to the origins of life, to name a few. Here, we report a cost-effective, disulfide-containing solid-support linkage that enables the on-column synthesis of nucleic acids with 3′-amino or 3′-phosphate modifications. The orthogonality of this solid-support linker facilitates an on-column protecting group strategy, enabling the synthesis of DNA and RNA containing 3′-amino-2′,3′-dideoxyribosides from commercial unprotected mononucleosides. Additionally, we present an on-column deprotection protocol for DNA and RNA, prior to cleavage from the solid support, eliminating the precipitation step typically required in conventional RNA workflows, leading to higher recovery for certain strands. Expanding on our previous work, we introduce a versatile modular tandem oligonucleotide synthesis (mTOS) approach, allowing selective release of downstream strands from the one directly bound to the solid-support via the disulfide-containing linker. Together, these advances in solid-support design and oligonucleotide synthesis unlock new opportunities in bioconjugation, biotechnology, and the study of prebiotic replication mechanisms, broadening the utility of chemically modified nucleic acids across research disciplines.

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