An Alternate Process for the Solid-Phase Synthesis and Solid-Phase Purification of Synthetic Nucleic Acid Sequences.

Abstract

The chemical synthesis of a riboside phosphoramidite has been achieved to provide a 5-O-capture linker and a 2-O-silyl ether protecting group with the intent of enabling an efficient solid-phase purification of synthetic DNA sequences. The riboside phosphoramidite has been incorporated into a DNA sequence while performing the penultimate automated solid-phase synthesis cycle of the sequence. The terminal 5-O-riboside moiety of the resulting DNA sequence is then conjugated to a capture linker to create an anchor for the solid-phase purification of the DNA sequence conjugate. Release of all DNA sequences from the synthesis support is achieved under standard basic conditions to yield a mixture of the desired DNA sequence conjugate along with unconjugated, shorter-than-full-length sequence contaminants. Upon exposure of all DNA sequences to a capture solid support, only the DNA sequence conjugate is chemoselectively captured, thereby allowing the unconjugated shorter-than-full-length DNA sequences to be efficiently washed away from the capture support. After 2-O-cleavage of the silyl ether protecting group from the terminal riboside ethylphosphate triester conjugate, the solid-phase-purified DNA sequence is efficiently released from the capture support through an innovative intramolecular cyclodeesterification of the ethylphosphate triester, prompted by the riboside's rigid cis-diol conformer, to provide a highly pure DNA sequence. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-1,4-anhydro-D-ribitol (3) Basic Protocol 2: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-3-O-[(N,N-diisopropylamino)ethyloxyphosphinyl]-1,4-anhydro-D-ribitol (6). Basic Protocol 3: Automated synthesis of the chimeric solid-phase-linked DNA sequence 8. Support Protocol: Preparation of 2-cyanoethyl-(5-oxohexyl)-N,N-diisopropylphosphoramidite (9). Basic Protocol 4: Solid-phase purification of the chimeric DNA sequence 10.