Journal Article

Harnessing sulfur-binding domains to separate Sp and Rp isomers of phosphorothioate oligonucleotides

He, Xinyi, Liu, Guang, Liu, Jinling, Wang, Yuli, Yu, Hao, Deng, Zixin, Ge, Fulin

2024

Overview

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of R p and S p diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many R p and S p diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in R p configuration, we developed a universal s eparation system for p hosphorothioate o ligonucleotide i somers using immobilized S BD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, R p isomers of the mixture can be completely bound by SBD and separated from S p isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60–90% of the starting R p isomers, with PS linkage not locating at 5’ or 3’ end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for R p and S p stereoisomers, respectively. Furthermore, as each R p phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform S p configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBD Ped , enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method. Key points • SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC. • SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers. • SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme. Graphical Abstract

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: Analysis / Binding / Biomedical and Life Sciences / Biotechnology / CD16 antigen / Chemical synthesis / Chromatography / Chromatography, High Pressure Liquid / Configurations / Deoxyribonucleic acid / Diastereoisomers / diastereomers / DNA / DNA, Single-Stranded - chemistry / DNA, Single-Stranded - metabolism / High-performance liquid chromatography / Identification and classification / Isomerism / Isomers / Life Sciences / Liquid chromatography / Liquid phases / liquids / magnetism / Methods / Methods and Protocols / Microbial Genetics and Genomics / Microbiology / Mixtures / Nitrilotriacetic acid / Oligonucleotides / Phosphates / Phosphorothioate / Phosphorothioate Oligonucleotides - chemistry / Phosphorothioate Oligonucleotides - isolation & purification / Phosphorothioate Oligonucleotides - metabolism / Proteins / RNA / Separation / Separation (Technology) / Single-stranded DNA / Snakes / Stereoisomerism / Stereoisomers / Substrates / Sulfur / Sulfur - chemistry / Sulfur - metabolism / synthesis