Harnessing sulfur-binding domains to separate Sp and Rp isomers of phosphorothioate oligonucleotides.
Appl Microbiol Biotechnol
; 108(1): 448, 2024 Aug 27.
Article
em En
| MEDLINE
| ID: mdl-39190037
ABSTRACT
Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp 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 Rp 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 Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, 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.
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MEDLINE
Assunto principal:
Enxofre
/
Oligonucleotídeos Fosforotioatos
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article