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

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  • Additional Information
    • Source:
      Publisher: Springer International Country of Publication: Germany NLM ID: 8406612 Publication Model: Electronic Cited Medium: Internet ISSN: 1432-0614 (Electronic) Linking ISSN: 01757598 NLM ISO Abbreviation: Appl Microbiol Biotechnol Subsets: MEDLINE
    • Publication Information:
      Original Publication: Berlin ; New York : Springer International, c1984-
    • Subject Terms:
      Phosphorothioate Oligonucleotides*/chemistry ; Phosphorothioate Oligonucleotides*/metabolism ; Phosphorothioate Oligonucleotides*/isolation & purification ; Sulfur*/chemistry ; Sulfur*/metabolism; Isomerism ; Stereoisomerism ; DNA, Single-Stranded/chemistry ; DNA, Single-Stranded/metabolism ; Chromatography, High Pressure Liquid
    • 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 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.
      (© 2024. The Author(s).)
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    • Grant Information:
      2020YFA0907200 National Key Research and Development Program of China; 32170047 National Natural Science Foundation of China; 31871250 National Natural Science Foundation of China; 31670034 National Natural Science Foundation of China; 21TQ1400204 Shanghai Pilot Program for Basic Research - Shanghai Jiao Tong University; 22ZR1430100 Natural Science Foundation of Shanghai
    • Contributed Indexing:
      Keywords: Affinity-based oligo purification; Microbial DNA binding protein; Phosphorothioate oligonucleotide; Protein-DNA interaction; Sulfur-binding domain; Thermolabile protein
    • Accession Number:
      0 (Phosphorothioate Oligonucleotides)
      70FD1KFU70 (Sulfur)
      0 (DNA, Single-Stranded)
    • Publication Date:
      Date Created: 20240827 Date Completed: 20240827 Latest Revision: 20240830
    • Publication Date:
      20240830
    • Accession Number:
      PMC11349849
    • Accession Number:
      10.1007/s00253-024-13283-3
    • Accession Number:
      39190037