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Synthesis of the 3′-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3′-S-phosphorothiolate linkage

Abstract

Oligodeoxynucleotides containing 3′-S-phosphorothiolate (3′-PS) linkages have become useful tools for probing enzyme-catalyzed cleavage processes in DNA. This protocol describes the synthesis of the phosphorothioamidite monomers derived from thymidine and 2′-deoxycytidine, and their application to a fully automated procedure for synthesising oligodeoxynucleotides containing 3′-PS linkages. The synthesis of the 5′-protected-3′-amidites is achievable in 2 weeks with the DNA synthesis and purification taking another 1 week.

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Figure 1
Figure 2: Synthesis of 3′-thionucleoside phosphorothioamidites.
Figure 3: A typical HPLC profile of the crude 4,4′-dimethyoxytrityl (5′-DMT)-protected ODN using HPLC 0–100 protocol described in Step 68.
Figure 4: A typical HPLC profile of a pure fully deprotected oligodeoxynucleotide using HPLC 0–40 protocol described in Step 75.
Figure 5: A typical mass spectrum profile of a pure fully deprotected oligodeoxynucleotide.

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Acknowledgements

The authors thank Alan Mills for obtaining mass spectra.

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Correspondence to James W Gaynor or Richard Cosstick.

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Gaynor, J., Bentley, J. & Cosstick, R. Synthesis of the 3′-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3′-S-phosphorothiolate linkage. Nat Protoc 2, 3122–3135 (2007). https://doi.org/10.1038/nprot.2007.451

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