UNIT 4.34 DNA Oligonucleotides Containing Stereodefined Phosphorothioate Linkages in Selected Positions
Published Online: 1 MAR 2009
Copyright © 2009 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Nucleic Acid Chemistry
How to Cite
Nawrot, B. and Rebowska, B. 2009. DNA Oligonucleotides Containing Stereodefined Phosphorothioate Linkages in Selected Positions. Current Protocols in Nucleic Acid Chemistry. 36:4.34:4.34.1–4.34.15.
- Published Online: 1 MAR 2009
- Published Print: MAR 2009
This unit describes a method for the synthesis of DNA chimeric PO/PS-oligonucleotides with a stereodefined phosphorothioate bond in the selected position. Diastereomerically pure 5′-O-DMTr-N-protected-deoxyribonucleoside-3′-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s obtained according to the previously described protocol (unit 4.17) are transformed via a stereospecific 1,3,2-oxathiaphospholane-ring opening condensation into the corresponding dinucleoside phosphorothioates. Such dimers cannot be introduced into an oligonucleotide chain via the phosphoramidite approach since the unprotected P-S− bond is easily oxidized during routine I2/Py/water oxidation of the phosphite function. In the methodology described here, the reversible alkylation of the PS function is applied. Subsequently, the 3′-phosphoramidites of such PS-protected dimers prepared in situ are used for routine synthesis of chimeric PO/PS-oligonucleotides according to the phosphoramidite method. The presence of the alkylated PS-function requires modified conditions for oligonucleotide deprotection and cleavage from the solid support. Detailed procedures for the synthesis of PS-dimers and their incorporation into an oligonucleotide chain, as well as deprotection/purification steps are presented. Curr. Protoc. Nucleic Acid Chem. 36:4.34.1-4.34.15. © 2009 by John Wiley & Sons, Inc.
- phosphorothioate oligonucleotide;
- stereodefined phosphorothioate;