Separation of RNA phosphorothioate oligonucleotides by HPLC

Phosphorothioate oligonucleotides are indispensable tools for probing nucleic acid structure and function and for the design of antisense therapeutics. Many applications involving phosphorothioates require site- and stereospecific substitution of individual pro-R(P) or pro-S(P) nonbridging oxygens. However, the traditional approach to phosphorothioate synthesis produces a mixture of R(P) and S(P) diastereomers that must be separated prior to use. High-performance liquid chromatography (HPLC) has proven to be a versatile method for effecting this separation, with both reversed phase (RP) and strong anion exchange (SAX) protocols yielding favorable results. In this chapter, we present several examples of successful separations of RNA phosphorothioate diastereomers by HPLC. We also report the use of complementary DNA oligonucleotides for the separation of poorly resolved phosphorothioate RNAs.