Method for chromatographic separation of synthetic phosphorothioate oligonucleotides
AssigneeApplied Biosystems, LLC
InventorB. John Bergot
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Abstract
A method is provided for the preparation and/or analysis or synthetic phosphorothioate and -dithioate oligonucleotides. In particular, the method permits separation of fully sulfurized phosphorothioate or -dithioate oligonucleotides from incompletely sulfurized defect species on strong-anion exchange HPLC columns using concentration gradients of novel "soft base" anionic eluents, such as bromide, thiocyanate, and the like.
Claims6
A method of separating completely sulfurized phosphorothioate or phosphorodithioate oligonucleotides from a mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides, the method comprising the steps of:
- (a) impregnating a strong anion exchanger with the mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides, the strong anion exchanger consisting of a mechanically rigid styrene-divinylbenzene matrix derivatized with a quaternary alkylammonium cation;
- (b) passing through the strong anion exchanger a buffer solution comprising a a soft-base counter ion at a concentration which monotonically increases with time to a sample-desorbing concentration, the soft-base counter ion being selected from the group consisting of bromide, thiocyanate, azide, iodide, and sulfide;
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- (d) recovering the eluate from step (b).
A method of measuring the fraction of completely sulfurized phosphorothioate or phosphorodithioate oligonucleotides in a mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides, the method comprising the steps of:
- (a) impregnating a strong anion exchanger with the mixture of completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides, the strong anion exchanger consisting of a mechanically rigid styrene-divinylbenzene matrix derivatized with a quaternary alkylammonium cation;
- (b) passing through the exchanger a buffer solution comprising a soft-base counter ion at a concentration which monotonically increases with time to a sample-desorbing concentration so that a chromatogram of the eluted completely sulfurized and incompletely sulfurized phosphorothioate and phosphorodithioate oligonucleotides is formed, the soft-base counter ion being selected from the group consisting of bromide, thiocyanate, azide, iodide, and sulfide;
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- (c) computing the ratio of the peak area corresponding to the completely sulfurized phosphorothioate or phosphorodithioate oligonucleotide on the chromatogram to the total area under all peaks corresponding to both completely sulfurized and incompletely sulfurized phosphorothioate or phosphorodithioate oligonucleotides on the chromatogram.
Dependencies are inferred from the claim text.