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Updated: May 28, 2026

Chemical Triphosphorylation of Oligonucleotides
Published on: June 2, 2022
Highly parallel oligonucleotide purification and functionalization using reversible chemistry
Kerri T York1, Ryan C Smith, Rob Yang
1Illumina, Inc., Advanced Research Group, San Diego, CA 92121, USA.
Abstract:
We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.
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