Optimized method for the synthesis and purification of adenosine – Folic acid conjugates for use as transcription initiators in the preparation of modified RNA

• Published in: Methods (San Diego, Calif.) Vol. 54; no. 2; pp. 260 - 266
• Main Authors: Laing, Brian M., Guo, Peixuan, Bergstrom, Donald E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2011
• Subjects: adenosine; adenosine monophosphate; Adenosine monophosphate (AMP); Adenosine Monophosphate - analogs & derivatives; Adenosine Monophosphate - chemical synthesis; Adenosine Monophosphate - isolation & purification; Affi-gel boronate; Chromatography, Affinity; Chromatography, High Pressure Liquid; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - chemistry; Folic acid; Folic Acid - analogs & derivatives; Folic Acid - chemical synthesis; Folic Acid - isolation & purification; gels; high performance liquid chromatography; In vitro transcription; ribonucleotides; RNA; RNA labeling; RNA Probes - biosynthesis; RNA Probes - chemical synthesis; T7 RNA polymerase; Transcription, Genetic; Viral Proteins - chemistry; In vitro transcription; RNA labeling; T7 RNA polymerase; Affi-gel boronate; Folic acid; Adenosine monophosphate (AMP)
• ISSN: 1046-2023; 1095-9130; 1095-9130
• DOI: 10.1016/j.ymeth.2010.12.007

We present an optimized synthetic strategy for the attachment of molecules to 5′-adenosine monophosphate (AMP), which can then be used to label the 5′-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH > 7.5 and releases them at pH < 6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.