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Assay of Ribozyme–Substrate Cleavage by Anion-Exchange High-Performance Liquid Chromatography

✍ Scribed by Ravi Vinayak; Alex Andrus; Nanda D. Sinha; Arnold Hampel

Publisher
Elsevier Science
Year
1995
Tongue
English
Weight
100 KB
Volume
232
Category
Article
ISSN
0003-2697

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✦ Synopsis

Methods

An HPLC procedure has been developed that can be RNA phosphoramidites and supports, 6-FAM phosused to monitor the rate of phosphodiester cleavage of phoramidite, and ancillary reagents for automated synan oligoribonucleotide substrate by an RNA ribozyme theses were obtained from Applied Biosystems. Triethwith catalytic activity. The method operates at high ylamine trihydrofluoride [Et 3 N(HF) 3 ], DMF, 1-butanol substrate concentrations (far beyond K m ), thus (HPLC grade), sodium acetate, and lithium perchlorate allowing the reactions to approach V max of the reacwere purchased from Aldrich. Concentrated ammotion. This method is an efficient alternative to radioisonium hydroxide (28-30%) was purchased from Baker. tope labeling methods.

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