Publication details

Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides

Authors

ZLOBINA Maria ŠEDO Ondrej CHOU Ming-Yuan SLEPÁNKOVÁ Lucia LUKAVSKY Peter

Year of publication 2016
Type Article in Periodical
Magazine / Source Biotechniques
MU Faculty or unit

Central European Institute of Technology

Citation
Web http://www.biotechniques.com/BiotechniquesJournal/2016/February/Efficient-large-scale-preparation-and-purification-of-short-single-stranded----RNA-oligonucleotides/biotechniques-362970.html
Doi http://dx.doi.org/10.2144/000114383
Field Genetics and molecular biology
Keywords single-stranded RNA; hammerhead ribozyme; isotope-labelled RNA; structural biology; RNA desalting
Description Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides. Hammerhead ribozyme RNAs self-cleave with over 95% efficiency during in vitro transcription as a function of magnesium concentration to produce high yields of the desired ssRNA products. The resulting ssRNAs can be purified from crude transcription reactions by denaturing anion-exchange chromatography and then desalted by weak anion-exchange chromatography using volatile ammonium bicarbonate buffer solutions. The ssRNA oligonucleotides produced this way are homogenous, as judged by mass spectrometry (MS), and are suitable for biochemical and biophysical studies. Moreover, for high-resolution NMR structure determination of RNA-protein complexes, our protocol enables efficient preparation of ssRNA oligonucleotides with various isotope-labeling schemes which are not commercially available.
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