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@article{1338674,
author = {Zlobina, Maria and Šedo, Ondrej and Chou, MingandYuan and Slepánková, Lucia and Lukavsky, Peter},
article_location = {New York},
article_number = {2},
doi = {http://dx.doi.org/10.2144/000114383},
keywords = {single-stranded RNA; hammerhead ribozyme; isotope-labelled RNA; structural biology; RNA desalting},
language = {eng},
issn = {0736-6205},
journal = {Biotechniques},
title = {Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides},
url = {http://www.biotechniques.com/BiotechniquesJournal/2016/February/Efficient-large-scale-preparation-and-purification-of-short-single-stranded----RNA-oligonucleotides/biotechniques-362970.html},
volume = {60},
year = {2016}
}
TY - JOUR
ID - 1338674
AU - Zlobina, Maria - Šedo, Ondrej - Chou, Ming-Yuan - Slepánková, Lucia - Lukavsky, Peter
PY - 2016
TI - Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides
JF - Biotechniques
VL - 60
IS - 2
SP - 75-83
EP - 75-83
PB - Biotechniques Office
SN - 07366205
KW - single-stranded RNA
KW - hammerhead ribozyme
KW - isotope-labelled RNA
KW - structural biology
KW - RNA desalting
UR - http://www.biotechniques.com/BiotechniquesJournal/2016/February/Efficient-large-scale-preparation-and-purification-of-short-single-stranded----RNA-oligonucleotides/biotechniques-362970.html
L2 - http://www.biotechniques.com/BiotechniquesJournal/2016/February/Efficient-large-scale-preparation-and-purification-of-short-single-stranded----RNA-oligonucleotides/biotechniques-362970.html
N2 - 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.
ER -
ZLOBINA, Maria, Ondrej ŠEDO, Ming-Yuan CHOU, Lucia SLEPÁNKOVÁ a Peter LUKAVSKY. Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides. \textit{Biotechniques}. New York: Biotechniques Office, 2016, roč.~60, č.~2, s.~75-83. ISSN~0736-6205. Dostupné z: https://dx.doi.org/10.2144/000114383.
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