J 2016

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

ZLOBINA, Maria, Ondrej ŠEDO, Ming-Yuan CHOU, Lucia SLEPÁNKOVÁ, Peter LUKAVSKY et. al.

Basic information

Original name

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

Authors

ZLOBINA, Maria (643 Russian Federation, belonging to the institution), Ondrej ŠEDO (203 Czech Republic, belonging to the institution), Ming-Yuan CHOU (158 Taiwan, belonging to the institution), Lucia SLEPÁNKOVÁ (40 Austria, belonging to the institution) and Peter LUKAVSKY (40 Austria, guarantor, belonging to the institution)

Edition

Biotechniques, New York, Biotechniques Office, 2016, 0736-6205

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 2.030

RIV identification code

RIV/00216224:14740/16:00087840

Organization unit

Central European Institute of Technology

DOI

UT WoS

000369476700005

Keywords in English

single-stranded RNA; hammerhead ribozyme; isotope-labelled RNA; structural biology; RNA desalting

Tags

Změněno: 27/4/2017 13:14, Mgr. Eva Špillingová

Abstract

V originále

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.

Links

ED1.1.00/02.0068, research and development project
Name: CEITEC - central european institute of technology
EE2.3.20.0042, research and development project
Name: Internacionalizace programu Strukturní biologie s důrazem na rozvoj nových směrů výzkumu
GA15-21122S, research and development project
Name: Strukturní podstata změn v sestřihu způsobující cystickou fibrózu
Investor: Czech Science Foundation
GBP206/12/G151, research and development project
Name: Centrum nových přístupů k bioanalýze a molekulární diagnostice
286154, interní kód MU
Name: SYLICA - Synergies of Life and Material Sciences to Create a New Future (Acronym: SYLICA)
Investor: European Union, Capacities
3014, interní kód MU
Name: Molecular basis of aberrant splicing of CFTR exon 9
Investor: EMBO (European Molecular Biology Organization)
630758, interní kód MU
Name: Aberrant Splicing of CFTR Exon 9 (Acronym: ASCE)
Investor: European Union, People