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

ZLOBINA, Maria, Ondrej ŠEDO, Ming-Yuan CHOU, Lucia SLEPÁNKOVÁ and Peter LUKAVSKY. Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides. Biotechniques. New York: Biotechniques Office, 2016, vol. 60, No 2, p. 75-83. ISSN 0736-6205. Available from: https://dx.doi.org/10.2144/000114383.

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

• Original language: English
• Type of outcome: Article in a journal
• Field of Study: Genetics and molecular biology
• Country of publisher: United States of America
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• Impact factor: Impact factor: 2.030
• RIV identification code: RIV/00216224:14740/16:00087840
• Organization unit: Central European Institute of Technology
• Doi: http://dx.doi.org/10.2144/000114383
• UT WoS: 000369476700005
• Keywords in English: single-stranded RNA; hammerhead ribozyme; isotope-labelled RNA; structural biology; RNA desalting
• Tags: CF NMR, CF PROT, rivok

Abstract

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