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

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.

Základní údaje

Originální název

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

Autoři

ZLOBINA, Maria (643 Rusko, domácí), Ondrej ŠEDO (203 Česká republika, domácí), Ming-Yuan CHOU (158 Tchaj-wan, domácí), Lucia SLEPÁNKOVÁ (40 Rakousko, domácí) a Peter LUKAVSKY (40 Rakousko, garant, domácí)

Vydání

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

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

Genetika a molekulární biologie

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 2.030

Kód RIV

RIV/00216224:14740/16:00087840

Organizační jednotka

Středoevropský technologický institut

DOI

http://dx.doi.org/10.2144/000114383

UT WoS

000369476700005

Klíčová slova anglicky

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

Štítky

CF NMR, CF PROT, rivok

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

Anotace

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.

Návaznosti

ED1.1.00/02.0068, projekt VaV

Název: CEITEC - central european institute of technology

EE2.3.20.0042, projekt VaV

Název: Internacionalizace programu Strukturní biologie s důrazem na rozvoj nových směrů výzkumu

GA15-21122S, projekt VaV

Název: Strukturní podstata změn v sestřihu způsobující cystickou fibrózu

Investor: Grantová agentura ČR, Strukturní podstata změn v sestřihu způsobující cystickou fibrózu

GBP206/12/G151, projekt VaV

Název: Centrum nových přístupů k bioanalýze a molekulární diagnostice

286154, interní kód MU

Název: SYLICA - Synergies of Life and Material Sciences to Create a New Future (Akronym: SYLICA)

Investor: Evropská unie, SYLICA - Synergies of Life and Material Sciences to Create a New Future, Kapacity

3014, interní kód MU

Název: Molecular basis of aberrant splicing of CFTR exon 9

Investor: EMBO (European Molecular Biology Organization), Molecular basis of aberrant splicing of CFTR exon 9

630758, interní kód MU

Název: Aberrant Splicing of CFTR Exon 9 (Akronym: ASCE)

Investor: Evropská unie, Aberrant Splicing of CFTR Exon 9, Lidé

Citovat

ZLOBINA, Maria, Ondrej ŠEDO, Ming-Yuan CHOU, Lucia SLEPÁNKOVÁ a Peter LUKAVSKY. Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides. 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.

@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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