One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry
Hannes FeyrerKatja Petzold

Abstract

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA.

References

Nov 11, 1987·Nucleic Acids Research·J F MilliganO C Uhlenbeck
Dec 31, 1997·Biotechnology Progress·J A Kern, R H Davis
Sep 17, 1999·Zeitschrift Für Naturforschung. C, a Journal of Biosciences·M G AbouHaidar, I G Ivanov
Jul 12, 2002·Nature·Jennifer A Doudna, Thomas R Cech
Nov 13, 2002·The Journal of Biological Chemistry·Iaroslav KuzmineCraig T Martin
Apr 16, 2005·Proceedings of the National Academy of Sciences of the United States of America·Jean GuillerezMarc Dreyfus
Nov 26, 2008·Oligonucleotides·Mark A Behlke
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Sep 8, 2012·Nature·Sarah DjebaliThomas R Gingeras
Aug 6, 2013·Journal of Molecular Biology·Marina Ostankovitch, Anna Marie Pyle
Sep 17, 2013·Methods in Enzymology·Julie L Brunelle, Rachel Green
Jan 15, 2015·Molecular Therapy. Nucleic Acids·Xingyu WangXingguo Liang
Jun 11, 2015·Cellular and Molecular Life Sciences : CMLS·Pak-Yin LuiNigel J Stevenson
Mar 22, 2016·Nature Communications·David W SalzmanJoanne B Weidhaas
Aug 27, 2016·Briefings in Functional Genomics·Taeyoung Koo, Jin-Soo Kim
Oct 5, 2016·Progress in Biophysics and Molecular Biology·Lizhe ZhuXuhui Huang
Dec 22, 2016·International Journal of Molecular Sciences·Qiulong ZhangGe Zhang
May 23, 2017·Genomics, Proteomics & Bioinformatics·Iain M Dykes, Costanza Emanueli
Sep 28, 2017·Nature Reviews. Molecular Cell Biology·Yigong Shi
Mar 14, 2018·Nucleic Acids Research·Xin MuSun Hur
Mar 17, 2018·Analytical and Bioanalytical Chemistry·Lorenzo BarontiKatja Petzold
May 31, 2018·International Journal of Biological Macromolecules·Subhomoi Borkotoky, Ayaluru Murali
Jun 9, 2018·Nucleic Acid Therapeutics·Ludger Johannes, Marco Lucchino
Dec 7, 2018·Pharmacology & Therapeutics·Ai-Ming YuMei-Juan Tu
Mar 25, 2019·Methods : a Companion to Methods in Enzymology·Marie FlammeMarcel Hollenstein
Jul 28, 2018·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Hui ChengRenjun Pei

❮ Previous
Next ❯

Methods Mentioned

BETA
in vitro transcription
electrophoresis

Software Mentioned

IDT

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.