title: Fast production of homogeneous recombinant RNA--towards large-scale production of RNA.
abstract: In the past decades, RNA molecules have emerged as important players in numerous cellular processes. To understand these processes at the molecular and atomic level, large amounts of homogeneous RNA are required for structural, biochemical and pharmacological investigations. Such RNAs are generally obtained from laborious and costly in vitro transcriptions or chemical synthesis. In 2007, a recombinant RNA technology has been described for the constitutive production of large amounts of recombinant RNA in Escherichia coli using a tRNA-scaffold approach. We demonstrate a general applicable extension to the described approach by introducing the following improvements: (i) enhanced transcription of large recombinant RNAs by T7 RNA polymerase (high transcription rates, versatile), (ii) efficient and facile excision of the RNA of interest from the tRNA-scaffold by dual cis-acting hammerhead ribozyme mediated cleavage and (iii) rapid purification of the RNA of interest employing anion-exchange chromatography or affinity chromatography followed by denaturing polyacrylamide gel electrophoresis. These improvements in the existing method pave the tRNA-scaffold approach further such that any (non-)structured product RNA of a defined length can cost-efficiently be obtained in (multi-)milligram quantities without in vitro enzymatic manipulations.
authors.affiliation: Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
authors.forename: Frank H T
authors.initials: FH
authors.lastname: Nelissen
authors.forename: Elizabeth H P
authors.initials: EH
authors.lastname: Leunissen
authors.forename: Linda
authors.initials: L
authors.lastname: van de Laar
authors.forename: Marco
authors.initials: M
authors.lastname: Tessari
authors.forename: Hans A
authors.initials: HA
authors.lastname: Heus
authors.forename: Sybren S
authors.initials: SS
authors.lastname: Wijmenga
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors; D019906:Nuclear Magnetic Resonance, Biomolecular; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D016337:RNA, Catalytic; D012357:RNA, Transfer, Lys; D014764:Viral Proteins; C427204:hammerhead ribozyme; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases