title: High-throughput assay and engineering of self-cleaving ribozymes by sequencing.
abstract: Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes.
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.
authors.forename: Shungo
authors.initials: S
authors.lastname: Kobori
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nomura
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.
authors.forename: Anh
authors.initials: A
authors.lastname: Miu
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA 95616, USA Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904 0495, Japan yoko@ucdavis.edu.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Yokobayashi
mesh_terms: D000494:Allosteric Regulation; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D059367:RNA Cleavage; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D016337:RNA, Catalytic
references.citation: Curr Opin Struct Biol. 2000 Jun;10(3):318-25
references.pmid: 10851196
references.citation: Proc Natl Acad Sci U S A. 2014 Sep 9;111(36):13028-33
references.pmid: 25157168
references.citation: Cell. 2003 May 30;113(5):577-86
references.pmid: 12787499
references.citation: Science. 1990 Aug 3;249(4968):505-10
references.pmid: 2200121
references.citation: Nature. 1990 Aug 30;346(6287):818-22
references.pmid: 1697402
references.citation: Biochemistry. 1993 Apr 27;32(16):4254-62
references.pmid: 8476853
references.citation: Chem Biodivers. 2007 Apr;4(4):633-55
references.pmid: 17443876
references.citation: Nucleic Acids Res. 2009 Jun;37(11):3545-57
references.pmid: 19357090
references.citation: J Am Chem Soc. 2009 Oct 7;131(39):13906-7
references.pmid: 19788322
references.citation: Science. 2009 Nov 13;326(5955):953
references.pmid: 19965505
references.citation: Science. 2010 Oct 15;330(6002):376-9
references.pmid: 20947767
references.citation: PLoS One. 2011;6(5):e19395
references.pmid: 21625587
references.citation: Biotechniques. 2011 Dec;51(6):413-6
references.pmid: 22150332
references.citation: PLoS One. 2011;6(12):e29604
references.pmid: 22242135
references.citation: RNA. 2012 May;18(5):871-85
references.pmid: 22454536
references.citation: FEBS Lett. 2012 Jul 16;586(15):2076-83
references.pmid: 22710175
references.citation: RNA Biol. 2011 Sep-Oct;8(5):719-27
references.pmid: 21734469
references.citation: Methods. 2012 Oct;58(2):164-70
references.pmid: 22885844
references.citation: Nucleic Acids Res. 2013 Feb 1;41(3):1873-84
references.pmid: 23241386
references.citation: Proc Natl Acad Sci U S A. 2013 Sep 10;110(37):14984-9
references.pmid: 23980164
references.citation: Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18460-5
references.pmid: 24167271
references.citation: Curr Opin Biotechnol. 2015 Feb;31:35-41
references.pmid: 25146171
references.citation: Nat Chem Biol. 2014 Jan;10(1):56-60
references.pmid: 24240507
references.citation: Nucleic Acids Res. 2014 Jan;42(2):1303-10
references.pmid: 24157838
references.citation: FEMS Microbiol Lett. 2014 Feb;351(2):162-9
references.pmid: 24372686
references.citation: ACS Synth Biol. 2013 Dec 20;2(12):684-9
references.pmid: 23697539
references.citation: Nat Methods. 2014 Jun;11(6):683-8
references.pmid: 24809628
references.citation: PLoS One. 2014;9(6):e100572
references.pmid: 24963654
references.citation: Nat Chem Biol. 2014 Sep;10(9):739-44
references.pmid: 25038788
references.citation: Curr Opin Biotechnol. 2002 Feb;13(1):31-9
references.pmid: 11849955