The Continuous Evolution In Vitro Technique

Carolina Díaz Arenas1, Niles Lehman1

1 Portland State University, Portland, Oregon
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 9.7
DOI:  10.1002/0471142700.nc0907s40
Online Posting Date:  March, 2010
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In vitro experimentation techniques were developed in response to the necessity of exploring new molecular structures and functions and to better understand evolutionary phenomena that shape organismal and molecular populations. The advancement of these techniques has allowed further exploration of more complicated evolutionary dynamics. One such technique is the continuous evolution in vitro (CE) method, to which this unit is devoted. The CE method is characterized by continuous cycles of amplification of RNA molecules that occur without much participation of the researcher. This feature allows us to evolve lineages in which the evolutionary phenomena occurring at the molecular level more closely mimic what happens in organismal populations in the present, or what may have happened in RNA populations during the RNA world stage of life. Curr. Protoc. Nucleic Acid Chem. 40:9.7.1‐9.7.16. © 2010 by John Wiley & Sons, Inc.

Keywords: evolution; in vitro; mutation; RNA

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Table of Contents

  • Introduction
  • Basic Protocol 1: Continuous Evolution In Vitro (CE) Experiments
  • Alternate Protocol 1: Continuous Evolution In Vitro (CE) Experiments Using a Microfluidics System
  • Support Protocol 1: Determining the Dilution and Amplification Scheme
  • Support Protocol 2: Preparation of Ligase Ribozymes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Continuous Evolution In Vitro (CE) Experiments

  • Stock ligase ribozymes [50 µM] (prepared in advance; see protocol 4)
  • CE buffer (prepared in advance; see recipe)
  • NTP mix (see recipe)
  • DEPC‐treated and/or RNase‐free water
  • PE mix (see recipe)
  • Ice bucket
  • 600‐µL and 1.5‐mL tubes
  • Timers
  • Benchtop cooler
  • 37°C heating block
  • Quick‐spin minifuge (ISC BioExpress, cat. no. C1301)
  • Additional reagents and equipment for carrying out PCR (e.g., Kramer and Coen, ) and agarose gel electrophoresis (e.g., Voytas, )

Alternate Protocol 1: Continuous Evolution In Vitro (CE) Experiments Using a Microfluidics System

  • α‐32P‐ATP (10 µCi/µL)
  • 2× XC (bromphenol blue with no xylene cylenol added) acrylamide gel‐loading dye
  • 5% polyacrylamide/8 M urea gel
  • Phosphorimager
  • Additional reagents and equipment for carrying out denaturing PAGE (e.g., see appendix 3A)
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Literature Cited

Literature Cited
   Ancel, L.W. and Fontana, W. 2000. Plasticity, evolvability, and modularity in RNA. J. Exp. Zool. 288:242‐283.
   Bartel, D.P. and Szostak, J.W. 1993. Isolation of new ribozymes from a large pool of random sequences. Science 261:1411‐1418.
   Beaudry, A.A. and Joyce, G.F. 1992. Directed evolution of an RNA enzyme. Science 257:635‐641.
   Breaker, R.R. and Joyce, G.F. 1994. Emergence of a replicating species from an in vitro RNA evolution reaction. Proc. Natl. Acad. Sci. U.S.A. 91:6093‐6097.
   Caldwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28‐33.
   Cech, T.R. 1987. The chemistry of self‐splicing RNA and RNA enzymes. Science 236:1532‐1539.
   Davidson, E.A., Dlugosz, P.J., Levy, M., and Ellington, A.D. 2009. Directed evolution of proteins in vitro using compartmentalization in emulsions. Curr. Protoc. Mol. Biol. 87:24.6.1‐24.6.12.
   Díaz Arenas, C. and Lehman, N. 2009a. Darwin's concept in a test tube: Parallels between organismal and in vitro evolution. Int. J. Biochem. Cell Biol. 41:266‐273.
   Díaz Arenas, C. and Lehman, N. 2009b. Quasispecies behavior observed in RNA populations evolving in a test tube. BMC Evol. Biol. Submitted.
   Ellinger, T., Ehricht, R., and McCaskill, J.S. 1998. In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system. Chem. Biol. 5:729‐741.
   Ellington, A.D. and Szostak, J.W. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346:818‐822.
   Ellington, A.D., Chen, X., Robertson, M., and Syrett, A. 2009. Evolutionary origins and directed evolution of RNA. Int. J. Biochem. Cell Biol. 41:254‐265.
   Guatelli, J.C., Whitfield, K.M., Kwoh, D.Y., Barringer, K.J., Richman, D.D., and Gingeras, T.R. 1990. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. U.S.A. 87:1874‐1878.
   Ikawa, Y., Tsuda, K., Matsumura, S., and Inoue, T. 2004. De novo synthesis and development of an RNA enzyme. Proc. Natl. Acad. Sci. U.S.A. 101:13750‐13755.
   Johns, G.C. and Joyce, G.F. 2005. The promise and peril of continuous in vitro evolution. J. Mol. Evol. 61:253‐263.
   Johnston, W.K., Unrau, P.J., Lawrence, M.S., Glasner, M.E., and Bartel, D.P. 2001. RNA‐catalyzed RNA polymerization: Accurate and general RNA‐templated primer extension. Science 292:1319‐1325.
   Joyce, G.F. 1989. Amplification, mutation and selection of catalytic RNA. Gene 82:83‐87.
   Joyce, G.F. 2004. Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem. 73:791‐836.
   Joyce, G.F. 2007. Forty years of in vitro evolution. Angew. Chem. Int. Ed. 46:2‐19.
   Kimura, M. 1983. The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Kun, Á., Santos, M., and Szathmáry, E. 2005. Real ribozymes suggest a relaxed error threshold. Nat. Genet. 37:1008‐1011.
   Lehman, N. 2004. Assessing the likelihood of recurrence during RNA evolution in vitro. Artif. Life 10:1‐22.
   Lehman, N. and Joyce, G.F. 1993. Evolution in vitro of an RNA enzyme with altered metal dependence. Nature 361:182‐185.
   Lehman, N., Delle‐Donne, M., West, M., and Dewey, G. 2000. The genotypic landscape during in vitro evolution of a catalytic RNA: Implication for genotypic buffering. J. Mol. Evol. 50:481‐490.
   Levisohn, R. and Spiegelman, S. 1969. Further extracellular Darwinian experiments with replicating RNA molecules; diverse variants isolated under different selective conditions. Proc. Natl. Acad. Sci. U.S.A. 63:805‐811.
   McGinness, K.E., Wright, M.C., and Joyce, G.F. 2002. Continuous in vitro evolution of a ribozyme that catalyzes three successive nucleotidyl addition reactions. Chem. Biol. 9:585‐596.
   Mills, D.R., Peterson, R.L., and Spiegelman, S. 1967. An extracellular Darwinian experiment with a self‐duplicating nucleic acid molecule. Proc. Natl. Acad. Sci. U.S.A. 58:217‐224.
   Orgel, L.E. 1979. Selection in vitro. Proc. R. Soc. Lond., B, Biol. Sci. 205:435‐442.
   Paegel, B.M. and Joyce, G.F. 2008. Darwinian evolution on a chip. PLoS Biol. 6:900‐906.
   Paegel, B.M., Grover, W.H., Skelley, A.M., Mathies, R.A., and Joyce, G.F. 2006. Microfluidic serial dilution circuit. Anal. Chem. 78:7522‐7527.
   Piasecki, S.K., Hall, B., and Ellington, A.D. 2009. Nucleic acid pool preparation and characterization. Methods Mol. Biol. 535:3‐18.
   Saffhill, R., Schneider‐Bernloehr, H., and Orgel, L.E. 1970. In vitro selection of bacteriophage Qβ ribonucleic acid variants resistant to ethidium bromide. J. Mol. Biol. 51:531‐539.
   Schmitt, T. and Lehman, N. 1999. Non‐unity heritability demonstrated by continuous evolution in vitro. Chem. Biol. 6:857‐869.
   Seelig, B. and Jäschke, A. 1999. A small catalytic RNA with Diels‐Alderase activity. Chem. Biol. 6:167‐176.
   Soll, S., Díaz Arenas, C., and Lehman, N. 2007. Accumulation of deleterious mutations in small abiotic populations of RNA. Genetics 175:267‐275.
   Tarasow, T.M., Tarasow, S.L., and Eaton, B.E. 1997. RNA‐catalysed carbon‐carbon bond formation. Nature 389:54‐57.
   Vartanian, J.‐P., Henry, M., and Wain‐Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversion. Nucleic Acids Res. 14:2627‐2631.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Voytek, S.B. and Joyce, G.F. 2007. Emergence of a fast reacting ribozyme that is capable of undergoing continuous evolution. Proc. Natl. Acad. Sci. U.S.A. 104:15288‐15293.
   Voytek, S.B. and Joyce, G.F. 2009. Niche partitioning in the coevolution of 2 distinctive RNA enzymes. Proc. Natl. Acad. Sci. U.S.A. 106:7780‐7785.
   Whitesides, G.M. 2006. The origins and the future of microfluidics. Nature 442:368‐373.
   Wilson, C. and Szostak, J.W. 1999. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68:611‐647.
   Wright, M.C. and Joyce, G.F. 1997. Continuous in vitro evolution of catalytic function. Science 276:614‐617.
   Wright, S. 1931. Evolution in Mendelian populations. Genetics 16:97‐159.
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