Compartmentalized Self‐Tagging for In Vitro‐Directed Evolution of XNA Polymerases

Vitor B. Pinheiro1, Sebastian Arangundy‐Franklin2, Philipp Holliger2

1 Birkbeck College, University of London, London, 2 MRC Laboratory of Molecular Biology, Cambridge
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 9.9
DOI:  10.1002/0471142700.nc0909s57
Online Posting Date:  June, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Template‐dependent synthesis of xenobiotic nucleic acids (XNAs) is an essential step for the development of functional XNA molecules, as it enables Darwinian evolution to be carried out with novel genetic polymers. The extraordinary substrate specificity of natural DNA polymerases greatly restricts the spectrum of XNAs available, thus making it necessary to identify DNA polymerase variants capable of incorporating a wider range of substrates. This unit summarizes compartmentalized self‐tagging (CST), a directed evolution strategy developed for the selection of DNA polymerase variants capable of XNA synthesis. Curr. Protoc. Nucleic Acid Chem. 57:9.9.1‐9.9.18. © 2014 by John Wiley & Sons, Inc.

Keywords: DNA polymerases; in vitro‐directed evolution; synthetic nucleic acids; XNA; compartmentalized self‐tagging; CST

PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • Plasmid harboring DNA polymerase gene (assembled in‐house from amplified genomic material)
  • 10× Expand High‐Fidelity buffer (with MgCl 2) and enzyme mix (Roche)
  • 10 µM forward primer (designed in‐house and commercially synthesized)
  • 10 µM reverse primer (designed in‐house and commercially synthesized)
  • 10 mM nucleotide triphosphates (GE Healthcare)
  • Formamide (molecular biology grade; Sigma‐Aldrich)
  • Deionized water, sterile
  • Agarose gel extraction kit (e.g., Qiagen's QIAquick)
  • 10× elution buffer (EB) stock solution: 100 mM Tris·Cl, pH 8.5; sterilize by passing through a 0.22‐µm filter; store up to 2 weeks at room temperature; dilute as required with sterile deionized, distilled water
  • 10× CutSmart buffer (NEB)
  • DpnI (NEB)
  • BsaI (NEB)
  • 10× T4 DNA ligase buffer and T4 ligase (NEB)
  • Electrocompetent E. coli cells (e.g., 10‐β cells, NEB)
  • 2×TY liquid medium supplemented with 100 µg/mL ampicillin (see recipe), or other appropriate liquid medium with selection agent
  • 2×TY plates, supplemented with 100 µg/mL ampicillin (see recipe), or other appropriate solid medium with selection agent
  • Storage buffer: 50% (v/v) glycerol (molecular biology grade; Sigma‐Aldrich)/50% 2×TY stock solution supplemented with 100 µg/mL ampicillin (see recipe); prepare fresh before use; sterilize by passing through a 0.22‐µm filter
  • 100% ethanol
  • Dry ice
  • 2.0 mg/mL anhydrotetracycline/dimethylformamide (DMF, molecular biology grade; Sigma‐Aldrich)
  • 10× ThermoPol buffer (NEB): dilute to 1× with sterile deionized, distilled water, as required
  • 50% (v/v) glycerol (molecular biology grade; Sigma-Aldrich): dilute with deionized, distilled water, and sterilize by passing through a 0.22‐µm filter; store up to 1 month at 4°C
  • 25 and 30 mM MnCl 2
  • 100 µM biotinylated DNA primer (Integrated DNA Technologies)
  • 100 mM dithiothreitol (DTT, molecular biology grade; Sigma‐Aldrich)
  • 10 mg/mL bovine serum albumin (BSA, NEB)
  • 2.5 mM XNA nucleotide triphosphates (custom synthesized by P. Herdewijn)
  • Oil phase for emulsion (see recipe)
  • TBT2: dilute from 10× TBT2 stock solution (see recipe)
  • Water‐saturated 1‐hexanol
  • MyOne C1 streptavidin‐coated paramagnetic beads (Life Technologies)
  • BWBS: dilute from 2× BWBS stock solution (see recipe)
  • 10× FASTstart buffer and Taq enzyme (Roche)
  • ExoSAP‐IT (USB)
  • 10× CutSmart buffer (NEB)
  • 10× Expand High‐Fidelity buffer (with MgCl 2) and enzyme mix (Roche)
  • Recombinant shrimp alkaline phosphatase (rSAP, NEB)
  • 1.5‐mL and 2‐mL microcentrifuge tubes
  • Thermocycler (e.g., Tetrad2, Bio‐Rad)
  • PCR purification kit with purification columns (e.g., QiAquick, Qiagen)
  • 37°C incubator
  • 20 × 20–cm square plates (Corning)
  • Cell scraper (e.g., ThermoFisher Scientific)
  • 50‐mL centrifuge tube (Fakon)
  • Vortexer
  • Spectrophotometer
  • 2‐mL round‐bottom microcentrifuge tubes with lids (Eppendorf)
  • 5‐mm steel beads (Qiagen)
  • Tissuelyser II (Qiagen)
  • 0.5‐mL or 0.2‐mL PCR tubes, strips or plates (Starlab)
  • S400 gel filtration columns (Illustra MicroSpin, GE Life Sciences)
  • Test tube rotator (Grant Instruments)
  • Magnetic stand (Promega)
  • Kingfisher mL magnetic particle processor (Thermo Scientific)
  • Additional reagents and equipment for carrying out agarose gel electrophoresis (Voytas, ), isolation of DNA by phenol‐chloroform extraction ( appendix 2A), electroporation (Seidman et al., ), and low‐volume spectrometry (Desjardins and Conklin, )
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Boudou, V., Kerremans, L., De Bouvere, B., Lescrinier, E., Schepers, G., Busson, R., Van Aerschot, A., and Herdewijn, P. 1999. Base pairing of anhydrohexitol nucleosides with 2,6‐diaminopurine, 5‐methylcytosine and uracil as base moiety. Nucleic Acids Res. 27:1450‐1456.
  Burmeister, P.E., Lewis, S.D., Silva, R.F., Preiss, J.R., Horwitz, L.R., Pendergrast, P.S., McCauley, T.G., Kurz, J.C., Epstein, D.M., Wilson, C., and Keefe, A.D. 2005. Direct in vitro selection of a 2′‐O‐methyl aptamer to VEGF. Chem. Biol. 12:25‐33.
  Desjardins, P.R. and Conklin, D.S. 2011. Microvolume quantitation of nucleic acids. Curr. Protoc. Mol. Biol. 93:A.3J.1‐A.3J.16.
  Diehl, F., Li, M., He, Y., Kinzler, K.W., Vogelstein, B., and Dressman, D. 2006. BEAMing: Single‐molecule PCR on microparticles in water‐in‐oil emulsions. Nat. Methods 3:551‐559.
  Engler, C., Kandzia, R., and Marillonnet, S. 2008. A one pot, one step, precision cloning method with high‐throughput capability. PloS One 3:e3647.
  Ghadessy, F.J., Ramsay, N., Boudsocq, F., Loakes, D., Brown, A., Iwai, S., Vaisman, A., Woodgate, R., and Holliger, P. 2004. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Nat. Biotechnol. 22:755‐759.
  Jager, S. and Famulok, M. 2004. Generation and enzymatic amplification of high‐density functionalized DNA double strands. Angew Chem. Int. Ed. Engl. 43:3337‐3340.
  Loakes, D. and Holliger, P. 2009. Polymerase engineering: Towards the encoded synthesis of unnatural biopolymers. Chem. Commun. 31:4619‐4631.
  Ochman, H., Gerber, A.S., and Hartl, D.L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120:621‐623.
  Pinheiro, V.B., Ong, J.L., and Holliger, P. 2012. Polymerase engineering: From PCR and sequencing to synthetic biology. In Protein Engineering Handbook Vol. III (S. Lutz & U.T. Bornscheuer, eds.) pp. 279‐302. Wiley‐VCH. Weinheim, Germany.
  Pinheiro, V.B., Taylor, A.I., Cozens, C., Abramov, M., Renders, M., Zhang, S., Chaput, J.C., Wengel, J., Peak‐Chew, S.Y., McLaughlin, S.H., Herdewijn, P., and Holliger, P. 2012. Synthetic genetic polymers capable of heredity and evolution. Science 336:341‐344.
  Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37:1.8.1‐1.8.10.
  Skerra, A. 1994. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151:131‐135.
  Vant‐Hull, B., Gold, L., and Zichi, D.A. 2000. Theoretical principles of in vitro selection using combinatorial nucleic acid libraries. Curr. Protoc. Nucl. Acid. Chem. 0:9.1.1‐9.1.16.
  Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
PDF or HTML at Wiley Online Library