Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect
ACS Publications. Most Trusted. Most Cited. Most Read
My Activity
CONTENT TYPES

Figure 1Loading Img

Evolving a Polymerase for Hydrophobic Base Analogues

View Author Information
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom, and Department of Chemistry, Stanford University, Stanford, California 94305
†MRC Laboratory of Molecular Biology.
‡Present address: Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46012 Valencia, Spain.
§Stanford University.
Cite this: J. Am. Chem. Soc. 2009, 131, 41, 14827–14837
Publication Date (Web):September 24, 2009
https://doi.org/10.1021/ja9039696
Copyright © 2009 American Chemical Society

    Article Views

    2516

    Altmetric

    -

    Citations

    LEARN ABOUT THESE METRICS
    Other access options
    Supporting Info (1)»

    Abstract

    Abstract Image

    Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson−Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: ϕ (abasic site), d5NI: ϕ, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.

    Recommended

    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. You can change your affiliated institution below.

    Supporting Information

    ARTICLE SECTIONS
    Jump To

    Protein sequences of selected polymerases (including 5D4), additional data on kinetic constants, substrate specificity, primer extension, NMR spectroscopy, reversion mutant activity, SCA and FoldX analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

    Cited By

    This article is cited by 70 publications.

    1. Mohammed Elias, Xiangying Guan, Devin Hudson, Rahul Bose, Joon Kwak, Ioanna Petrounia, Kenza Touah, Sourour Mansour, Peng Yue, Gauthier Errasti, Thomas Delacroix, Anisha Ghosh, Raj Chakrabarti. Evolution of Organic Solvent-Resistant DNA Polymerases. ACS Synthetic Biology 2023, 12 (11) , 3170-3188. https://doi.org/10.1021/acssynbio.2c00515
    2. Maximilian Gantz, Stefanie Neun, Elliot J. Medcalf, Liisa D. van Vliet, Florian Hollfelder. Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments. Chemical Reviews 2023, 123 (9) , 5571-5611. https://doi.org/10.1021/acs.chemrev.2c00910
    3. Tushar Aggarwal, William A. Hansen, Jonathan Hong, Abir Ganguly, Darrin M. York, Sagar D. Khare, Enver Cagri Izgu. Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign. ACS Chemical Biology 2022, 17 (7) , 1924-1936. https://doi.org/10.1021/acschembio.2c00373
    4. Miglena Manandhar, Eugene Chun, Floyd E. Romesberg. Genetic Code Expansion: Inception, Development, Commercialization. Journal of the American Chemical Society 2021, 143 (13) , 4859-4878. https://doi.org/10.1021/jacs.0c11938
    5. Gillian Houlihan, Sebastian Arangundy-Franklin, and Philipp Holliger . Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering. Accounts of Chemical Research 2017, 50 (4) , 1079-1087. https://doi.org/10.1021/acs.accounts.7b00056
    6. Hayley J. Schultz, Andrea M. Gochi, Hannah E. Chia, Alexie L. Ogonowsky, Sharon Chiang, Nedim Filipovic, Aurora G. Weiden, Emma E. Hadley, Sara E. Gabriel, and Aaron M. Leconte . Taq DNA Polymerase Mutants and 2′-Modified Sugar Recognition. Biochemistry 2015, 54 (38) , 5999-6008. https://doi.org/10.1021/acs.biochem.5b00689
    7. Dianjie Hou and Marc M. Greenberg . DNA Interstrand Cross-Linking upon Irradiation of Aryl Halide C-Nucleotides. The Journal of Organic Chemistry 2014, 79 (5) , 1877-1884. https://doi.org/10.1021/jo4028227
    8. Yin Nah Teo and Eric T. Kool . DNA-Multichromophore Systems. Chemical Reviews 2012, 112 (7) , 4221-4245. https://doi.org/10.1021/cr100351g
    9. Amit Ketkar, Maroof K. Zafar, Surajit Banerjee, Victor E. Marquez, Martin Egli, and Robert L. Eoff . A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota. Journal of the American Chemical Society 2012, 134 (25) , 10698-10705. https://doi.org/10.1021/ja304176q
    10. Renatus W. Sinkeldam, Nicholas J. Greco and Yitzhak Tor. Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications. Chemical Reviews 2010, 110 (5) , 2579-2619. https://doi.org/10.1021/cr900301e
    11. Nicola Ramsay, Ann-Sofie Jemth, Anthony Brown, Neal Crampton, Paul Dear and Philipp Holliger . CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase. Journal of the American Chemical Society 2010, 132 (14) , 5096-5104. https://doi.org/10.1021/ja909180c
    12. Hui Ding and Marc M. Greenberg. DNA Damage and Interstrand Cross-Link Formation upon Irradiation of Aryl Iodide C-Nucleotide Analogues. The Journal of Organic Chemistry 2010, 75 (3) , 535-544. https://doi.org/10.1021/jo902071y
    13. Arghya Sett, Manoj Gadewar, M. Arockia Babu, Amrita Panja, Punya Sachdeva, Abdulmajeed G. Almutary, Vijay Upadhye, Saurabh Kumar Jha, Niraj Kumar Jha. Orchestration and theranostic applications of synthetic genome with Hachimoji bases/building blocks. Chemical Biology & Drug Design 2024, 103 (1) https://doi.org/10.1111/cbdd.14378
    14. Fatima Akram, Fatima Iftikhar Shah, Ramesha Ibrar, Taseer Fatima, Ikram ul Haq, Waqas Naseem, Mahmood Ayaz Gul, Laiba Tehreem, Ghanoor Haider. Bacterial thermophilic DNA polymerases: A focus on prominent biotechnological applications. Analytical Biochemistry 2023, 671 , 115150. https://doi.org/10.1016/j.ab.2023.115150
    15. Leping Sun, Xingyun Ma, Binliang Zhang, Yanjia Qin, Jiezhao Ma, Yuhui Du, Tingjian Chen. From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma. RSC Chemical Biology 2022, 3 (10) , 1173-1197. https://doi.org/10.1039/D2CB00116K
    16. Trevor A. Christensen, Kristi Y. Lee, Simone Z. P. Gottlieb, Mikayla B. Carrier, Aaron M. Leconte. Mutant polymerases capable of 2′ fluoro-modified nucleic acid synthesis and amplification with improved accuracy. RSC Chemical Biology 2022, 3 (8) , 1044-1051. https://doi.org/10.1039/D2CB00064D
    17. Jia Wei Siau, Samuel Nonis, Sharon Chee, Li Quan Koh, Fernando J Ferrer, Christopher J Brown, Farid J Ghadessy. Directed co-evolution of interacting protein–peptide pairs by compartmentalized two-hybrid replication (C2HR). Nucleic Acids Research 2020, 48 (22) , e128-e128. https://doi.org/10.1093/nar/gkaa933
    18. Karen Duffy, Sebastian Arangundy-Franklin, Philipp Holliger. Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biology 2020, 18 (1) https://doi.org/10.1186/s12915-020-00803-6
    19. Zahra Ouaray, Steven A. Benner, Millie M. Georgiadis, Nigel G.J. Richards. Building better polymerases: Engineering the replication of expanded genetic alphabets. Journal of Biological Chemistry 2020, 295 (50) , 17046-17059. https://doi.org/10.1074/jbc.REV120.013745
    20. Jan Špaček, Nilesh Karalkar, Miroslav Fojta, Joseph Wang, Steven A. Benner. Electrochemical reduction and oxidation of eight unnatural 2′-deoxynucleosides at a pyrolytic graphite electrode. Electrochimica Acta 2020, 362 , 137210. https://doi.org/10.1016/j.electacta.2020.137210
    21. Anna V. Yudkina, Dmitry O. Zharkov. Protein Engineering of DNA-Dependent Enzymes. 2020, 19-33. https://doi.org/10.1007/978-3-030-41283-8_2
    22. Elizabeth C. Gardner, Ella J. Watkins, Jimmy Gollihar, Andrew D. Ellington. Emulsion-based directed evolution of enzymes and proteins in yeast. 2020, 87-110. https://doi.org/10.1016/bs.mie.2020.04.053
    23. Stefanie Neun, Paul J. Zurek, Tomasz S. Kaminski, Florian Hollfelder. Ultrahigh throughput screening for enzyme function in droplets. 2020, 317-343. https://doi.org/10.1016/bs.mie.2020.06.002
    24. Ali Nikoomanzar, Nicholas Chim, Eric J. Yik, John C. Chaput. Engineering polymerases for applications in synthetic biology. Quarterly Reviews of Biophysics 2020, 53 https://doi.org/10.1017/S0033583520000050
    25. Govindan Raghunathan, Andreas Marx. Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. Scientific Reports 2019, 9 (1) https://doi.org/10.1038/s41598-018-37233-y
    26. S. A. Zhukov, A. A. Fokina, D. A. Stetsenko, S. V. Vasilyeva. Methods for Molecular Evolution of Polymerases. Russian Journal of Bioorganic Chemistry 2019, 45 (6) , 726-738. https://doi.org/10.1134/S1068162019060426
    27. Timothy A. Coulther, Hannah R. Stern, Penny J. Beuning. Engineering Polymerases for New Functions. Trends in Biotechnology 2019, 37 (10) , 1091-1103. https://doi.org/10.1016/j.tibtech.2019.03.011
    28. Anna J. Simon, Simon d’Oelsnitz, Andrew D. Ellington. Synthetic evolution. Nature Biotechnology 2019, 37 (7) , 730-743. https://doi.org/10.1038/s41587-019-0157-4
    29. Zhanar Abil, Andrew D. Ellington. Compartmentalized Self‐Replication for Evolution of a DNA Polymerase. Current Protocols in Chemical Biology 2018, 10 (1) , 1-17. https://doi.org/10.1002/cpch.34
    30. Gillian Houlihan, Sebastian Arangundy-Franklin, Philipp Holliger. Engineering and application of polymerases for synthetic genetics. Current Opinion in Biotechnology 2017, 48 , 168-179. https://doi.org/10.1016/j.copbio.2017.04.004
    31. Joos Aschenbrenner, Andreas Marx. DNA polymerases and biotechnological applications. Current Opinion in Biotechnology 2017, 48 , 187-195. https://doi.org/10.1016/j.copbio.2017.04.005
    32. Zhanar Abil, Jared W Ellefson, Jimmy D Gollihar, Ella Watkins, Andrew D Ellington. Compartmentalized partnered replication for the directed evolution of genetic parts and circuits. Nature Protocols 2017, 12 (12) , 2493-2512. https://doi.org/10.1038/nprot.2017.119
    33. Rubén Agudo, Patricia A. Calvo, María I. Martínez-Jiménez, Luis Blanco. Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase. Nucleic Acids Research 2017, 45 (15) , 9046-9058. https://doi.org/10.1093/nar/gkx633
    34. Sydney L. Rosenblum, Aurora G. Weiden, Eliza L. Lewis, Alexie L. Ogonowsky, Hannah E. Chia, Susanna E. Barrett, Mira D. Liu, Aaron M. Leconte. Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates. ChemBioChem 2017, 18 (8) , 816-823. https://doi.org/10.1002/cbic.201600701
    35. Tadas Povilaitis, Gediminas Alzbutas, Rasa Sukackaite, Juozas Siurkus, Remigijus Skirgaila. In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique. Protein Engineering, Design and Selection 2016, 29 (12) , 617-628. https://doi.org/10.1093/protein/gzw052
    36. María A. Dellafiore, Javier M. Montserrat, Adolfo M. Iribarren. Modified Nucleoside Triphosphates for In-vitro Selection Techniques. Frontiers in Chemistry 2016, 4 https://doi.org/10.3389/fchem.2016.00018
    37. Doug Millar, Yonka Christova, Philipp Holliger. A polymerase engineered for bisulfite sequencing. Nucleic Acids Research 2015, 43 (22) , e155-e155. https://doi.org/10.1093/nar/gkv798
    38. Pierre-Yves Colin, Anastasia Zinchenko, Florian Hollfelder. Enzyme engineering in biomimetic compartments. Current Opinion in Structural Biology 2015, 33 , 42-51. https://doi.org/10.1016/j.sbi.2015.06.001
    39. Andrew Currin, Neil Swainston, Philip J. Day, Douglas B. Kell. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chemical Society Reviews 2015, 44 (5) , 1172-1239. https://doi.org/10.1039/C4CS00351A
    40. Hiroto Fujita, Kohsuke Nakajima, Yuuya Kasahara, Hiroaki Ozaki, Masayasu Kuwahara. Polymerase-mediated high-density incorporation of amphiphilic functionalities into DNA: Enhancement of nuclease resistance and stability in human serum. Bioorganic & Medicinal Chemistry Letters 2015, 25 (2) , 333-336. https://doi.org/10.1016/j.bmcl.2014.11.037
    41. Roberto Laos, J. Michael Thomson, Steven A. Benner. DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides. Frontiers in Microbiology 2014, 5 https://doi.org/10.3389/fmicb.2014.00565
    42. Tingjian Chen, Floyd E. Romesberg. Directed polymerase evolution. FEBS Letters 2014, 588 (2) , 219-229. https://doi.org/10.1016/j.febslet.2013.10.040
    43. Sachin A. Ingale, Peter Leonard, Haozhe Yang, Frank Seela. 5-Nitroindole oligonucleotides with alkynyl side chains: universal base pairing, triple bond hydration and properties of pyrene “click” adducts. Org. Biomol. Chem. 2014, 12 (42) , 8519-8532. https://doi.org/10.1039/C4OB01478B
    44. Kang Lan Tee, Tuck Seng Wong. Polishing the craft of genetic diversity creation in directed evolution. Biotechnology Advances 2013, 31 (8) , 1707-1721. https://doi.org/10.1016/j.biotechadv.2013.08.021
    45. S.-i. Nakano, Y. Uotani, Y. Sato, H. Oka, M. Fujii, N. Sugimoto. Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules. Nucleic Acids Research 2013, 41 (18) , 8581-8590. https://doi.org/10.1093/nar/gkt608
    46. Samantha A. Wynne, Vitor B. Pinheiro, Philipp Holliger, Andrew G. W. Leslie, . Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA. PLoS ONE 2013, 8 (8) , e70892. https://doi.org/10.1371/journal.pone.0070892
    47. Wei-Cheng Lu, Andrew D. Ellington. In vitro selection of proteins via emulsion compartments. Methods 2013, 60 (1) , 75-80. https://doi.org/10.1016/j.ymeth.2012.03.008
    48. Vitor B. Pinheiro, David Loakes, Philipp Holliger. Synthetic polymers and their potential as genetic materials. BioEssays 2013, 35 (2) , 113-122. https://doi.org/10.1002/bies.201200135
    49. Feng Liang, Ying-Zhu Liu, Peiming Zhang. Universal base analogues and their applications in DNA sequencing technology. RSC Advances 2013, 3 (35) , 14910. https://doi.org/10.1039/c3ra41492b
    50. John C. Chaput, Hanyang Yu, Su Zhang. The Emerging World of Synthetic Genetics. Chemistry & Biology 2012, 19 (11) , 1360-1371. https://doi.org/10.1016/j.chembiol.2012.10.011
    51. Balint Kintses, Christopher Hein, Mark F. Mohamed, Martin Fischlechner, Fabienne Courtois, Céline Lainé, Florian Hollfelder. Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution. Chemistry & Biology 2012, 19 (8) , 1001-1009. https://doi.org/10.1016/j.chembiol.2012.06.009
    52. Markus Schmidt, Víctor de Lorenzo. Synthetic constructs in/for the environment: Managing the interplay between natural and engineered Biology. FEBS Letters 2012, 586 (15) , 2199-2206. https://doi.org/10.1016/j.febslet.2012.02.022
    53. Bastian Holzberger, Julian Strohmeier, Vanessa Siegmund, Ulf Diederichsen, Andreas Marx. Enzymatic synthesis of 8-vinyl- and 8-styryl-2′-deoxyguanosine modified DNA—novel fluorescent molecular probes. Bioorganic & Medicinal Chemistry Letters 2012, 22 (9) , 3136-3139. https://doi.org/10.1016/j.bmcl.2012.03.056
    54. Vitor B. Pinheiro, Alexander I. Taylor, Christopher Cozens, Mikhail Abramov, Marleen Renders, Su Zhang, John C. Chaput, Jesper Wengel, Sew-Yeu Peak-Chew, Stephen H. McLaughlin, Piet Herdewijn, Philipp Holliger. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336 (6079) , 341-344. https://doi.org/10.1126/science.1217622
    55. Lesley R. Rutledge, Stacey D. Wetmore. A computational proposal for the experimentally observed discriminatory behavior of hypoxanthine, a weak universal nucleobase. Physical Chemistry Chemical Physics 2012, 14 (8) , 2743. https://doi.org/10.1039/c2cp23600a
    56. D.M. Perrin. Lifelike but Not Living. 2012, 3-33. https://doi.org/10.1016/B978-0-444-53349-4.00220-X
    57. Alicja K Antonczak, Josephine Morris, Eric M Tippmann. Advances in the mechanism and understanding of site-selective noncanonical amino acid incorporation. Current Opinion in Structural Biology 2011, 21 (4) , 481-487. https://doi.org/10.1016/j.sbi.2011.04.004
    58. David Loakes. Nucleotides and nucleic acids; oligo- and polynucleotides. 2011, 139-216. https://doi.org/10.1039/BK9781849731386-00139
    59. Claudia Baar, Marc d’Abbadie, Alexandra Vaisman, Mercedes E. Arana, Michael Hofreiter, Roger Woodgate, Thomas A. Kunkel, Philipp Holliger. Molecular breeding of polymerases for resistance to environmental inhibitors. Nucleic Acids Research 2011, 39 (8) , e51-e51. https://doi.org/10.1093/nar/gkq1360
    60. Peter Walde, Zengwei Guo. Enzyme-catalyzed chemical structure-controlling template polymerization. Soft Matter 2011, 7 (2) , 316-331. https://doi.org/10.1039/C0SM00259C
    61. Christopher J. Hipolito, Marcel Hollenstein, Curtis H. Lam, David M. Perrin. Protein-inspired modified DNAzymes: dramatic effects of shortening side-chain length of 8-imidazolyl modified deoxyadenosines in selecting RNaseA mimicking DNAzymes. Organic & Biomolecular Chemistry 2011, 9 (7) , 2266. https://doi.org/10.1039/c0ob00595a
    62. Yasuyuki Hirama, Noriaki Minakawa, Akira Matsuda. Synthesis and characterization of oligodeoxynucleotides containing a novel tetraazabenzo[cd]azulene:naphthyridine base pair. Bioorganic & Medicinal Chemistry 2011, 19 (1) , 352-358. https://doi.org/10.1016/j.bmc.2010.11.023
    63. Christian Jäckel, Donald Hilvert. Biocatalysts by evolution. Current Opinion in Biotechnology 2010, 21 (6) , 753-759. https://doi.org/10.1016/j.copbio.2010.08.008
    64. Rafael Giraldo. Amyloid Assemblies: Protein Legos at a Crossroads in Bottom‐Up Synthetic Biology. ChemBioChem 2010, 11 (17) , 2347-2357. https://doi.org/10.1002/cbic.201000412
    65. Ramon Kranaster , Andreas Marx. Engineered DNA Polymerases in Biotechnology. ChemBioChem 2010, 11 (15) , 2077-2084. https://doi.org/10.1002/cbic.201000215
    66. Aaron M. Leconte, Maha P. Patel, Lauryn E. Sass, Peter McInerney, Mirna Jarosz, Li Kung, Jayson L. Bowers, Philip R. Buzby, J. William Efcavitch, Floyd E. Romesberg. Directed Evolution of DNA Polymerases for Next‐Generation Sequencing. Angewandte Chemie International Edition 2010, 49 (34) , 5921-5924. https://doi.org/10.1002/anie.201001607
    67. Aaron M. Leconte, Maha P. Patel, Lauryn E. Sass, Peter McInerney, Mirna Jarosz, Li Kung, Jayson L. Bowers, Philip R. Buzby, J. William Efcavitch, Floyd E. Romesberg. Directed Evolution of DNA Polymerases for Next‐Generation Sequencing. Angewandte Chemie 2010, 122 (34) , 6057-6060. https://doi.org/10.1002/ange.201001607
    68. Agne Tubeleviciute, Remigijus Skirgaila. Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding. Protein Engineering, Design and Selection 2010, 23 (8) , 589-597. https://doi.org/10.1093/protein/gzq032
    69. Christian Gloeckner, Ramon Kranaster, Andreas Marx. Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries. Current Protocols in Chemical Biology 2010, 2 (2) , 89-109. https://doi.org/10.1002/9780470559277.ch090183
    70. Markus Schmidt. Xenobiology: A new form of life as the ultimate biosafety tool. BioEssays 2010, 32 (4) , 322-331. https://doi.org/10.1002/bies.200900147