DOCSLIB.ORG

Artificial DNA in Living Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Artificial DNA in Living Cells Artificial DNA in Living Cells Floyd E. Romesberg, Ph.D. National Academy of Sciences Forum on Synthetic Biology 19 September 2014 Washington, DC Chemical Biology and Biophysics • Efforts to expand the genetic alphabet by unnatural base pair design and polymerase evolution • Novel evolution-inspired approaches to antibiotic discovery • Biophysical characterization of how protein dynamics are evolved for biological function Expansion of the Genetic Alphabet/Code B DNA Polymerase B RNA Polymerase B B AA* AA* B B Seminal work of Steve Benner (FAME) Peter Schultz (TSRI) Predominantly Hydrophobic Nucleotides (sugar & phosphate not shown) S O NH N N N HO OH BTp BFr IN BTz NAP 2MN 3MN DMN 1HN 2HN O O S N O S O N N N N N N N O N S N O N O N O N S P7AI PM7AI PPP 7OFP 4TFP 4OFP 7OTP 4OTP 7TFP N N N N N N N N N N N N N N N N N Neb M7AI 4M7AI 7AI PP ImPy Np 8Q N N O N O N O N O N O N O PICS PIM PNICS ICS 5MICS MICS N N N N O N O N S N S N S NICS oNICS SNICS SoNICS SICS Assays: Thermostability and Kinetics 5’-GCGTACXCATGCG-3’ 3’-CGCATGYGTACGC-5’ 0.39 Incorporation 0.38 Extension A 0.37 260 0.36 0.35 0.34 X N 0.33 0 40 50 60 70 80 90 T, ºC Structure of a First Generation Hydrophobic UBP With D.Wemmer (UC Berkeley) and P. Schultz (TSRI) Second Generation Hydrophobic Nucleotides BEN MM1 MM2 MM3 DM2 DM3 DM4 DM5 DM TM TM2 F F F F F F F F F F F F F F TMB 2FB 3FB 4FB 3,5DFB 2,3DFB 3,4DFB 2M3FB 3F4MB TFB Br CN Br CN N N N Br CN N O N O 2Br 3Br 4Br 2CN 3CN 4CN 2Py 3Py 4Py PYR 3MP O F O F F Cl N O N O N O N O N O N O N O O 4MP 5MP 3MOP 4MOP 3FP 4FP 5FP 3CB 2OMB O O O O O S S S S O O O O 3OMB 4OMB MMO1 MMO2 DMO Tp MTp ETp MOTp MFr DMFr SAR Issue dYTP insertion X Efficient dYTP Insertion: dYTP–minor groove hydrophobic group template dX–minor groove hydrophobic group dNTP (extension dY:dX) Y X Efficient extension: Primer terminus dY–minor groove hydrogen-bond acceptor Template dX–minor groove hydrophobic group Two Screens of 3600 Candidate UBPs Extension Screen Full Length Screen SYBR +dNTPs +SYBR X X X +dNTPs dYTP Y +SYBR Y X X SYBR A Family of UBPs that are Efficiently Replicated and Transcribed in Vitro << < Sequencing Analysis of Replication Fidelity Calibration Curve for d5SICS-dNaM Replication 1 0 % natu ral L R OneTaq PCR with d5SICS-dNaM DNA Amplification Efficiency (%) Fidelity (%) ACT b GTG 2.5×1012 97 99.945 ± 0.016 GTC b GGT 1.5×1012 95 99.874 ± 0.035 AGC b CGT 3.5×1012 96 99.930 ± 0.003 CCG b GAA 8.1×1012 >99 99.664 ± 0.037 GTA b TGT 3.1×1012 95 99.987 ± 0.021 AGA b AGT 8.5×1012 >99 >99.98 CCT b AAA 8.4×1012 >99 99.866 ± 0.014 GGT b TCC 2.6×1012 94 99.958 ± 0.012 ACT b b GTG 3.8×1012 96 ≈ 99.5 ACT b A b GTG 2.7×1012 94 ≈ 99.5 ACT b GTGACT b GTG 2.0×1012 93 99.47 NNN b NNN 4.8×1012 97 99.925 ± 0.008 PCR Selection X = 5SICS Y = NaM Single Nucleotide Frequency dNaM dNaM dNaM dNaM Nucleotide position relative to dNaM Single Nucleotide Frequency 24.7% 18.7% dNaM dNaM dNaM dNaM Nucleotide position relative to dNaM Correlation Analysis dNaM dNaM dNaM dNaM Nucleotide position relative to dNaM Dinucleotide Frequency X XX X X Dinucleotide Frequency X X 2.3% 3.5% X X Structural Studies of d5SICS-dNaM With Tammy Dwyer (U. San Diego) Structural Studies of d5SICS-dNaM: Two Questions Structural Studies of d5SICS-dNaM With K. Betz & A. Marx Structural Studies of d5SICS-dNaM Expansion of the Genetic Alphabet/Code B DNA Polymerase B RNA Polymerase B B AA* AA* B B Extracellular Degradation of dNaM and d5SICS Activity of Phaeodactylum tricornutum NTT (PtNTT2) in E. coli (dATP uptake) Transporter 1 Transporter 2 Transporter 3 Transporter 4 Transporter 5 Transporter 6 Transporter 7 Transporter 8 PtNTT2-Mediated Uptake in E. coli pACS and pINF (X=NaM, Y=TPT3) d5SICS-dNaM and dTPT3-dNaM The Experiment Transform E. coli with pACS, induce PtNTT2, add dXTPs, transform with pINF, grow, recover pINF and characterize Controls: transform with pUC19 instead of pINF do not induce PtNTT2 do not add dXTPs Analysis of pINF After Growth in E. coli (15 h, 22 Doublings, 107-Fold Amplification) Sequencing Analysis of pINF After Growth in E. coli (15 h, 22 Doublings, 107-Fold Amplification) Global Nucleoside Content in pINF and pUC19 via LC-MS/MS With Ivan Correa, Nan Dai, and Jeremy Foster (NEB) Expansion of the Genetic Alphabet/Code B DNA Polymerase B RNA Polymerase B B AA* AA* B B Reddit AMA • 773 comments; score of 3056 (93% upvoted) • Much of the dialogue was technical, unlike what was generated by the NYT and NPR stories Jim Thomas Comments on Legal/Regulatory Implications “The arrival of this unprecedented ‘alien’ life form could in time have far-reaching ethical, legal and regulatory implications,” Jim Thomas of the ETC Group, a Canadian advocacy organization, said in an email. “While synthetic biologists invent new ways to monkey with the fundamentals of life, governments haven’t even been able to cobble together the basics of oversight, assessment or regulation for this surging field.” Comments in Response to Pollack’s NYT Article Comments in Response to Pollack’s NYT Article Comments in Response NPR Health Blog Objections to Comparison with Nature Imagine writing a story with a language with only four letters. A fifth and sixth letter would let you write more Interesting stories. Objections to Comparison with Nature Imagine writing a story with a language with only four letters. A fifth and sixth letter would let you write more Interesting stories. play .
Load more

Recommended publications
  • Addressing Evolutionary Questions with Synthetic Biology
    Addressing evolutionary questions with synthetic biology Florian Baier and Yolanda Schaerli Department of Fundamental Microbiology, University of Lausanne, Biophore Building, 1015 Lausanne, Switzerland Correspondence: [email protected]; [email protected] Abstract Synthetic biology emerged as an engineering discipline to design and construct artificial biological systems. Synthetic biological designs aim to achieve specific biological behavior, which can be exploited for biotechnological, medical and industrial purposes. In addition, mimicking natural systems using well-characterized biological parts also provides powerful experimental systems to study evolution at the molecular and systems level. A strength of synthetic biology is to go beyond nature’s toolkit, to test alternative versions and to study a particular biological system and its phenotype in isolation and in a quantitative manner. Here, we review recent work that implemented synthetic systems, ranging from simple regulatory circuits, rewired cellular networks to artificial genomes and viruses, to study fundamental evolutionary concepts. In particular, engineering, perturbing or subjecting these synthetic systems to experimental laboratory evolution provides a mechanistic understanding on important evolutionary questions, such as: Why did particular regulatory networks topologies evolve and not others? What happens if we rewire regulatory networks? Could an expanded genetic code provide an evolutionary advantage? How important is the structure of genome and number of chromosomes? Although the field of evolutionary synthetic biology is still in its teens, further advances in synthetic biology provide exciting technologies and novel systems that promise to yield fundamental insights into evolutionary principles in the near future. 1 1. Introduction Evolutionary biology traditionally studies past or present organisms to reconstruct past evolutionary events with the aim to explain and predict their evolution.
    [Show full text]
  • Efficient and Sequence-Independent Replication of DNA Containing A
    Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet Denis A. Malysheva, Kirandeep Dhamia, Henry T. Quacha, Thomas Lavergnea, Phillip Ordoukhanianb, Ali Torkamanic, and Floyd E. Romesberga,1 aDepartment of Chemistry, bCenter for Protein and Nucleic Acid Research, and cThe Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037 Edited* by Jack W. Szostak, The Howard Hughes Medical Institute and Massachusetts General Hospital, Boston, MA, and approved June 6, 2012 (received for review March 27, 2012) The natural four-letter genetic alphabet, comprised of just two were hindered by low-fidelity replication and chemical instability base pairs (dA-dT and dG-dC), is conserved throughout all life, and (23), but modifications have been found more recently that its expansion by the development of a third, unnatural base pair overcome these limitations, and DNA containing such unnatural has emerged as a central goal of chemical and synthetic biology. pairs has been amplified by PCR (24). The amplification of DNA We recently developed a class of candidate unnatural base pairs, containing multiple, contiguous unnatural base pairs has also exemplified by the pair formed between d5SICS and dNaM. Here, been shown (10), but the more general effects of sequence context we examine the PCR amplification of DNA containing one or more have yet to be reported. The second strategy, originally pursued d5SICS-dNaM pairs in a wide variety of sequence contexts. Under by our group (25), and in part inspired by the observation in the standard conditions, we show that this DNA may be amplified work by Kool and coworkers (26) that H-bonds are not absolutely with high efficiency and greater than 99.9% fidelity.
    [Show full text]
  • UNIVERSITY of CALIFORNIA, SAN DIEGO the Mechanism Of
    UNIVERSITY OF CALIFORNIA, SAN DIEGO The mechanism of recognition and processing of DNA damage and modifications by RNA polymerase II A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Ji Hyun Shin Committee in Charge: Professor Dong Wang, Chair Professor James Kadonaga, Co-Chair Professor Shannon Lauberth 2016 The Thesis of Ji Hyun Shin is approved and it is acceptable in quality and form for publication and on microfilm and electronically: Chair University of California, San Diego 2016 iii TABLE OF CONTENTS Signature Page .……………………………………………………………………. iii Table of Contents …………………………………………………………………. iv List of Figures ………………………………………………………..…….............v List of Tables ………………………………………………………………………vii Acknowledgements ………………………………………………………………...viii Vita …………………………………………………………………………..…….. ix Abstract of the Thesis ……………………………………………………………... x Introduction ………………………………………………………………………...1 Project Design and Rationale……………………………………………….…........ 8 Chapter 1: The recognition of unnatural nucleoside triphosphates by RNA Pol II………………………………………………..………….10 Chapter 2: RNA Pol II transcriptional arrest and bypass of regioisomeric ethylated thymidine lesions …………………………...... 30 Chapter 3: RNA Pol II transcriptional arrest by non-covalent minor groove binders: Py-Im polyamides ……………………………... 40 Summary & Future Implications …………………………………………………...49 References …………………………………………………………………………. 53 iv LIST OF FIGURES Figure i. Structure of RNA polymerase II elongation complex.……………...…… 2 Figure
    [Show full text]
  • De Novo Nucleic Acids: a Review of Synthetic Alternatives to DNA and RNA That Could Act As † Bio-Information Storage Molecules
    life Review De Novo Nucleic Acids: A Review of Synthetic Alternatives to DNA and RNA That Could Act as y Bio-Information Storage Molecules Kevin G Devine 1 and Sohan Jheeta 2,* 1 School of Human Sciences, London Metropolitan University, 166-220 Holloway Rd, London N7 8BD, UK; [email protected] 2 Network of Researchers on the Chemical Evolution of Life (NoR CEL), Leeds LS7 3RB, UK * Correspondence: [email protected] This paper is dedicated to Professor Colin B Reese, Daniell Professor of Chemistry, Kings College London, y on the occasion of his 90th Birthday. Received: 17 November 2020; Accepted: 9 December 2020; Published: 11 December 2020 Abstract: Modern terran life uses several essential biopolymers like nucleic acids, proteins and polysaccharides. The nucleic acids, DNA and RNA are arguably life’s most important, acting as the stores and translators of genetic information contained in their base sequences, which ultimately manifest themselves in the amino acid sequences of proteins. But just what is it about their structures; an aromatic heterocyclic base appended to a (five-atom ring) sugar-phosphate backbone that enables them to carry out these functions with such high fidelity? In the past three decades, leading chemists have created in their laboratories synthetic analogues of nucleic acids which differ from their natural counterparts in three key areas as follows: (a) replacement of the phosphate moiety with an uncharged analogue, (b) replacement of the pentose sugars ribose and deoxyribose with alternative acyclic, pentose and hexose derivatives and, finally, (c) replacement of the two heterocyclic base pairs adenine/thymine and guanine/cytosine with non-standard analogues that obey the Watson–Crick pairing rules.
    [Show full text]
  • Direct Detection of Unnatural DNA Nucleotides Dnam and D5sics Using the Mspa Nanopore
    RESEARCH ARTICLE Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore Jonathan M. Craig, Andrew H. Laszlo, Ian M. Derrington, Brian C. Ross, Henry Brinkerhoff, Ian C. Nova, Kenji Doering, Benjamin I. Tickman, Mark T. Svet, Jens H. Gundlach* Department of Physics, University of Washington, Seattle, Washington, United States of America * [email protected] Abstract Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here OPEN ACCESS we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore Citation: Craig JM, Laszlo AH, Derrington IM, Ross sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and BC, Brinkerhoff H, Nova IC, et al. (2015) Direct Hel308 helicase are capable of controlling the motion of DNA containing dNaM and d5SICS Detection of Unnatural DNA Nucleotides dNaM and through the pore and that single reads are sufficient to detect the presence and location of d5SICS using the MspA Nanopore. PLoS ONE 10 (11): e0143253. doi:10.1371/journal.pone.0143253 dNaM and d5SICS within single molecules. Editor: Floyd Romesberg, The Scripps Research Institute, UNITED STATES Received: August 26, 2015 Accepted: November 2, 2015 Introduction Published: November 20, 2015 Nature uses the four natural nucleotides, A, C, G and T, to encode genetic information. How- Copyright: © 2015 Craig et al. This is an open ever, Escherichia coli was recently shown to be capable of propagating an expanded genetic access article distributed under the terms of the alphabet that includes the unnatural nucleotides dNaM and d5SICS (Fig 1A)[1].
    [Show full text]
  • A Semisynthetic Organism Engineered for the Stable Expansion of the Genetic Alphabet
    A semisynthetic organism engineered for the stable expansion of the genetic alphabet Yorke Zhanga,1, Brian M. Lamba,1, Aaron W. Feldmana, Anne Xiaozhou Zhoua, Thomas Lavergneb, Lingjun Lic, and Floyd E. Romesberga,2 aDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; bDépartement de Chimie Moléculaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5250, Université Grenoble Alpes, F-38000 Grenoble, France; and cSchool of Chemistry and Chemical Engineering, Henan Normal University, Henan 453007, People’s Republic of China Edited by Clyde A. Hutchison III, The J. Craig Venter Institute, San Diego, CA, and approved December 21, 2016 (received for review October 6, 2016) All natural organisms store genetic information in a four-letter, two- (dNaMTP and d5SICSTP), and provided with a plasmid-encoded base-pair genetic alphabet. The expansion of the genetic alphabet nucleoside triphosphate transporter (NTT2) from Phaeodactylum with two synthetic unnatural nucleotides that selectively pair to tricornutum (which we denote as PtNTT2)(13),isabletoimport form an unnatural base pair (UBP) would increase the information the unnatural triphosphates and replicate a single dNaM-d5SICS storage potential of DNA, and semisynthetic organisms (SSOs) that UBP on a second plasmid (14). stably harbor this expanded alphabet would thereby have the Although this first SSO represented an important proof-of- potential to store and retrieve increased information. Toward this concept, the generality of the expanded genetic alphabet remained goal, we previously reported that Escherichia coli grown in the unclear, as retention of the UBP was explored at only a single locus presence of the unnatural nucleoside triphosphates dNaMTP and and in only a single sequence context.
    [Show full text]
  • 12.12.2017 1 CHAPTER 1 OLIGONUCLEOTIDES Part 2
    12.12.2017 Canonical nucleobase pairing and alternative base pairs CHAPTER 1 OLIGONUCLEOTIDES Part 2 – noncanonical nucleobases Natural and non-natural base pairs that function in polymerase reactions Self-assembly of whole genes and DNA nanostructures The technology tested by assembly of the kanamycin-resistance gene and growing the bacteria in the environment containing kanamycin after assembly and conversion of that gene. S. Benner et al., Beilstein J. Org. Chem. 2014, 10, 2348–2360. doi:10.3762/bjoc.10.245 1 12.12.2017 AEGIS – Artificially Expanded Genetic Information System AEGIS – Artificially Expanded Genetic Information System ZP CG S. Benner et al., Beilstein J. Org. Chem. 2014, 10, 2348–2360. doi:10.3762/bjoc.10.245 S. Benner et al., J. Am. Chem. Soc., 2011 , 133 (38), pp 15105–15112 AEGIS – Permanent orthogonal nucleobases surviving PCR ACGTZP-DNA crystal structures Electron density presented to the minor groove recognition site by polymerases „ minor groove scanning hypothesis” Error rate 0,2% per a PCR cycle – both removal and incorporation of Z and P the artificial genetic system capable to evolve. Pol: Deep Vent – 2 Z/P, Taq/Phu – 3-4 Z/P 18-mers: 2+2 Z:P pairs B-DNA dZTP ( deprotonated ) at higher pH pairs slightly with G 6 consecutive Z:P A-DNA loss of some Z, but also gain of some new Z mutants. 0,1 nm wider, but otherwise alike G:C pairs S. Benner et al., J. Am. Chem. Soc., 2011 , 133 (38), pp 15105–15112 S. Benner et al., J. Am. Chem. Soc., 2015, 137, pp 6947–6955 2 12.12.2017 Unnatural aminoacid incorporation using a noncanonical base pair (A) The coupled transcription–translation system using the nonstandard codon– anticodon interaction for the site-specific incorporation of 3-chlorotyrosine into the Ras protein.
    [Show full text]
  • Duplex Stability Introduction
    Duplex Stability Introduction Many molecular biology applications require oligonucleotide primers and/or probes with enhanced duplex stability, that is, a higher affinity for their complementary sequences. Examples include antisense, siRNA, and SNP detection experiments. For these and other applications, enhancing primer/probe-target duplex stability increases both the selectivity and sensitivity of the oligo for its target. An oligo's binding affinity for its target can be manipulated by incorporating various modifications into it, either on the ends or at an internal position (1). Examples include 2'-OMethyl base, 2-Amino-dA, 5-methyl-dC and 2'-Fluoro bases. Changes in duplex stability incurring from such modifications is indicated by changes in the melting temperature (Tm) of the duplex formed between the modified oligo and its target over that formed by the unmodified oligo. Depending on the modification, Tm can be increased over a range of 0.5C to 3.0C per base substitution. 8 Westchester Plaza, Suite 130, Elmsford, NY 10523 | Tel: 914-769-1192 | Fax: 914-769-1193 | www.genelink.com | Page 1 Duplex Stability Design Protocols Modulating Oligonucleotide Duplex Stability--Design Considerations. Specific and stable hybridization of an oligo to its complementary sequence is the desired outcome of a successful hybridization protocol. The melting temperature (Tm) of an oligo indicates the strength of the affinity, and thus the stability, of the hybridization. Manipulation of the oligo sequence to increase its duplex stability or, in some cases, to decrease the duplex stability in certain loop structures will lead to oligos with increased affinity for the target molecule. Duplex stability can be modulation by incorporation of modifications that are altered on the nucleotide base (e.g., 5-methyl-dC) or sugar (e.g., 2’-F or 2’-O-Methyl RNA bases).
    [Show full text]
  • Efficient and Sequence-Independent Replication of DNA Containing A
    Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet Denis A. Malysheva, Kirandeep Dhamia, Henry T. Quacha, Thomas Lavergnea, Phillip Ordoukhanianb, Ali Torkamanic, and Floyd E. Romesberga,1 aDepartment of Chemistry, bCenter for Protein and Nucleic Acid Research, and cThe Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037 Edited* by Jack W. Szostak, The Howard Hughes Medical Institute and Massachusetts General Hospital, Boston, MA, and approved June 6, 2012 (received for review March 27, 2012) The natural four-letter genetic alphabet, comprised of just two were hindered by low-fidelity replication and chemical instability base pairs (dA-dT and dG-dC), is conserved throughout all life, and (23), but modifications have been found more recently that its expansion by the development of a third, unnatural base pair overcome these limitations, and DNA containing such unnatural has emerged as a central goal of chemical and synthetic biology. pairs has been amplified by PCR (24). The amplification of DNA We recently developed a class of candidate unnatural base pairs, containing multiple, contiguous unnatural base pairs has also exemplified by the pair formed between d5SICS and dNaM. Here, been shown (10), but the more general effects of sequence context we examine the PCR amplification of DNA containing one or more have yet to be reported. The second strategy, originally pursued d5SICS-dNaM pairs in a wide variety of sequence contexts. Under by our group (25), and in part inspired by the observation in the standard conditions, we show that this DNA may be amplified work by Kool and coworkers (26) that H-bonds are not absolutely with high efficiency and greater than 99.9% fidelity.
    [Show full text]
  • MOLECULAR DYNAMICS STUDY of NON-HYDROGEN-BONDING BASE-PAIR DNA DUPLEX D(GTCD NAM GCGCCGTGGC)
    International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 10, Issue 7, 2018 Original Article MOLECULAR DYNAMICS STUDY OF NON-HYDROGEN-BONDING BASE-PAIR DNA DUPLEX d(GTCD NAM GCGCCGTGGC). d(GCCACGGCGCD 5SICS GAC) RAKESH KUMAR TIWARI DDU Gorakhpur University, Gorakhpur Email: [email protected] Received: 18 Jan 2018 Revised and Accepted: 15 Jun 2018 ABSTRACT Objective: The objective of the study was to elucidate the structural activity of a natural DNA sequence modified by a hydrophobic base-pair which didn’t form Watson-Crick (W-C) hydrogen-bond. The modified unnatural base-pair (DNAM-D5SICS) was introduced in DNA sequences of 14-mer for molecular dynamics study in water solution. Methods: A 200 ns molecular-dynamics-simulation in orthogonal-box-water-solvent, using Particle-mesh-Ewald-method (PME) within periodic- boundary-condition (PBC) was performed by using AMBER-14 code. The force-field-ff12SB-force-field was used during the simulation while the force-field-parameters of modified base-pair, compatible to ff12SB-force-field, were calculated by Gaussian-09-code using ab-inito /Hartree-Fock- methodology. The code CPPTRAJ, (a module of AMBER-14) CURVE and Chimera were used in the analysis of the data. Results: Root mean square deviation (RMSD) of heavy atoms of the trajectory revealed that the structure of the equilibrated duplex was stable, sequence-dependent and had mixed DNA-conformation. A little distortion near to the neighbor of the modified base-pair in the duplex strand was observed. However, we got a stabilized structure of such type of duplex if we placed modified base-pair after the third place in the strand.
    [Show full text]
  • Hydrophobic Unnatural Base Pairs and the Expansion of the Genetic Alphabet Aaron W
    LETTER LETTER REPLY TO HETTINGER: Hydrophobic unnatural base pairs and the expansion of the genetic alphabet Aaron W. Feldmana, Michael P. Ledbettera, Yorke Zhanga, and Floyd E. Romesberga,1 We have recently reported the successful creation of a inversion turns out to be more significant than the ki- semisynthetic organism that has an expanded genetic netic data predict, once translation is established selec- alphabet by virtue of the retention on a plasmid of either tion pressure may be used to prevent inversion, just as the dNaM-d5SICS or dNaM-dTPT3 unnatural base pair deleterious natural base mutations are eliminated by (UBP), the latter of which can be retained at natural-like negative selection. Alternatively, if the default condi- levels with the use of a Cas9 editing system (1, 2). De- tion extends to transcription and translation, inversion spite these demonstrations that a cell can retain a UBP in would be eliminated as an issue, even if it occurs, be- its DNA, Hettinger (3) now concludes that such hydro- cause either nucleotide in a codon would selectively phobic UBPs are not suitable for the expansion of the pair with either nucleotide in the anticodon, but neither genetic alphabet. Hettinger cites Hirao and Kimoto (4), would pair with a natural nucleotide. who note that self-pairing may be a problem inherent to Hettinger (3) also suggests that sequences with hydrophobic UBPs, and Hettinger (3) refers to their mode consecutive UBPs are too destabilizing, with the ob- of pairing as a “default condition,” meaning they simply jection presumably being that they will not be repli- exclude pairing with hydrophilic, natural nucleotides, cated.
    [Show full text]
  • Synthetic Fictions: Turning Imagined Biological Systems Into Concrete Ones
    Synthese https://doi.org/10.1007/s11229-020-02567-6 UNREALISTIC MODELS Synthetic fictions: turning imagined biological systems into concrete ones Tarja Knuuttila1 · Rami Koskinen1 Received: 28 February 2019 / Accepted: 3 February 2020 © The Author(s) 2020 Abstract The recent discussion of fictional models has focused on imagination, implicitly con- sidering fictions as something nonconcrete. We present two cases from synthetic biology that can be viewed as concrete fictions. Both minimal cells and alternative genetic systems are modal in nature: they, as well as their abstract cousins, can be used to study unactualized possibilia. We approach these synthetic constructs through Vai- hinger’s notion of a semi-fiction and Goodman’s notion of semifactuality. Our study highlights the relative existence of such concrete fictions. Before their realizations nei- ther minimal cells nor alternative genetic systems were any well-defined objects, and the subsequent experimental work has given more content to these originally schematic imaginings. But it is as yet unclear whether individual members of these heteroge- neous groups of somewhat functional synthetic constructs will eventually turn out to be fully realizable, remain only partially realizable, or prove outright impossible. Keywords Fiction · Models · Synthetic biology · Modality · Semifactuality · Unrealisticness 1 Introduction Scientific models are unrealistic in various ways. The epistemic status of these sim- plified and highly idealized, and thus strictly speaking false depictions of reality,
    [Show full text]