Evolution of Novel Biopolymers

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

Every organism’s blueprint is stored as genetic information encoded in DNA. Before a cell can divide, its genetic information needs to be copied and duplicated. This process is carried out by sophisticated enzymes called polymerases. The astonishing ability of polymerases to both “read” and “write” genetic information is vital to all life on earth and central to modern biology and medicine enabling genome sequencing and the polymerase chain reaction. Our work is aimed at the engineering of novel polymerases capable of activity in unfavourable conditions, and utilising unnatural building blocks. Such "designer" polymerases will be key enabling technologies for the generation of next-generation nucleic acid drugs, sensors and nanomaterials.

Technical Summary

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction (i.e. resynthesis) rather than deconstruction (analysis). Synthesis thus complements reductionist and analytic studies of life, and allows novel approaches towards fundamental biological questions.

We have been exploiting the synthesis paradigm to explore the chemical etiology of the genetic apparatus shared by all life on earth. Specifically, we ask why information storage and propagation in biological systems is based on just two types of nucleic acids, DNA and RNA. Is the chemistry of life’s genetic system based on chance or necessity? Does it reflect a "frozen accident", imposed at the origin of life, or are DNA and RNA functionally superior to simple alternatives.

We have developed advanced generation selection strategies, that allow the evolution of polymerases capable of utilizing exclusively unnatural nucleotide substrates enabling the enzymatic synthesis, replication and evolution of entirely synthetic genetic polymers (XNA). Such systems expand the central dogma and will conclusively address questions such as the capacity of genetic polymers other than DNA and RNA for information storage and propagation (i.e. heredity). Beyond heredity, we have demonstrated a capacity for Darwinian evolution by the de novo selection of specific ligands (XNA aptamers) and catalysts (XNAzymes) based on these entirely synthetic backbones. Thus, key hallmarks of living systems, including heredity and evolution are not limited to DNA and RNA but can be implemented in synthetic polymers and are likely to be emergent properties of polymers capable of information storage.

The further exploration of the informational, structural and catalytic potential of synthetic genetic polymers, should advance our understanding of the parameters of chemical information encoding, and will provide a source of novel ligands, catalysts and nanostructures with tailor-made chemistries for applications in biotechnology and medicine.

We have also leveraged our selection technology to make progress in the engineering and evolution of RNA polymerase ribozymes towards a general polymerase and self-replication capacity We have discovered RNA polymerase ribozymes that are capable of the templated synthesis (i.e. transcription) of another simple ribozyme or RNA oligomers exceeding their own size (>200 nts), a key milestone on the road to self-replication.

Publications

10 25 50
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Pinheiro VB (2012) The XNA world: progress towards replication and evolution of synthetic genetic polymers. in Current opinion in chemical biology

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Pinheiro VB (2012) Synthetic genetic polymers capable of heredity and evolution. in Science (New York, N.Y.)

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Pinheiro VB (2013) Synthetic polymers and their potential as genetic materials. in BioEssays : news and reviews in molecular, cellular and developmental biology

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Pinheiro VB (2014) Compartmentalized Self-Tagging for In Vitro-Directed Evolution of XNA Polymerases. in Current protocols in nucleic acid chemistry

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Ramsay N (2010) CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase. in Journal of the American Chemical Society

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Smith DA (2012) Reversible fluorescence photoswitching in DNA. in The journal of physical chemistry. B

 
Description Royal Society Policy seminar on "Synthetic Biology"
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Royal Society of Chemistry Replicating and Learning from Nature Exploration Workshop
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description 14-ERASynBio INTENSIFYBBSRC
Amount £287,638 (GBP)
Funding ID BB/M005623/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2014 
End 07/2017
 
Description Carlsberg Foundation
Amount £50,000 (GBP)
Organisation Carlsberg Foundation 
Sector Academic/University
Country Denmark
Start 10/2018 
End 09/2019
 
Description DFG Fellowship (Baar)
Amount £48,000 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 01/2007 
End 12/2008
 
Description DFG, Fellowship
Amount £37,805 (GBP)
Organisation German Research Foundation 
Sector Public
Country Germany
Start 11/2004 
End 01/2006
 
Description ERASynBio2 - Orthogonal biosystems based on phosphonate XNAs
Amount £250,283 (GBP)
Funding ID UCL Ref 168510 BB/N01023X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 04/2019
 
Description EuroSynBio - SYNAPTA
Amount £334,000 (GBP)
Funding ID BB/I004793/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2010 
End 04/2013
 
Description FEBS Fellowship
Amount £78,000 (GBP)
Organisation Federation of European Biochemical Societies (FEBS) 
Sector Charity/Non Profit
Country European Union (EU)
Start 06/2012 
End 06/2015
 
Description FP6 NEST Pathfinder
Amount £403,381 (GBP)
Organisation Sixth Framework Programme (FP6) 
Sector Public
Country European Union (EU)
Start 05/2006 
End 07/2009
 
Description HFSP Fellowship
Amount £93,780 (GBP)
Funding ID LT000045/2012 
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 01/2013 
End 12/2015
 
Description Herchel Smith Studentship Christopher Wan
Amount £180,000 (GBP)
Organisation University of Cambridge 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2021
 
Description Junior Research Fellowship
Amount £55,000 (GBP)
Organisation University of Cambridge 
Sector Academic/University
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description MRC/AZ CRISPR-X: Leveraging XNA technology for advanced genome editing
Amount £206,423 (GBP)
Funding ID BSF35 
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 05/2019 
End 04/2020
 
Description MRC/AZ Deep screening: Leveraging NextGen sequencing technology for a quantum leap in ligand discovery
Amount £263,000 (GBP)
Funding ID BSF24 
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 04/2017 
End 03/2019
 
Description MRC/AZ Explore XNA Aptamer technology for in vivo selection of targeting moieties and oligonucleotide delivery
Amount £215,642 (GBP)
Funding ID BSF01 
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 06/2015 
End 06/2019
 
Description MRC/AZ In Cellulo target discovery using XNA technology
Amount £305,948 (GBP)
Funding ID BSF11 
Organisation AstraZeneca 
Sector Private
Country United Kingdom
Start 01/2016 
End 01/2019
 
Description MRCT DGF Project Grant
Amount £110,000 (GBP)
Funding ID A853-0137 
Organisation MRC-Technology 
Sector Academic/University
Country United Kingdom
Start 09/2011 
End 09/2014
 
Description NSERC Canada fellowship
Amount £40,000 (GBP)
Organisation Natural Sciences and Engineering Research Council of Canada (NSERC) 
Sector Public
Country Canada
Start 10/2007 
End 12/2008
 
Description Simons Collaboration on the Origins of Life Postdoctoral Fellowship
Amount $243,000 (USD)
Funding ID 293387 
Organisation Simons Foundation 
Sector Charity/Non Profit
Country United States
Start 11/2013 
End 10/2016
 
Description US Gates - Duffy
Amount £44,700 (GBP)
Organisation Higher Education Funding Council for England 
Department Gates Cambridge Scholarship
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Wellcome Trust Internship
Amount £1,360 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2006 
End 08/2006
 
Title CSR 
Description polymerases with expanded substrate spectrum 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact licensing deals with New England Biolabs, Human Genetic Signatures 
 
Title CST 
Description novel polymerases capable of utilizing entirely unnatural orthogonal substrates 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact improved tools for e.g. generation of nucleic acid therapeutics 
 
Description CryoEM imaging of XNA nanotechnology objects 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact first structures of XNA nano-object
 
Description CyDNA in super-resolution microscopy 
Organisation University of Edinburgh
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact publication: 22861666 (August, 2012) Reversible Fluorescence Photoswitching in DNA.
 
Description DNA/XNA origami nanopores 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description Deep Screening 
Organisation ETH Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description Hydrophobic base analogues 
Organisation Stanford University
Department Department of Chemistry
Country United States 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description In vivo target discovery 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description LNA and UNA triphosphates as polymerase substrates 
Organisation University of Southern Denmark
Country Denmark 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description Mutant Polymerases 
Organisation Agilent Technologies
Country United States 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Mutant Polymerases 
Organisation Exiqon
Country Denmark 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Mutant Polymerases 
Organisation Helicos Biosciences
Country United States 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Mutant Polymerases 
Organisation Life Technologies
Department Applied Biosystems
Country United States 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Mutant Polymerases 
Organisation Life Technologies
Department Invitrogen
Country United States 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Mutant Polymerases 
Organisation New England Biolabs
Country United States 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Mutant Polymerases 
Organisation Solexa
Country United States 
Sector Private 
PI Contribution generation of novel polymerases with potential biotechnological impact
Collaborator Contribution non-exclusive license for both polymerases and CSR selection technology potential for collaboration
Impact generation of licensing revenue for MRC
 
Description Oligonucleotide arrays 
Organisation Agilent Technologies
Country United States 
Sector Private 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description Phage Display 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution consulting for GSK/ Domantis dispute with AbLynx
Collaborator Contribution GSK/ Domantis dispute with AbLynx
Impact contribution to settlement
Start Year 2008
 
Description Polymerase Evolution 
Organisation Domantis Ltd
Country United Kingdom 
Sector Private 
PI Contribution development of novel directed evolution methodologies for proteins and in particular polymerases
Collaborator Contribution Collaboration agreement with Domantis funded 2 postdoctoral scientists in my group
Impact funding of postdoctoral researchers, cost of patent prosecution, consumables; Collaboration agreement with Domantis funded 2 postdoctoral scientists in my group
 
Description Polymerases for Bisulfite Sequencing 
Organisation Human Genetic Signatures
Country Australia 
Sector Private 
PI Contribution evaluation agreement
Collaborator Contribution evaluation agreement
Impact evaluation agreement
Start Year 2008
 
Description SHAPE mapping of XNAs and RNAs 
Organisation University of Notre Dame
Department Department of Chemistry and Biochemistry
Country United States 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact publication: 25470036 (February, 2015) Catalysts from synthetic genetic polymers.
 
Description Structural studies of XNA polymerases. 
Organisation University of Konstanz
Department Department of Chemistry
Country Germany 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact 23940661 (, 2013) Structures of an apo and a binary complex of an evolved archeal B family DN...
 
Description TNA triphosphates as polymerase substrates. 
Organisation Arizona State University
Department School of Molecular Sciences
Country United States 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description Translesion synthesis of XNA polymerases 
Organisation Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
Country United States 
Sector Public 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description X-ray crystallography of mutant DNA polymerases. 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description XNA nucleotide and solid-phase synthesis. 
Organisation University of Leuven
Department Rega Institute for Medical Research
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Description XNA side-chain modification chemistry 
Organisation University of Bonn
Country Germany 
Sector Academic/University 
PI Contribution Scientific
Collaborator Contribution Scientific
Impact N/A
 
Title Arabino-DNA polymerase 
Description polymerase for the synthesis of synthetic genetic polymers based on arabino-nucleotides 
IP Reference WO2013156786 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact none
 
Title Artificial genetic systems 
Description An artificial genetic system based on polymerase engineering enabling the discovery of novel biopolymer therapeutics 
IP Reference WO2011135280 
Protection Patent application published
Year Protection Granted 2011
Licensed No
Impact NA
 
Title CST Emulsions 
Description A novel method for the selection of nucleic acid processing and other enzymes and in particular polymerases 
IP Reference WO2005045072 
Protection Patent granted
Year Protection Granted
Licensed Yes
Impact A novel method for the selection of nucleic acid processing and other enzymes based on the compartmentalized self-tagging technique
 
Title CST2.0 
Description improved methodology for CST emulsions, filed as a continuation in part 
IP Reference US20100184071 
Protection Patent granted
Year Protection Granted 2010
Licensed No
Impact Significant improvement on CST (compartmentalised self tagging) method which is already patented and relates to screening of polymerases with specific properties. The method allows for selection of only true positives which was frequently not the case with the original CST.
 
Title Directed Evolution Method 
Description directed evolution method based on the novel principle of compartmentalized self-replication 
IP Reference WO2002022869 
Protection Patent granted
Year Protection Granted 2007
Licensed Yes
Impact Method to select for proteins with altered characteristics. The protein of interest is randomly mutated and variants of it undergo selection based on presence or absence of nucleic acid polymerase activity.
 
Title E10 polymerase 
Description A polymerase for the high-density incorporation of fluorescent-dye labeled nucleotides into DNA 
IP Reference US2010035767 
Protection Patent granted
Year Protection Granted 2010
Licensed Yes
Impact improved microarray protocols, novel diagnostics
 
Title Emulsion Compositions 
Description novel inert oxidant-free emulsion formulation 
IP Reference US2008187968 
Protection Patent granted
Year Protection Granted 2008
Licensed Yes
Impact Invention describes improved chemical composition of two phase emulsions. Such emulsions are of use for large scale screening
 
Title Hu1 polymerase 
Description polymerases with a resitance to common environmental inhibitors e.g. humic acid 
IP Reference US2009305345 
Protection Patent granted
Year Protection Granted 2009
Licensed Yes
Impact improved diagnostic & forensic PCR assays, ancient DNA research
 
Title M4 Emulsions 
Description polymerases with increased ability for mismatch extension, enhanced processivity and expanded substrate spectrum 
IP Reference AU2010200031 
Protection Patent granted
Year Protection Granted 2010
Licensed Yes
Impact Method for the generation of DNA polymerases which exhibit relaxed substrate specificity
 
Description BBC Discovery program, World service 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Radio interview

NA
Year(s) Of Engagement Activity 2010
 
Description BBC4 radio program 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Radio science program

NA
Year(s) Of Engagement Activity 2010
 
Description Cambridge Science Society talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Gave talk at Cambridge Scientific Society with more than 1000 permanent members (and a much larger email database). The Society is a prestigious platform for the first public exposure of new research and findings. Past speakers include Nobel Laureates James Watson, Sir Tim Hunt and Sir John Walker, eminent scientists Sir Roger Penrose, Sir Richard Friend, Sir John Beddington and Peter Atkins, popular author Matt Ridley, and former Universities and Sciences minister David Willetts among many others.

Tlk was well received by ca. 100 attendees and prompted many discussions and online Tweets.
Year(s) Of Engagement Activity 2016
URL http://www.scisoc.com/talks/
 
Description New polymerases for old DNA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact talk on "Second Nature", Nature publishings virtual web conference site in Second Life

follow up questions from public by e-mail
Year(s) Of Engagement Activity 2008
 
Description Orthosome 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact describe FP6 funded project : orthosome on website

feedback on project, attract students
Year(s) Of Engagement Activity 2007
 
Description Press article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact phone interview for Guardian article "Biologists join the race to create synthetic life"

article
Year(s) Of Engagement Activity 2008
 
Description Public lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact New Scientist Instant Expert: Origins and Evolution of Life event at The Royal College of General Practitioners
Year(s) Of Engagement Activity 2017
URL https://www.nature.com/natureevents/science/events/58969-New_Scientist_Instant_Expert_The_Origins_of...
 
Description RSC Policy seminar 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact seminar to public / policymakers about impact and future of synthetic biology

interviews with journalists e.g. Today program, Guardian science podcast etc.
Year(s) Of Engagement Activity 2008
 
Description RSC discussion meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact RSC 2 day discussion meeting for ca. 120 leading academics, policymakers and other stakeholders

RSC published summary booklet
Year(s) Of Engagement Activity 2008
 
Description Radio interview (Deutschlandfunk 2013) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact radio interview
Year(s) Of Engagement Activity 2013
URL http://www.dradio.de/dlf/sendungen/forschak/2171572/
 
Description Royal Society Summer Science Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Members of my lab were involved in presenting a stand at the Royal Society Summer Science Exhibition
Year(s) Of Engagement Activity 2019
 
Description Talk , LMB Alumni symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact talk sparked question and discussions afterwards

increased interest in my research
Year(s) Of Engagement Activity 2014
URL http://www2.mrc-lmb.cam.ac.uk/alumnisymposium/programme.php
 
Description Talk for non-scientific staff 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact talk sparked questions and discussion afterwards

better understanding of my research by support staff
Year(s) Of Engagement Activity 2014