14-ERASynBio: BioMolecular Origami

Lead Research Organisation: University of Bristol
Department Name: Chemistry

Abstract

The overall objective of synthetic biology is to make the engineering of biological systems easier, more predictable, and, ultimately, applicable to real-life applications. Molecular structures assembled from biopolymers, such as proteins and nucleic acids represent the basic functional units crucial for all branches of synthetic biology. Whilst, many approaches in this field use "plug-and-play" strategies to engineer naturally evolved biological parts (primarily genes and functional protein domains), the grand challenge of synthetic biology is not only to combine the existing natural structures but to assemble de novo molecular structures unseen in nature that could embody new functions and be produced sustainably for different applications. To realize this challenge we need to develop the fundamental tools to program the sequences of nucleic acids and polypeptides to to control self-assembly into defined three-dimensional (3D) nanostructures. The primary objectives of this proposal are to develop an understanding of these basic processes and tools to apply it. We will concentrate on the most versatile biomolecules, polypeptides, which are also the most difficult to control. We will test and demonstrate tools for the design and engineering of new polypeptide and nucleic acid-polypeptide hybrid systems. This will pave the way to unprecedented control over the construction of, and applications for, biomolecule-based nanostructures and materials.

Biopolymers can self-assemble into complex structures defined at the nanometer scale. There is also a considerable potential for their sustainable large-scale production in cell factories. Both properties make them highly desirable for diverse technological applications. More specifically, proteins provide masterful examples of complex self-assembling nanostructures that have versatile functionalities beyond the reach of any manmade materials, including catalysis, molecular recognition, assembly of cellular scaffolds and many others[1]. However, our ability to engineer native protein-like structure and function de novo, although improving, is limited. In contrast, in recent decades, DNA has been spectacularly repurposed by bioengineers to form designed structures based on complementary base pairing. Now, we can design nucleotide sequences to form almost any 2D or 3D nanoscale structures, from boxes to spheres, with a feature resolution of few nanometers. Although engineered nucleic acid-based nanostructures have been functionalized via chemical modifications,[2] compared to proteins their range of functionalities is extremely limited. This proposal aims to combine the advantages of polypeptide and nucleic-acid systems and circumvent the limits of both. We have now reached the threshold of considerable advances in synthetic biology to harvest the potentials of polypeptide-based design of nano-scale structures. In order to achieve this goal, this project aims to translate concepts and technologies between different subfields of synthetic biology, combining methods of structural biology, DNA nanotechnology, mathematics and large scale gene synthesis.

The BioOrigami consortium comprises seven groups that are pioneers in molecular synthetic biology based on nucleic acids, peptides and proteins. We plan to progress the design of protein structures beyond folds present in nature and bridge the gap between the elegant, but largely non-functional self-assembled nucleic-acid nanostructures and the exquisite functionality and scalability of protein assemblies. This project is supported by the power of large-scale gene synthesis and screening.

Technical Summary

Biological organisms are capable of producing chemicals, materials and molecular machines that far exceed our engineering capabilities. Underlying these abilities are the unique properties of proteins, exquisitely evolved for function, allowing precise positioning of atoms and chemistries. Designing novel proteins is difficult because of our still incomplete understanding of how proteins fold for a given primary amino-acid sequence. Here we apply principles of synthetic biology to define and modularize building blocks that can be combined in rational ways to give us control of 3D positioning in designed macromolecular structure. Members of the consortium have advanced design and engineering principles for polypeptide- and DNA-based nanostructures (Goodman et al. Science 2005, King et al. Science 2012, Sobczak et al. Science 2012, Fletcher et al. Science 2013, Gradisar et al. Nat Chem Biol 2013), and developed next-generation gene synthesis to facilitate high-throughput approaches (Kosuri et al. PNAS 2013). We will build on these foundations to engineer bio-macromolecular assemblies with shapes and functions of unprecedented complexity. This approach of structural synthetic biology represents a leap from traditional protein engineering, analogous to how engineering of organisms by synthetic biology extends traditional genetic engineering. Our approach borrows and learns from the DNA assembly field, bringing in the advantages of the wider chemical functionalities and potential for industrial-level production of polypeptides. We will deliver an expanded toolbox of polypeptide building elements; rules, design principles and methods for constructing complex bionanostructures; and routes to nucleic acid/ polypeptide-hybrid platforms for the community of synthetic biology. The project will expand the limits of the designed polypeptide and nucleic acid/protein hybrid providing a platform to facilitate their use in a wide range of healthcare and industrial applications.

Planned Impact

5.1. Impact on scientific advances
a. Conceptual advances in new types of designed bionanostructures
b. Development of an extended toolbox for nanostructure self-assembly available to the synthetic-biology community
c. Generation of new tools for the design of biomolecular self-assembly

5.2. Potentials for economic applications
The developed designed nanostructures will represent scaffolds for hosting different functionalities with nanoscale resolution. A characteristic feature of the developed designed polypeptide assemblies (WP2) in contrast to most native proteins is that they enclose the internal cavity of defined size that is amenable to engineering. Design of polypeptide vertices will provide tools for regulated assembly/ disassembly with straightforward applications for encapsulation and delivery of cargo, such as drugs or reaction products or engineering of catalytic centers with applications in health and biosynthesis. Expansion of the toolbox of orthogonal building elements (WP1) will allow engineering of cells for the formation of specific interactions for the formation of microtissues for cell-based therapy. Introduction of folded domains into vertices (WP2) will provide the foundation of designed vaccines, while DNA-protein hybrids (WP4) have interesting potentials for nanoscale sensors, multi-enzymatic catalytic platforms and compartments and also for biomineralization and interface with electronic elements. Development of pores from DNA-peptide hybrids in WP4 opens exciting potentials for sensing, therapy, DNA sequencing. Designed nanostructures will also provide a powerful scientific tool as structural elements in actuators, sensors and support structure determination.

5.3. Sustainable production technology
Designed peptide- and nucleotide-based nanostructures have been mainly produced using chemically synthesized building blocks. This project will support the shift of the polypeptide and DNA-based nanotechnology from chemical synthesis towards biotechnological production, which is a very efficient and sustainable production technology. Application of biotechnological production techniques will also move the current boundary of investigated structures beyond the limitations of chemical synthesis in terms of complexity, cost, environmental impact and sustainability.

5.4. Increasing cooperation and training - the need for the international perspective
The underlying interdisciplinarity of the problem requires a transnational interdisciplinary consortium which could not have been achieved at the national level. The project consortium represents strengthens the cohesion and critical mass of competences in the rapidly developing area of structural synthetic biology. The project will contribute to the training of new generations of structural synthetic biologists. Dissemination will include exchange training of junior researchers through scientific exchange visits. The project will support scientific training of >10PhD students and postdoctoral researchers.

5.5. Dissemination and exploitation of results, data sharing and management of IP
The project will disseminate results through scientific publications, exchange scientific visits between partners intended primarily for junior researchers, presentation at scientific conferences and to the general public. The bulk of results of the project will be made available to the general scientific community through publications in international peer-reviewed high-ranking journals, public lectures and participation at to international meetings (at least 8 annual presentations at international conferences and 5 publications in high-ranked journals). Consortium will organize an international conference on the advances in structural synthetic biology at the end of the project, aiming at disseminating results to scientists, industrial and medical users of technology and other stakeholders. Synthetic biology will also be promoted to the general public.

Publications

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Baker EG (2017) Miniprotein Design: Past, Present, and Prospects. in Accounts of chemical research

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Batchelor M (2019) Dynamic ion pair behavior stabilizes single a-helices in proteins. in The Journal of biological chemistry

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Wood CW (2018) CCBuilder 2.0: Powerful and accessible coiled-coil modeling. in Protein science : a publication of the Protein Society

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Woolfson DN (2017) How do miniproteins fold? in Science (New York, N.Y.)

 
Description BioMolecular Origami: Progress Against University of Bristol and Oxford Objectives

The stated aims for this ERASynBio grant are listed below in blue, our progress against these is given below in black, and then our plans for the next 6 - 12 months are listed in red.

Objectives of WP1 (Bristol):
• Develop a platform for coiled-coil design, CC-Builder, using Crick parameterization; combinatorial sequence searching and Rosetta modeling and scoring;
• Use CC-Builder to design parallel and antiparallel orthogonal heterodimers;
• *Design and screen an extensive synthetic library of coiled-coil building elements using next-generation DNA synthesis and screening.

WP1 Deliverables
D1.1: Consortium (M12) and publicly (M24) available CC-Builder program;
D1.2: First-generation designs of anti-parallel coiled coils (M12) for use in other WPs, and later publication (M24 - 36);
*D1.3: Report on the development of the NGB2H screening method (M12);
*D1.4: Validated combinatorial expression of orthogonal building elements (M24 - 36);

WP1 Milestones
M1.1: First-generation parallel and antiparallel dimers available to consortium (M06);
M1.2: Fully functional version of CC-Builder available to consortium (M12);
*M1.3: Next-generation DNA synthesis and screening of coiled-coil candidates (M12);
*M1.4: Second-generation parallel and antiparallel dimers available to consortium (M18).

*These are objectives for the UCLA and Ljubljana groups and are in progress there.

Progress at Bristol:
The majority of the Bristol-based objectives for this WP have been met. These cut across the whole consortium but mainly with the Oxford, UCLA, and Ljubljana collaborations. Specifically:

D.1.1 - CCBuilder has been improved, distributed to our collaborators, made available on the web, and published (Wood, CW. et al. (2014) Bioinformatics 30: 3029-3035.). This is being widely used by the community, and particularly by the Ljubljana group. Moreover, we have developed two other computational resources, CCScanner and ISAMBARD (DOI: 10.5281/zenodo.345289) for computational design. These are being used by us and by the Ljubljana group to design new sets of coiled coils for supramolecular assembly.

D.1.2 - New coiled-coil building blocks. We have generated in silico a large number of new designs for obligate coiled-coil heterodimers required by the UCLA and Ljubljana teams. These are now being used and tested by them in vivo and in supramolecular assemblies, respectively.

We have also generated an entirely new set of neutral-charged coiled-coil heterodimers needed for the work with Oxford (see below). This was unexpected and proved essential as some of our other heterodimers bound non-specifically to DNA thwarting attempts to make DNA-peptide conjugates.

The design and construction of antiparallel coiled coils has been more troublesome. However, aided by the ISAMBARD computational design suite, we now have several working examples that are fully characterised in solution and that we are taking into X-ray crystallography. Moreover, we have successfully developed completely orthogonal helix:helix interactions comprising an a-helix and a polyproline II helix in the antiparallel orientation. This is accepted for publication in Nature Chemical Biology (Baker, EG et al. (2017) Nature Chem Biol In Press.)

Plans for Bristol on WP1 for the next 6 - 12 month.
• Publish CCBuilder2.0/CCScanner and ISAMBARD; in preparation and submitted, respectively.
• Complete the biophysical characterization of "neutral coiled-coiled heterodimers dimers" and published with the Oxford group (see below).
• Complete the characterization of antiparallel coiled-coil motifs and distribution to the consortium.


Objectives of WP3 (Oxford lead, in collaboration with Bristol):
• Use DNA-peptide hybrids to drive and study peptide-peptide interactions;
• Create a new class of templated peptide nanostructure;
• Deliver functional assemblies that span membranes for applications in sensing.

WP3 Deliverables
D3.1: Publication of DNA-templated hexameric a-helical coiled-coil barrel (M14);
D3.2: Report on general design and engineering strategies for a-helical barrels directed by DNA templating (M30);
D3.3: DNA-peptide hybrid membrane nanopores (M36);

WP3 Milestones
M3.1: DNA-templated hexameric alpha helical coiled coil barrel (M10);
M3.2: Library of DNA-peptide conjugates (M14);
M3.3: Novel hexameric barrel (M18);
M3.4: DNA-hydrophobic peptide conjugates (M23);
M3.5: Electrophysiology measurements of DNA-templated peptide pore (M28);
M3.6: Demonstration of switchable pore (M36);

Progress at Oxford and Bristol
Since writing the ERASynBio proposal, we have discovered new a-helical barrels (Thomson, AR et al. (2014) Science 346: 485-488): this has allowed us to change and improve our plans for DNA-directed peptide assembly. However, attempts to couple some of the new barrel peptides to DNA oligonucleotides revealed technical problems that we had not envisaged, partly because there is no literature precedent for this type of work. This includes incompatibility of standard click chemistry with DNA-peptide conjugation, and promiscuous binding of cationic peptides to DNA. The two post-docs on the grant, Drs Emily Baker (Bristol) and Juan Jin (Oxford), worked extremely hard and well together to overcome these difficulties by developing methods for metal-free click reactions, and by designing and characterising an entirely new series of neutral heterodimeric coiled coils (see above). In addition, they have developed a completely unprecedented DNA-peptide hybrid system in which peptide interactions control the assembly of large DNA-origami nanostructures, allowing the sensitive quantification of peptide-peptide interactions using electron microscopy and fluorescence correlation microscopy. This work is being prepared for ACS Nano. They also have the other key peptide pieces in place for the following investigations.

(1) To study enzyme-like barrel assemblies, we have adapted our recently published peptides that make a homo-heptameric barrel incorporating 7 catalytic esterase triads (Burton et al. (2016) Nature Chemistry 8: 837-844). Emily and Juan have made shortened versions of these peptides that harbour the triad but do not assemble in water, and are, therefore, inactive. Our objective is to assemble these via new DNA templates, which they have designed, and to test whether the hybrids have catalytic activity. We will use the unique capacity of DNA-templated assembly to control the assembly of heteromeric systems to improve catalytic efficiency through control of peptide sequence in ways not possible in purely peptide systems.

(2) Similarly, we have recently made peptides that assemble to form ion-channel a-helical barrels in membranes (Mahendran et al. (2016) Nature Chemistry In press). However, these only insert and conduct under applied voltage, and we have no control over oligomeric state. We will use DNA templates similar to those developed for (1) to drive the assembly of membrane-spanning peptides that insert spontaneously into the membrane. These hybrids will be tested by electrophysiology experiments now available in Bristol and described in the Mahendran paper.

Plans for Oxford and Bristol on WP3 for the next 6 - 12 month.
• Publish our data showing that DNA-peptide hybrids can be made, assembled into DNA origamis, and used to direct the assembly of origamis (in preparation).
• Demonstrate that DNA templating can assemble catalytically active a-helical barrels.
• Alter the composition of templated peptide barrels to improve catalysis.
• Publish "DNA-templated catalytic a-helical barrels" in a high-profile journal.
• Initiate work on templating membrane-active a-helical barrels with DNA assemblies.
Exploitation Route We are already building new proteins and protein assemblies with the peptide modules that we have designed and characterised.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title CCBuilder 
Description A computational method and GUI for design and engineering of coiled-coil assemblies of all oligomer states. 
Type Of Technology Software 
Year Produced 2014 
Open Source License? Yes  
Impact Being used and adopted by national and international research groups. 
URL http://coiledcoils.chm.bris.ac.uk/app/cc_builder/
 
Title CCBuilder 2.0 
Description Builds protein models. Can be used easily and freely over the WWW and by non-experts. 
Type Of Technology Webtool/Application 
Year Produced 2017 
Impact Widely used by structural biologists and protein designers/engineers. 
URL http://coiledcoils.chm.bris.ac.uk/ccbuilder2/builder
 
Description Pint of Science Festival: Dark side of protein science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact As part of the Pint of Science Festival, researchers from BrisSynBio participated in the 'Dark Matters' event. The event was held in Friska café, Bristol, and involved scientific crafts and discussions between researchers and the public.
Director of BrisSynBio, Professor Dek Woolfson, along with Gail Bartlett, Jack Heal, Drew Thomson and Chris Wood organised the event 'Dark Matters'. Analogous to the idea of dark matter, protein science focuses on the protein structures that could theoretically exist but are not present in natural biology.
Year(s) Of Engagement Activity 2015
URL http://www.bristol.ac.uk/publicengagementstories/stories/2015/dark-side-protein-science.html
 
Description Pint of Science, Bristol, UK, May 2015, "From galaxies of stars to a new universe of proteins" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Part of the Pint of Science 2015 Programme in Bristol. About 60 people attended.
Year(s) Of Engagement Activity 2015
URL https://pintofscience.co.uk/event/dark-matters/
 
Description RSC: Synthetic Biology: The Free Edinburgh Festival Fringe Show (Heal) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Supported by an award from the Royal Society of Chemistry,the Edinburgh Fringe Festival hosted its first science stand-up on the subject of synthetic biology. Jack Heal's 'Do Scientists Dream of Synthetic Sheep?' show took a comedic approach to genome engineering, de-extinction and more - with the crowd helping to shape its direction with questions and discussion. The show considered questions from artificial life to Jurassic Park, and ran for 21 days.
Purpose: To interest the public in science.
Outcome: The comic felt freshly enthused about doing [synthetic biology] research.
Reflection: Free shows encourage people to take risks in their choices of which shows to see. This spirit is perfect for science outreach events which have to try hard to avoid becoming 'by scientists, for scientists'.

None yet.
Year(s) Of Engagement Activity 2014,2015
URL http://www.bristol.ac.uk/publicengagementstories/stories/2016/jack-heal.html?platform=hootsuite
 
Description Synthetic proteins for a synthetic biology: faster, fitter, stronger 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Better Humans Science Café, Bristol, UK, October 5 2016, "Synthetic proteins for a synthetic biology: faster, fitter, stronger"
Year(s) Of Engagement Activity 2016
 
Description We the Curious, Bristol, UK, September 2019, Futures 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Woolfson gave an interactive talk on protein design and synthetic biology to a general as part of Bristol Futures 2019 at We the Curious, Bristol, UK, in September 2019.
Year(s) Of Engagement Activity 2019
URL https://www.futures2019.co.uk/events/we-the-curious/