14-ERASynBio: BioMolecular Origami

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.

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.

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.