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Modifying nucleic acid nanostructures using triplex formation

Reference	BB/H019219/1
Principal Investigator / Supervisor	Professor Keith R Fox
Co-Investigators / Co-Supervisors	Professor Tom Brown, Dr David Rusling
Institution	University of Southampton
Department	Centre for Biological Sciences
Funding type	Research
Value (£)	346,022
Status	Completed
Type	Research Grant
Start date	01/08/2010
End date	31/01/2014
Duration	42 months

Abstract

By exploiting simple Watson-Crick base-pairing rules it is now possible to 'program' DNA and RNA strands to self-assembly into precise three-dimensional objects, arrays and devices. Several useful applications have arisen from this technology, including the construction of molecular sensors, the encapsulation of molecules for drug delivery, and DNA-based molecular lithography. Despite these advances, a universal method for positioning functional groups, proteins and other molecules at specific positions within these complexes has yet to be achieved. One suitable method for achieving this goal would be to exploit the triplex approach to DNA sequence recognition. This approach would rely on forming a triplex at specific duplex regions within a nanostructure using third strands tagged with a appropriate molecules. This method would be particular useful as binding of the third strand is within the major groove and does not require duplex denaturation, leading to little disruption to the overall nanostructure. Binding of the third strand could also be reversed by exploiting the pH dependence of certain triplexes. Moreover, triplex 'building blocks' are likely to be more rigid, with a greater persistence length and better connectivity than their duplex counterparts. In our previous work we have designed and synthesised novel nucleotide analogues that can be used to increase the stability of DNA triplexes and we are well placed to exploit these for preparing novel DNA nano-structures. In this work we will explore the rules governing triplex formation within DNA and RNA nanostructures and use their formation as a means of introducing different chemical and biological molecules within these structures. We will then use these findings to explore novel applications of these complexes, such as in the purification and structure determination of different nanostructures, and their use in arranging repeating molecules for crystallography.

Summary

In addition to its natural role in the storage of genetic information DNA is also an excellent material for constructing precise three-dimensional objects and assemblies on the nano-scale. By using the well-known base pairing of G with C and A with T, sequences of DNA can be easily designed so that they assemble into these structures forming precise shapes. In some cases it is possible to achieve nearly 100% yield by simply heating the sequences together in salty water and allowing the solution to cool back to room temperature. As a construction material DNA is also relatively easy and cheap to make, as well as being biodegradable and non-toxic. DNA has been used to construct containers capable of holding drugs and other biological molecules as well as devices capable of responding to different molecular signals. The next step is to develop a method of introducing different chemical or biological molecules within these DNA nano-structures. This would be useful, for example, in positioning different components within a DNA assembly to generate tiny 'nano' or 'bio' chips or to introduce a molecule onto a DNA cage capable of targeting it to a specific cell type, where it could then dispense its cargo. In this work we will develop such a method by exploiting the capacity of DNA to form three-stranded structures. These triplex structures are simply formed by adding a third strand of DNA to an existing double-stranded structure, where it attaches to the normal base pairs using specific triplet combinations. As the majority of DNA nano-structures are constructed from double-stranded segments it is possible to recognise these regions using a third strand of DNA, by generating a such three-stranded structure. Triplex formation is exquisitely specific and the third strand can therefore be designed to recognise a single region within a nanostructure. By attaching other molecules to this 'extra' strand it will be possible to position these at precise locations. The strategy canalso be used to join nano-structures together, or to increase the rigidity of an existing structure. These complexes can also be easily disassembled by changing the conditions (i.e. increasing the pH). Our approach will offer a new general platform for producing many nanometer-scale structures and devices and will enable the design and synthesis of new supramolecular structures and materials.

Impact Summary

This application is requesting funding for research that will provide new insights into the formation of molecular nanostructures. 'Nanoscience' is a relatively new area of research, with a number of potential applications, most of which have yet to be realised. Nanomaterials made from biological building blocks, such as nucleic acids as outline in this proposal, provide a link between synthetic biology and bionanotechnology and will provide nanostructures as components for building synthetic biosystems. The results from this research programme will therefore add to basic scientific knowledge in this area which will benefit researchers in academia and in the commercial sectors. The self-assembly of nanostructures will generate artificial materials with new properties; in order to achieve this it will be necessary to be able to connect nanostructures and build large assemblies by guided self-assembly. Our approach will offer a generic platform for producing new nanometer-scale structures and devices. The project will lead to development of fundamental knowledge of intermolecular interactions which will impact on the design and synthesis of new supramolecular structures and materials with significant potential. DNA is a versatile construction material. Its self-assembly properties can generate molecular nanostructures, it can be used as a molecular glue and as a fuel for molecular engines. Oligonucleotides bind together by their complementary sequences with simple design features. Many different applications have been proposed for DNA-based nanostructures including: the creation of crystalline 2D and 3D DNA arrays for organizing proteins for structure determination. Porous 3D DNA nanostructures can encapsulate a variety of proteins and have potential applications to drug delivery. DNA nanotubes have been used to template the growth of metallic nanowires and have potential applications in molecular electronics. They may also be employed for DNA-based computers. The main beneficiaries from this research will be those involved in fundamental research into self-assembling nanostructures in both the academic and commercial sectors. The results of this research programme will be disseminated through the usual range of outputs (e.g. peer-reviewed scientific journals, general-interest publications, university seminars, conference lectures and posters, webpages etc.). The University has a dedicated Media Centre on to aid with public announcements and production of newsletters, web pages and social networking blogs to further inform the public of the research. The applicants will engage with the public through lectures, University Science and Engineering days, open days and visit days. The activities are supported by all members of the research team including PhD students and postdoctoral researchers. We will seek to enhance the public¿s understanding of DNA and the potential offered by nanoscience. The applicants have also been involved in visits to local schools to give lectures on a wide variety of topics relating to DNA. Exploitation of any results will be coordinated through the University's Research and Innovation Services (R&IS). This supports the world-class research by focusing on its impact on national and global economies and society. It has a good track record of developing and maintaining relationships with external partners and exploiting research outputs for the benefit of society. This is an internationally competitive field and it is essential to maintain good contacts with others working in related areas. As well as presentation to international conferences we have arranged for the research co-investigator to spend several w

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Structural Biology, Synthetic Biology, Technology and Methods Development
Research Priority	Nanotechnology
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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