Modifying nucleic acid nanostructures using triplex formation

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

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 can also 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.

Technical Summary

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.

Planned Impact

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

Publications

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Rusling D (2012) DNA Conjugates and Sensors -

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Rusling DA (2012) Triplex-directed covalent cross-linking of a DNA nanostructure. in Chemical communications (Cambridge, England)

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Rusling DA (2014) Functionalizing designer DNA crystals with a triple-helical veneer. in Angewandte Chemie (International ed. in English)

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Rusling DA (2014) Sequence-specific recognition of DNA nanostructures. in Methods (San Diego, Calif.)

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Abdallah HO (2016) Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking. in Chemical communications (Cambridge, England)

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Chandrasekaran AR (2018) Triplex-forming oligonucleotides: a third strand for DNA nanotechnology. in Nucleic acids research

 
Description We have shown that it is possible to target DNA nanostructures using triplex helix formation. This enables the spatial positioning of molecular components on DNA arrays with nanometre precision. The third strand can be targeted different regison of the array (to both crossover and non-crossover strands and across the region spanning the crossover itself). By conjugating biotin to the end of the oligonucleotide we are able to recruit streptavidin molecules to precise locations within a DNA array
Exploitation Route The formation of precise molecular nanostructures is an area of considerable academic interest, for which the practical applications are yet to be developed. Triple helix formation within these structures offer the opportunity to attach further components to these with precision. Ability to target a macroscopic 3D crystal based on tensegrity triangle offers possibilities for positioning molecules for structural analysis.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Presentation at Southampton CafĂ© Scientifique 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Dr R talked about the importance of DNA nanostructures and our research in this area the Southampton Cafe Scientifique (Feb 2013) Presentation on DNA nanostructures to Southampton Cafe Scientifique

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description RSC Nucleic Acids Group meeting - Birmingham 2012 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Presentation at the Royal Society of Chemistry Nucleic Acids Group meeting in Birmingham

no actual impacts realised to date
Year(s) Of Engagement Activity 2012