Control of G-quadruplex self-assembly through glycosidic bond angle

In our planet nature has evolved for millions of years adapting and evolving. Whilst doing that it created and utilized codes to do so. So far as we can ascertain DNA sequence is the code for life as we see it. Currently various groups make use of this understanding of how nature uses its molecules to perform functions to develop highly organized nano-size materials, motors, and other tools with various functions both inside and outside cells. DNA is one of these molecules. The double-helical structure of DNA is not the only structure that it adopts. There is a particular architecture DNA folds into that looks more like a knot- it is known as G-quadruplex structure. DNA can thus also fold into many different forms of this architecture. Many of these knots are unexpectedly resistant to high temperatures and to degradation by exposure to enzymes. These properties, together with our understanding of other highly fine-tuned properties derived from evolution, make DNA a molecule that can be utilized for technological purposes. However, in order to realize their potential, we have to understand how to use them for design purposes. In this context, a fundamental question is what type of knots/forms can DNA adopt and how do we control their formation? We know that one of the four bases that make up DNA is mostly responsible for adoption of these knots; G. Sequential segments of Gs make up the knots. The G can be locked into two different positions. Utilizing this understanding we have previously established that there are 26 theoretically possible knots. Better yet, we have predicted that the context of the presence of the two states of G in the DNA molecule will control the precise knot DNA folds into. In this project our aim is to verify experimentally our prediction that it is possible to utilize the two states of G in the DNA sequence to control the folding of the 26 knots. Indeed, it is also currently known that the folding into these knots can be influenced by very small chemical modification of this G base. We will also systematically measure some of the properties that make these knots of interest as materials and tools. We will measure both their temperature stability and their resistance to enzymes. Lack of good performance in these two domains is usually the cause for not using molecules from cells for technological purposes. Our proposal is thus fundamental to the future use of DNA knots for technological purposes. There are two significant benefits of the proposed work. Firstly, we hope to establish that it is experimentally feasible to design these knots. This is important for making nanomaterials and biotechnological tools. Secondly, data generated from this work has the potential to predict knots formation from the DNA sequence. This is relevant since it allows for developing a method for finding these knots in the genome. Currently, this is of great interest since these knots happen to have controlling functions in the genome. Thus the association of type of knot with function will be made feasible.

In order to realize programmed build up of DNA objects, devices, and materials, a systematization of the principles at the basis of control of the construction process is necessary. Much progress has been made in self-assembly of complementary (Watson-Crick) DNA. Here we propose establishing the rules for a rational approach towards the design of quadruplex topologies. Utilizing the formalism of quadruplex folding, based on a two-state disposition of the glycosidic bond angle, we have derived the sequence of stacking tetrad GBA combinations feasible for anti-parallel quadruplexes of three loops. In order to provide proof of concept validating this axiomatic approach we experimentally determined one of the hypothesized topologies. The control of the fold of this representative novel topology of three-stacked tetrads containing all three loop types was exerted through the length of the loops bridging the quadruplex stem. A single sequence was tried to achieve this topology. However, not all topologies can be discriminated through their loop length alone. Several issues may impede successful design; e.g. loop composition and its interactions may influence the topology, or the assembly of quadruplex entities may result in multistranded architectures, or both 5' and 3' ends may have to be modified to stabilize single folds. More often than not, the sum of these factors in tandem determines the final topology. To minimize these effects it is currently feasible to control the fold of quadruplexes by forcing the GBA of selected guanosines of the quadruplex stem to adopt the desired conformation. Therefore, knowledge of the sequence of GBA that a quadruplex stem is able to adopt will, in principle, enable the rational design of quadruplex topologies. NMR spectroscopy is the most appropriate technique to perform this study, since it allows for the rigorous assessment of the self-assembled topologies.

DNA G-quadruplexes are unusually temperature and enzyme resistant. These properties make them suitable candidates for biotechnological use. This project creates a knowledge and understanding platform that may enable the development of various pharmaceutical and biotechnological products. Who would be the users and beneficiaries of this research outside the research community? Some obvious industrial beneficiaries are: (i) Companies with an interest in developing molecular electronics based on DNA. (ii) Companies interested in developing DNA technology tools (iii) Companies interested in the development of DNAzymes and chemical biology tools. (iv) Existing companies interested in developing aptamers to target diseases and disease states. (v) Companies interested in selecting G-quadruplex targets for drug discovery. Efforts by the PI to engage industrial stakeholders In the biotechnological context, the PI has been involved in the European Science Foundation COST Action MP0802, 'Self-assembled guanosine structures for molecular electronic devices' (06/2008 - 06/2012). The Action includes the participation of various industrial stakeholders and has as its main objective the development of molecular electronics based on quadruplex nucleic acids. We have been voted coordinator for one of the four Working Groups- 'Biochemical & Biorecognition Properties'. The first of the stated objectives of this working group is develop understanding of the rules for G-quadruplex unimolecular and macromolecular self-assembly- the overall objective of this proposal. The Action has started recently. The PI expects to have direct access to, and feed-back from industrial stakeholders involved once opportunities arise. Thus mechanisms for identifying opportunities are in place. As one of two country representatives the PI is in the process of recruiting more industrial participants from the UK to this Action. Still in the biotechnological context the PI is invited speaker to the European Science Foundation research conference on 'Self-assembly of Guanosine Derivatives: from Biological Systems to Nanotechnological Applications', Innsbruck, Austria, to be held on 20-25/06/2009. This meeting includes the participation of various industrial stakeholders. In it the PI is going to present results of the application of the formalism of quadruplex folding to inform these stakeholders of the potential benefits in applying its principles in their developments of technological tools. These initial steps should pay off once the outcomes of this project become known. The PI would then be in a position of applying control of quadruplex folding to any specific industrial partner's aims identified. Realization of the Potential Results of this Proposal by the PI In the pharmaceutical context, by developing this platform knowledge on control of quadruplex folding we intend to pursue specific projects towards generating patents. The University of Ulster has schemes in place to convert potential ideas and results from research into products of industrial value. Thus any exploitable results will first be communicated to the Office of Innovation. At the University of Ulster the Office of Innovation includes an expert team that is responsible for the capture, protection and commercialisation of Ulster's intellectual capital by providing support for intellectual property management, technology licensing, Spin-Out Company Creation and Industrial Collaborations. At any time if an opportunity is identified by researchers at Ulster University one may contact directly a member of this team by phone or through a dedicated webpage. To support the clustering of knowledge-based companies, the University of Ulster has also established various Science Research Parks. Thus, methods for communication and engagement with industrial stake holders are current practice by experienced staff that is available to the PI of this proposal.