Investigating the stability and function of i-motif DNA

It is assumed that DNA exists as a double helix, the structure first described by Watson and Crick in 1953. However, it is lesser-known that DNA can adopt many different types of structure. DNA is composed of four different "building blocks" called bases: adenine, guanine, thymine and cytosine. DNA sequences rich in cytosine can form alternative secondary structures called i-motifs, first discovered in 1993. Sequences of this type are widespread throughout our genome and may play a role in gene expression, but there are many fundamental questions unanswered. The stability of i-motif structures is highly dependent on the particular sequence of bases. To understand which sequences are more likely to form i-motifs in biology, an understanding of their stability is required. This project proposes a systematic investigation of a library of different DNA sequences to determine the optimum sequence requirements for stable i-motif formation. This will allow us to define rules to predict the stability of i-motif structure based on the type of DNA sequence. We will then use this information for computational analysis of the human genome to estimate the number of sequences which could potentially form i-motif structures, and where these reside in the human genome. Using a mixture of molecular biology techniques, we will investigate the impact of stabilisation of i-motif structure on transcription and de-convolute the interplay between the dynamics of i-motifs and G-quadruplexes (structures which have previously been well-characterised) on gene expression. This proposal will reveal which i-motif sequences are most stable and potentially new targets for drug design, therapeutics and structural investigation.

This proposal is about the investigation of the stability and potential biological functions of i-motif DNA secondary structures. The project will use a range of biophysical and molecular biological techniques to answer the following key questions:
1) Which types of DNA sequences form the most stable i-motif structures?
2) How does epigenetic modification change the stability and dynamics of these structures?
3) Can targeting i-motifs with small molecule ligands interfere with gene expression?
4) Is there any interplay between the position of i-motif and G-quadruplex structures and the effects on gene expression and are there any synergistic effects between them?
Biophysical analysis will be performed on C-rich DNA libraries. UV melting and annealing experiments will be used to determine the thermal stability of the structures and insight into the kinetics of folding/unfolding processes. UV thermal difference spectroscopy will provide indicative spectra to characterise the type of DNA secondary structure formed. CD spectroscopy will provide further insight on the type of secondary structure in solution and will be used to determine the transitional pH of the sequence to give an indication of the pH dependent stability of the structure. Footprinting will be used to give insight into folding and topology. In vitro analysis will be performed using an RNA polymerase stop assay to indicate whether DNA secondary structures disrupt transcription. In vivo methodology will include cloning of plasmids for a luciferase based reporter gene assay for assessment of the effect of DNA secondary structure on transcription. Further experiments will utilise quantitative real time PCR, which measures the quantity of mRNA produced from chromosomal DNA. Both in vitro and in vivo experiments will be investigated using previously characterised i-motif and G-quadruplex interacting ligands as chemical tools.

The findings from this study will develop an understanding of the stability of different types of DNA sequences which are able to form i-motif secondary structures and also their potential effects in biology. The work will directly impact the post-doctoral research assistant working on the project, who will be trained in both biophysical and bioinformatic techniques. This will contribute towards not only their education and professional development but also the education economy for UEA, Norwich Research Park, East Anglia and the UK.

The identification of regions in the human genome where the most stable i-motif forming sequences are present will identify novel targets for further structural investigation and targets for drug design. This will directly affect interest in the design of small molecule ligands to interact with i-motif DNA. The number of stable potential i-motif forming sequences in the human genome is expected to be much lower compared to other types of secondary structures such as G-quadruplexes; this will allow higher chances of achieving high specificity. The work will indicate whether it is possible to affect transcription by targeting i-motif DNA. From this, work can be built on the potential of small molecule ligands to stabilise or destabilise i-motif structures for use as nanotechnological and chemical biological tools, or in the development of therapeutics for genetic disease. In this aspect, industry would benefit, for example, pharmaceutical companies involved in transcriptional-related drug discovery. This will also impact the applicant's research projects involving design and development of small molecules to target i-motif DNA, and consequently will aid applications for financial support for these studies.

The outcomes of this work will increase the opportunity for development of further national and international collaborations. For example, there are a large number of world-leading researchers who are working on DNA secondary structure and the results from the work in this proposal would inspire many of these researchers to consider working on the i-motif forming sequence in their DNA or gene of interest. This work would have the opportunity to impact a very board range of researchers, from those working on i-motif and/or G-quadruplexes, DNA based nanotechnology, genome bioinformatics, gene expression and epigenetics (hence also the pharmaceutical industry) to those interested in DNA-based self assembly and nanotechnology (and therefore the material sciences and photonics industry).

Finally, the general public would benefit from this work as it signifies hope for further understanding of how genes are read and potential novel targets for therapeutics for genetic diseases. Consequently, we would expect that there will be a wide range of beneficiaries: academic scientists, industrial enterprise (from pharmaceuticals to nanotechnology) and the general public.