i-Motifs: Sequence, Structure and Function in Ageing

DNA is often assumed to be a double helix, the "twisted ladder" structure which was first proposed by Watson and Crick in 1953. However, it is less well known that DNA can adopt different shapes and these can be used as switches to control how it works.

DNA is comprised of four bases, often described as the "building blocks" for life because they encode all the information required to build and maintain an organism. The sequence of these four bases (adenine, guanine, thymine and cytosine) is what defines us as humans and what makes us different to bacteria, yeast and plants. DNA sequences which contain lots of the base cytosine can form alternative secondary structures which instead of appearing like the normal "twisted ladder" of two strands, are a very tightly packed "knot" of four strands of DNA. We call these structures i-motifs. Sequences of this type have been used as pH-dependent switches in nanotechnology but are also widespread throughout the human genome, exist in cells and have been shown to play a role in gene expression and defining how long our cells live. Despite these recent advances, we lack the detail about how these structures work in the body. We know that for some regions of DNA, these types of sequences may play a role in our predisposition to getting certain diseases, such as Diabetes. We also know that these sequences are actively mutated and deleted as we age and in diseases such as Cancer. To be able to understand the effects of these structures have on disease, we need to understand how they can be changed and what difference this makes to how they work in biology. This could potentially give us ways to diagnose or treat certain genetic diseases.

The central aim of this proposal is to investigate the relationship between sequence, structure and function of DNA i-motif structures in switching genes on and off and how this changes during ageing. We will examine this using a wide range of computational, biological and biophysical techniques. Our previous work has given us an understanding of which types of sequences could potentially fold into i-motif structures. Using biophysical and molecular biology methods, we will investigate the importance of the structure of i-motif in humans and their precise influence in controlling gene switching. This will give us information about how important the structure of i-motif is to function. We have preliminary data to show that i-motif forming sequences are mutated and deleted as we age, and this can affect the progression of disease. We aim to decipher whether there are "hot spots" in critical regions of the genome that are critically affected by mutations. Finally, we will perform a global study of where i-motif structures are present in human cells and observe whether their distribution changes as cells age. This will involve development of a new technique, based on looking at a "footprint" these structures have.

The project will advance our understanding of how i-motifs work in biology and how they are controlled by mutations affected by the ageing process. The outcomes of the work will also improve our understanding about the folding of i-motifs under different conditions, allowing better prediction of regulating properties based on DNA sequence. This will impact the design and creation of DNA/RNA based nanotechnologies. The development of a new tool to study the prevalence of i-motif structures in human cells will be able to be applied to any other organism, which will expand the scope of our research to plant scientists and microbiologists.

C-rich regions of DNA can form alternative secondary structures called i-motifs, which have not only shown utility as bio-compatible pH-responsive materials in nanotechnology, but also have been shown to exist and act as molecular switches in cells. Although there have been significant advances in this area, we are still yet to understand their prevalence in the human genome and extent of their roles in biology.

Our hypothesis is that iM structures form in vivo but natural mutations will affect the function of these structures and could result in age-related diseases and conditions. The project will test this using a range of bioinformatic, biological, biophysical and molecular biology techniques.

In this proposal, we will test the importance of the structural features in i-motif and their effect on biological function using cell-based reporter gene assays. We will systematically mutate cytosines and loops out of the sequence of interest and correlate between the underlying C-rich sequence, resulting structure and gene function. The proposal aims to reveal the global presence of i-motifs in cells. This will involve developing a method, based on similar principles to SHAPE chemical probing in RNA and G-quadruplex-seq methods. It will reveal the presence of i-motif structures and can be able to be applied in vitro, in cells or in vivo to any organism. We will bring together the new understanding of i-motifs to investigate the effects of ageing on these structures, their potential changes in function and how this may play a role in age-related diseases and conditions.