Biological relevance and targeting of i-motif DNA – iCARE

Non-canonical nucleic acids secondary structures have emerged during the last decades as critical factors to modulate DNA and RNA transactions. Among them, i-motifs (i-DNA) represent unusual, four-stranded DNA structures in which cytosines are intercalated via a stack of hemi-protonated C–C base pairs (CH+:C) that may form at C-rich genomic tracks present in promoters or at telomeres. Some of these i-DNA structures have been well characterized in vitro, but our knowledge on their biological relevance is still limited, due to an a priori concerning their formation only at low pH. i-DNA may mirror other four-stranded G-rich structures (G-quadruplexes), but it is unknown whether, and to which extent, the two structures can coexist at the same loci. The rules governing the formation of i-DNA are also unclear and several example in the literature suggest that some i-DNA may be formed at neutral pH and that the algorithms predicting G-quadruplexes could not be directly used to predict i-DNA structure formation. Recent reports using immunostaining with a specific antibody (iMab) and in-cell NMR demonstrated that i-DNA structures are able to form and are stable in the nuclei of human cells. In contrast to G-quadruplexes where the biological relevance has been brought using antibodies and highly specific chemical probes, relatively few molecules have been reported to interact with i-DNA, and a controversy concerning their binding mode, selectivity and affinity persists in the literature. The dynamics of i-DNA structures (i.e., their ability to fold or unfold) may be relevant for mediating key biological processes. The discovery of iMab serves as a proof of principle suggesting that selective targeting of these structures can be achieved. While a number of proteins have been shown to interact with G-quadruplexes, few examples of i-DNA interacting proteins have been reported so far. Furthermore, it is not clear whether these proteins recognize i-DNA structures, cytosine-rich sequences, or nucleotides in i-DNA loops.
In this context, the consortium iCARE relies on a multidisciplinary expertise combining chemists, biophysicists, biochemists and biologists, that have already proven to work successfully together (as evidenced by joint publications from chemistry to biology). iCARE aims to address the following scientific goals:
- Understand the rules that govern the formation of i-DNA, their pH dependency, thermodynamics and folding kinetics, and to understand their biological relevance versus G4 (WP 1);
- Identify unambiguous (i.e., affine and specific) i-DNA-interacting ligands using a novel strategy to lock i-DNA in a folded state stable at physiological pH, and to explore their cellular effects as i-DNA chemical probes or therapeutic candidates (WP 2);
- Identify the proteins interacting with i-DNA (using an original pull-down strategy coupled to proteomic analysis) and to determine their cellular relevance to act at putative i-DNA genomic sites (WP 3).
In particular, the use of constrained i-DNA as a pH-independent substrate in WP2 and WP 3 represents a key distinctive feature of this project compared with all previously used approaches, allowing to overcome the current limitations to screen specific ligands at neutral pH, and to identify the i-DNA-protein interactome.
Achievement of the project’s goals will improve our understanding of biological functions of i-DNA at the genome-wide level and its cellular druggability. Indeed, drugs that directly target i-DNA or interfere with its processing have strong therapeutic potential. Finally, the accumulation of data in vitro, combined with the i-DNA interactome will greatly help to improve genome-wide predictions, not only in humans but also for additional applications in other organisms.