SEA-iDNA

The consortium's main goal is to identify highly specific i-motif DNA antibodies through phage display technology, leveraging an innovative chemical tool - a constrained i-motif structure - and to validate their efficacy in cellular environments. I-motifs are four-stranded DNA structures formed by the stacking of hemiprotonated C-C base pairs (CH+:C) within cytosine-rich regions, often found in gene promoters or telomeres. A distinctive feature of i-motifs is their strong pH dependence: they typically form in vitro under acidic conditions, raising questions about their stability and existence in cells. However, the discovery of the iMab antibody provided initial evidence of i-motif structures in human cells using immunofluorescence. Despite this breakthrough, concerns have emerged regarding iMab’s low selectivity, as it also binds to cytosine-rich sequences that do not adopt an i-motif conformation, limiting its reliability in immunodetection techniques such as immunofluorescence and CUT&TAG. A key challenge in i-motif research lies in the acidic conditions required for their formation, which may lead to protonation of ligands, including antibodies, and thus increase non-specific DNA interactions. Recent studies by Partner 1 have confirmed that the only commercially available i-motif antibody lacks specificity, as it also binds to non-folded C-rich sequences. This underscores the urgent need for antibodies that can selectively recognize the folded i-motif conformation, providing the scientific community with a more reliable tool to study its biological role. To address this challenge, we will use constrained i-motif structures, which have demonstrated stability under physiological conditions, as selective targets for phage display screening. The resulting antibody candidates will undergo rigorous evaluation using ELISA and BioLayer Interferometry (BLI) to identify those with high affinity for i-motifs while exhibiting minimal or no binding to other DNA structures, particularly unfolded C-rich sequences. The most promising antibodies will then be assessed in a cellular context to determine their ability to detect i-motif DNA sequences in vivo. This approach will pave the way for the development of highly specific antibodies, ultimately advancing the study of i-motif DNA and its potential biological functions.