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Project

Nanobodies to recognize non-canonical DNA structures and regulate their formation in vivo (OZRIFTM7)

In 1953, Watson and Crick presented their iconic structure of double-stranded DNA. Since then, this structure has been firmly etched into humanity's cultural memory. But DNA is also capable of adopting other structures that only appear in certain contexts. Widely known, for example, are the so-called G- quadruplexes (G4), formed from G-rich sequences, and i-motifs, formed from the complementary strand. Although these structures have been extensively studied, their role in gene regulation and their link to diseases such as cancer, Alzheimer's disease, and diabetes is still the subject of extensive research. Traditionally, non-canonical DNA structures were studied in vitro and their existence in cells was controversial. This issue was clarified by pioneering studies based on the detection of G4 and i motifs with specific antibodies developed using synthetic antibody libraries.

Recently, a new fold of a non-canonical DNA structure called AGCGA quadruplex was discovered. At the sequence level, these structures adhere to a repeat of the AGCGA motif. At the structural level, these quadruplexes are stabilized by G-A and G-C base pairs that form GAGA - and GCGC-quadruplexes. Unlike G-quadruplexes, these structures are not stabilized by specific cation binding and are thus insensitive to the nature of the cation. The bioinformatics study has identified AGCGA quadruplex sequence motifs in over 40 human genes that are associated with the regulation of basic cellular processes, neurological disorders, cancer, and abnormalities in bone and cartilage development, while mutations of the motifs have been linked to autism.


The main goal of the proposed project is to determine whether AGCGA quadruplexes are formed in vivo. This requires the development of detection tools, specific ligands, that recognize AGCGA quadruplexes in the cellular native context. We plan to develop a panel of nanobodies (single-chain camelid antibodies) that specifically recognize AGCGA quadruplexes. The nanobody-AGCGA-quadruplex interactions will be studied in terms of affinity, specificity, selectivity, and structural aspects of recognition, leading to a set of well-characterized reagents for AGCGA-quadruplex detection. Ultimately, these nanobodies will be used for the detection of AGCGA quadruplex in cells by immunoimaging techniques. In parallel, we propose to investigate the stability of AGCGA-quadruplex in vitro using biophysical techniques to determine how solution conditions (cation concentration, water activity, molecular displacement) affect its stability. This will corroborate the results of the in vivo studies and provide a complementary approach to assess whether AGCGA quadruplexes are stable in vivo. Establishing the potential existence of a new type of non-canonical DNA structure will provide a strong boost to the field of non-canonical DNA biology, while the development of AGCGA quadruplex-specific antibodies will provide new tools for the scientific community to further research.
Date:1 Oct 2021 →  Today
Keywords:Structural Biology, Molecular biophysics, nanobody, non-canonical DNA, Quadruplex DNA, i-motif
Disciplines:Other biological sciences not elsewhere classified