Informace o publikaci
SG4: A Nanobody for Specific Recognition of G-Quadruplex Structures
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| Rok publikování | 2025 |
| Druh | Konferenční abstrakty |
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| Popis | G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures formed by guanine-rich sequences. These structures play critical roles in fundamental biological processes, including DNA replication, transcriptional regulation, telomere maintenance, and immunoglobulin class switching. Enriched in regulatory and disease-associated genomic regions, G4s have garnered attention as potential therapeutic targets, particularly in cancer, neurodegenerative disorders, and viral infections [1-3]. In this study, we investigate the molecular interaction between G4 DNA and SG4, a newly developed single-domain nanobody engineered for high specificity and affinity toward G-quadruplex structures [4]. Our aim was to characterize the binding behaviour and structural impact of SG4 on G4 DNA, providing insight into its potential as a molecular tool or therapeutic agent. Using an integrative biophysical approach, we employed Electrophoretic Mobility Shift Assay (EMSA), nano Differential Scanning Fluorimetry (nanoDSF), Microscale Thermophoresis (MST), and Circular Dichroism (CD) spectroscopy to assess the binding dynamics, thermal stability, and conformational integrity of G4 structures upon interaction with SG4. EMSA confirmed selective binding of SG4 to G4-forming sequences, with no detectable interaction with non-G4 controls. CD spectroscopy further demonstrated that SG4 binding preserves the native topology of the G4 structures, without inducing conformational rearrangements. The nanoDSF and MST analyses revealed high-affinity binding, characterized by a notable increase in G4 melting temperature, indicating significant thermal stabilization. Our findings establish SG4 as a selective and stabilizing binder of G-quadruplex DNA. Unlike traditional small-molecule G4 ligands, which often cause structural perturbations or exhibit limited specificity, SG4 offers a biologically compatible, highly specific tool for probing G4 dynamics in vitro and potentially in vivo. These properties position SG4 as a promising candidate for future applications in G4-targeted diagnostics and therapeutics. Keywords: Electrophoretic Mobility Shift Assay (EMSA), G-quadruplexes, Microscale Thermophoresis (MST), Nanobody, nano Differential Scanning Fluorimetry (nanoDSF) References: (1) Maizels, N., Nature structural & molecular biology, 2006, 13.12, 1055-1059. (2) Štefan, U., et al., International journal of biological macromolecules, 2024, 280, 136008. (3) Bartas, M., et al., Biochimie, 2018, 150, 70-75. (4) Galli, S., et al., Journal of the American Chemical Society, 2022, 144.50, 23096-23103. |