BrdU immuno-tagged G-quadruplex ligands: a new ligand-guided immunofluorescence approach for tracking G-quadruplexes in cells

Abstract

G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag. However, this approach is limited by a low-labeling degree impeding precise visualization in specific subcellular regions. Herein, we present a new visualization strategy based on the immuno-recognition of 5-bromo-2′-deoxyuridine (5-BrdU) modified G4 ligands, functionalized prior- or post-G4-target binding by CuAAC. Remarkably, recognition of the tag by antibodies leads to the detection of the modified ligands exclusively when bound to a G4 target both in vitro, as shown by ELISA, and in cells, thereby providing a highly efficient G4-ligand Guided Immunofluorescence Staining (G4-GIS) approach. The obtained signal amplification revealed well-defined fluorescent foci located in the perinuclear space and RNase treatment revealed the preferential binding to G4-RNA. Furthermore, ligand treatment affected significantly BG4 foci formation in cells. Our work headed to the development of a new imaging approach combining the advantages of immunostaining and G4-recognition by G4 ligands leading to visualization of G4/ligands species in cells with unrivaled precision and sensitivity.