Click-Chemistry-Based Biomimetic Ligands Efficiently Capture G-Quadruplexes In Vitro and Help Localize Them at DNA Damage Sites in Human Cells

Abstract

Interrogating G-quadruplex (G4) biology at its deepest roots in human cells relies on the design, synthesis, and use of ever smarter molecular tools. Here, we demonstrate the versatility of biomimetic G4 ligands referred to as TASQ (template assembled synthetic G-quartet) in which a biotin handle was incorporated for G4-focused chemical biology investigations. We have rethought the biotinylated TASQ design to make it readily chemically accessible via an efficient click-chemistry-based strategy. The resulting biotinylated, triazole-assembled TASQ, or BioTriazoTASQ, was thus shown to efficiently isolate both DNA and RNA G4s from solution by affinity purification protocols, for identification purposes. Its versatility was then further demonstrated by optical imaging that provided unique mechanistic insights into the actual strategic relevance of G4-targeting strategies, showing that ligand-stabilized G4 sites colocalize with and, thus, are responsible for DNA damage foci in human cells.

The multivalent G-quadruplex (G4)-ligands MultiTASQs allow for versatile click chemistry-based investigations

“…Nuclei were subsequently stained with DAPI and images were collected by confocal laser scanning microscopy. The pattern seen in Figure 3 corresponds to what has been described for the twice-as-smart probe N-TASQ (direct labelling) 35–36 and biotinylated TASQ (pre-targeted imaging): 22–23 a dense nucleolar staining (yellow arrow) along with smaller, discrete nucleoplasmic foci (white arrow) ascribed to G4 clusters that fold during DNA transactions as they localize where DNA is at work (no DAPI staining). This approach, though qualitative in nature, provides high-quality images (high brightness, signal-to-noise ratio and foci definition) that are currently being exploited to assess whether and how G4-interacting agents (stabilizers 37 or destabilizers) 34, 38 modulate G4 landscapes in cells.…”

“…The two solutions were then mixed and stirred at 25 °C for 1 h (an HPLC-MS monitoring allowed for assessing the efficiency of the CuAAC, if needed). Of note: i - the final proportion of 1-butanol is 2% only, which is compatible with the stability of G4s in the condition of the experiments; and ii - the demetallation of clicked MultiTASQ, usually performed with Na 2 S treatment, 23, 32 is avoided here for reproducibility issue related to the loss of material during the precipitation step (the presence of copper within the cyclene template did not affect the properties of the TASQ, Figure S11). az MultiTASQ was mixed with a slight excess of dibenzocyclooctyne (DBCO)-PEG4-biotin conjugate (1.1 mol.…”

“…Again, these results were in line with those of BioCyTASQ (averaged enrichment = 18.7- and 6.1-fold for DNA and RNA G4s, respectively) and BioTriazoTASQ. 23 Quite satisfyingly, none of them were able to pull dsDNA down (between 0.6 and 0.9-fold enrichment with F-duplex), confirming the excellent G4-selectivity of TASQs.…”

“…To further exploit the versatility of MultiTASQs, we used their clickable handle in situ click imaging protocols to image G4 landscapes by clicking TASQs, once in their cellular G4 sites, with fluorescent partners. This approach is different from the pre-targeted G4 imaging we previously reported on, 22–23 as the very nature of the clickable appendages of MultiTASQs (small size, no H-bonding ability) ensures that the target engagement of TASQs in cells is not biased, as it could be the case with the biotin appendage. To this end, we adapted the in situ click imaging protocol initially developed with PDS-α: 13 MCF7 human breast cancer cells were incubated either live (10 μM, 24 h) or after fixation (20 μM, 1 h) with MultiTASQ (to demonstrate the modularity of this approach).…”

“…These results were in line with what was obtained with the previously reported TASQs. 22–23 Their G4-affinity was confirmed via an equilibrium-binding assay that relies on the use of a G4 (here, Myc, Table S2) labeled with a Cy5 dye (on its 5’-end) that is quenched upon ligand binding. 31 This assay was calibrated with PhenDC3, 14 as it was used in the initial setup ( app K D = 29.6 ± 0.9 nM); in our hands (Figure 2B), the G4 affinity of PhenDC3 was confirmed ( app K D = 57.7 ± 0.2 nM), and that of MultiTASQs lower ( app K D = 1.44 ± 0.1 and 0.90 ± 0.1 μM for MultiTASQ and az MultiTASQ, respectively) and in line with that of BioCyTASQ ( app K D = 1.01 ± 0.1 μM).…”