Promoter G-Quadruplex Binding Styryl Benzothiazolium Derivative for Applications in Ligand Affinity Ranking and as Ethidium Bromide Substitute in Gel Staining

Abstract

Fluorescent compounds that can preferentially interact with certain nucleic acids are of great importance in new drug discovery in a multitude of functions including fluorescence-based displacement assays and gel staining. Here, we report the discovery of an orange emissive styryl-benzothiazolium derivative (compound 4) which interacts preferentially with Pu22 G-quadruplex DNA among a pool of nucleic acid structures containing G-quadruplex, duplex, and single-stranded DNA structures as well as RNA structures. Fluorescence-based binding analysis revealed that compound 4 interacts with Pu22 G-quadruplex DNA in a 1:1 DNA to ligand binding stoichiometry. The association constant (K a) for this interaction was found to be 1.12 (±0.15) × 106 M–1. Circular dichroism studies showed that the binding of the probe does not cause changes in the overall parallel G-quadruplex conformation; however, signs of higher-order complex formation were seen in the form of exciton splitting in the chromophore absorption region. UV–visible spectroscopy studies confirmed the stacking nature of the interaction of the fluorescent probe with the G-quadruplex which was further complemented by heat capacity measurement studies. Finally, we have shown that this fluorescent probe can be used toward G-quadruplex-based fluorescence displacement assays for ligand affinity ranking and as a substitute for ethidium bromide in gel staining.

Preferential Binding of a Red Emissive Julolidine Derivative to a Promoter G‐Quadruplex

“…Several laboratories, including us, have used this differential fluorescence response of a fluorophore to tentatively rank the preferred nucleic acid structures of the same. [59][60][61][62] Such screening results should always be accompanied by more rigorous binding studies to rule out any false leads as fluorescence response is prone to structural, protonation, and viscosity changes at the binding site microenvironment. Using this fluorescence emission-based screening method, initially, we recorded the fluorescence response of compounds 1, 2, and 3 with various oligonucleotides that consisted of a single strand, duplex, i-motif, and G-quadruplexes (See Table 1 for complete sequences).…”

Role of Alkali Cations in DNA–Thioflavin T Interaction

“…G4 is an intriguing DNA structure that interacts with dyes . Several fluorophores preferentially bind to G4 than double-stranded DNA (dsDNA) − and manifest enhanced fluorescence after binding, enabling the detection and study of G4 in various biological, chemical, and environmental contexts. − The interaction between dyes and G4 structures provides insight into their structure, topology, dynamics, and microenvironment. , Thioflavin T (ThT) is extensively used for probing G4 because it remarkably enhances fluorescence in the presence of G4 . ThT belongs to the group of molecular rotors that show strong emission when ring rotation is inhibited on adopting a specific chemical configuration .…”

Cavity Lasing Characteristics of Thioflavin T and Thioflavin X in Different Solvents and Their Interaction with DNA for the Controlled Reduction of a Light Amplification Threshold in Solid-State Biofilms

“…A defining characteristic of ThT, ThX, and other molecular rotors is their low fluorescence quantum yield in low-viscosity environments . Changes in the environment’s viscosity or binding to biomolecules, such as amyloid protein fibrils − or DNA, − results in significant emission enhancement. The underlying photophysical mechanism for this fluorescence boost is related to ultrafast torsional motion leading to nonradiative deactivation via twisted intramolecular charge transfer (TICT) .…”