Identification of optimal fluorescent probes for G-quadruplex nucleic acids through systematic exploration of mono- and distyryl dye libraries

Abstract

A library of 52 distyryl and 9 mono-styryl cationic dyes was synthesized and investigated with respect to their optical properties, propensity to aggregation in aqueous medium, and capacity to serve as fluorescence “light-up” probes for G-quadruplex (G4) DNA and RNA structures. Among the 61 compounds, 57 dyes showed preferential enhancement of fluorescence intensity in the presence of one or another G4-DNA or RNA structure, while no dye displayed preferential response to double-stranded DNA or single-stranded RNA analytes employed at equivalent nucleotide concentration. Thus, preferential fluorimetric response towards G4 structures appears to be a common feature of mono- and distyryl dyes, including long-known mono-styryl dyes used as mitochondrial probes or protein stains. However, the magnitude of the G4-induced “light-up” effect varies drastically, as a function of both the molecular structure of the dyes and the nature or topology of G4 analytes. Although our results do not allow to formulate comprehensive structure–properties relationships, we identified several structural motifs, such as indole- or pyrrole-substituted distyryl dyes, as well as simple mono-stryryl dyes such as DASPMI [2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide] or its 4-isomer, as optimal fluorescent light-up probes characterized by high fluorimetric response (I/I 0 of up to 550-fold), excellent selectivity with respect to double-stranded DNA or single-stranded RNA controls, high quantum yield in the presence of G4 analytes (up to 0.32), large Stokes shift (up to 150 nm) and, in certain cases, structural selectivity with respect to one or another G4 folding topology. These dyes can be considered as promising G4-responsive sensors for in vitro or imaging applications. As a possible application, we implemented a simple two-dye fluorimetric assay allowing rapid topological classification of G4-DNA structures.

Reversible photoswitching of the DNA-binding properties of styrylquinolizinium derivatives through photochromic [2 + 2] cycloaddition and cycloreversion

“…In contrast, the very low emission intensity of 3d did not change upon the addition of ct DNA. The fluorescence light-up effects of the ligands 3a-c upon association with DNA resembled the ones observed for other styryl-substituted quinolizinium derivatives [62][63][64][65][66][67]. Accordingly, the emission enhancement most likely resulted from the accommodation of the ligand in the constrained binding site of the DNA, which led to a restricted conformational flexibility.…”

“…As the quinolizinium ion has been established as a versatile platform for the development of DNA intercalators [60], we identified styryl-substituted quinolizinium derivatives as a promising basis for the search for photoswitchable DNA binders based on the photocycloaddition-photocycloreversion equilibrium. In fact, some selected styrylquinolizinium derivatives have already been shown to bind to DNA [61][62][63][64][65][66][67], however, their photocycloaddition reaction and the propensity of the corresponding photodimers to release the DNA-binding ligand have not been reported so far. Herein, we report on the photochemical and DNA-binding properties of the selected styrylquinolizinium derivatives 3a-d and demonstrate their ability to operate as photoswitchable DNA ligands.…”

Selective, pH‐Dependent Colorimetric and Fluorimetric Detection of Quadruplex DNA with 4‐Dimethylamino(phenyl)‐Substituted Berberine Derivatives

“…Moreover, this chemosensor and derivatives thereof have the potential to be applied for the fluorimetric detection of pH gradients and extracellular nucleic acids in biofilms, [31] that have been suggested just recently to contain G4‐DNA as functional component [60] . Although numerous fluorescent probes for G4‐DNA are known, [9–19] to the best of our knowledge, no examples have been reported so far with these particular features. Namely, only a few fluorescent probes for G4‐DNA have been reported that operate at lower pH, [61] but these probes do not exhibit the dual‐mode response of compound 3 a .…”

“…For an efficient detection, however, either the emission intensity of the otherwise non‐fluorescent ligand has to increase strongly or the emission energy has to shift distinctly. Thus, several organic fluorophores have been introduced that serve this purpose, for example benzimidazole, [9] benzindole, [10] triazine, [11] coumarin, [12] cyanine, [13] pyridinium, [14,15] quinolinium, [15,16] quinolizinium, [15,17] triangulenium [18] and benzothiazole [19] based dyes. Among the promising compound classes for this application are also DNA‐binding natural products with suitable fluorescence properties, because these compounds usually provide sufficient affinity, and often selectivity, towards DNA, along with the required biocompatibility.…”

“…In this context, quadruplex DNA has been identified as a very important analyte, because of its activity in many biological processes, such as transcription regulation [6,7] or telomerase inhibition [7,8] . As a result, fluorescent probes have been developed and employed widely for the selective detection of G4‐DNA structures [9–19] . Such probes usually consist of a G4‐DNA‐binding molecule whose emission properties change upon association to the target G4‐DNA.…”