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

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/I0 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.

The dark-yellow solution was stirred in the ice bath for 20 min, then at room temperature for 1 h and finally cooled again in the ice bath. A solution of 1- (2-methyl-1,3-dioxolan-2-yl)propan-2-one [6] (9.34 g, 64.8 mmol) in dry Et2O (12 mL) was added. The reaction mixture was stirred in the ice bath for 15 min and then at room temperature for 30 min and finally poured into an S4 ice-water mixture (100 mL). The organic phase was separated and the aqueous layer was extracted with MTBE (3 × 50 mL). The combined organic phases were washed with water and brine, dried over Na2SO4. The volatiles were removed in vacuo, yielding a brown residue containing the crude 2-methyl-1-(2-methyl-1,3-dioxolan-2-yl)-3-(pyridin-2-yl)propan-2-ol (4.44 g). The residue was dissolved in Ac2O (10 mL) and H2SO4 (96%, 0.50 mL) was carefully added. The mixture was heated at reflux (bath temp. 150 °C) for 3 h, then cooled to room temperature. Ice-water (20 mL) was subsequently added and the resulting mixture was left to stir overnight. Charcoal (1 g) was then added and the mixture was filtered. The filter cake was then rinsed with water. NH4PF6 (6.30 g, 38.6 mmol) dissolved in a small amount of water was added to the filtrate. The precipitated solid was collected by filtration, washed with water (3x), dried under vacuum and recrystallized from EtOH, to give I11 (1.82 g, 14% yield) as a white crystalline solid. 1 [7]: A mixture of 2aminopyridine (1.00 g, 10.6 mmol) and 2,4-pentanedione (1.31 mL, 1.28 g, 12.8 mmol) with polyphosphoric acid (10 mL) was stirred at 90 °C for 3 h and then poured into ice (60 g). A solution of NH4PF6 (6.93 g, 42.5 mmol) in a minimal volume of water was added to the melt.

General procedure for the synthesis of distyryl dyes by Knoevenagel condensation 1 :
A mixture of the heterocyclic salt I1-I16 (2.5 mmol), aldehyde (7.5 mmol, 3 molar equiv, unless otherwise stated) and piperidine (0.50 mL, 5 mmol, 2 molar equiv) in EtOH (25 mL) was heated under reflux for 2.5 h. After cooling to room temperature, the precipitated solid was collected by filtration, washed with EtOH (2 × 5 mL) and Et2O (2 × 5 mL) and dried. The crude iodide salt was either purified through a recrystallization from a suitable solvent (as indicated below) to give an analytically pure sample, or subjected to anion exchange to bromide or chloride, as described below.

S6
Procedure for anion exchange: Ion-exchange resin (Amberlite IRA-402, Clform, or Amberlite IRA-400, Brform, about 20 mmol equiv) was thoroughly rinsed with a mixture of MeCN and MeOH (1:1,v/v) and charged into a short glass column. The dye (iodide salt) was dissolved in a minimal amount of a mixture of MeCN and MeOH (1:1,v/v) and loaded in the column. The product was then eluted with the same solvent mixture (about 50 mL). The solvents were removed in vacuo and the residue was recrystallized from a suitable solvent (as indicated below), to give an analytically pure dye.
Fixed-wavelength fluorescence measurements. All fluorescence analyses were performed with a microplate reader (BMG FluoStar Omega), using a 96-well quartz plate with a transparent bottom (Hellma). Samples were prepared by mixing working solutions of DNA samples (5.6 μM or equivalent in K-100 buffer, 90 µL) or the buffer alone with working solutions of dyes in K-100 buffer (25 μM in K-100 buffer containing 2.5% v/v DMSO, 10 µL). The final concentrations were 2.5 μM for the dye and 5 μM (or equivalent) for DNA in a total volume of 100 μL per well. The plates were stirred for 3 min at 300 rpm and then left to equilibrate for 1 h at room temperature in the dark. Fluorescence emission was recorded by using a microplate reader, exciting each dye at the appropriate wavelength with the aid of appropriate filters (Table S1). The instrument gain was set for each channel and kept constant throughout all the analyses.
Multivariate analysis. The light-up data matrix reported in Table S1 was normalized to a scale of 0 to 1; normalization was always performed for each nucleic acid sequence separately.
Multivariate analysis (PCA) was performed with Origin Pro 2018b (OriginLab, Northampton, MA). The PCA data are presented as score plots of PC1 versus PC2.
Fluorescence quantum yield and brightness measurements. Dyes 1p, 1u, 17a and 18a were diluted in K-100 buffer at varying concentration (1.2-3 μM) alone or in the presence of G4 structures (c-myc or 22AG, 6 µM). Absorption and emission spectra (λex = 500 nm, slits width 5 nm, PMT voltage 550 V) of the resulting solutions were measured on a UV Cary-300 spectrophotometer and on a Cary Eclipse fluorimeter, respectively, using a transparent and asymmetric quartz cuvette (1 × 0.4 cm pathlength). Fluorescence emission spectra were integrated between 510 and 800 nm and the obtained values were plotted as a function of the absorbance at 500 nm for each dye. The slope of the resulting plots was used for the quantum yield (Φ) calculations, using the data recorded for Rhodamine 6G at identical settings as a reference (Φ = 0.95 in EtOH [14]): where Φ is fluorescence quantum yield, S is the slope obtained from the Area vs absorbance plots and n is the refractive index of the solvents; subscripts ref and sample denote rhodamine 6G and dye or dye-G4 complex, respectively.
Molar absorptivity coefficients for the complexes at the absorption maximum were calculated from the absorbance and concentration data obtained for each dye-G4 complex. Brightness S32 (B) of each complex and of the dyes alone was subsequently calculated through the following formula:

= ×
where Φ is fluorescence quantum yield and εmax is the molar absorptivity coefficient in the absorption maximum. As above, the subscript sample denotes the dye or dye-G4 complex.