Trianguleniums as Optical Probes for G-Quadruplexes: A Photophysical, Electrochemical, and Computational Study

Abstract

Nucleic acids can adopt non-duplex topologies, such as G-quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA-M2 as a unique fluorescence probe for such studies. This probe's emission lifetime is highly dependent on the topology of the DNA it interacts with opening up the possibility of carrying out live-cell imaging studies. Herein, we describe the origin of its fluorescence selectivity for G-quadruplexes. Cyclic voltammetry predicts that the appended morpholino groups can act as intra- molecular photo-induced electron transfer (PET) quenchers. Photophysical studies show that a delicate balance between this effect and inter-molecular PET with nucleobases is key to the overall fluorescence enhancement observed upon nucleic acid binding. We utilised computational modelling to demonstrate a conformational dependence of intra-molecular PET. Finally, we performed orthogonal studies with a triangulenium compound, in which the morpholino groups were removed, and demonstrated that this change inverts triangulenium fluorescence selectivity from G-quadruplex to duplex DNA, thus highlighting the importance of fine tuning the molecular structure not only for target affinity, but also for fluorescence response.

Keywords: DNA; nucleic acids; optical probes; quadruplexes; triangulenium.

Scheme 1

Synthetic route and chemical structure of trianguleniums studied in this work. (i) Py⋅HCl, 200 °C, 1 h; (ii) 4‐(2‐aminoethyl)morpholine, N‐methyl‐2‐pyrrolidone (NMP), room temperature, 2 h then NH₄PF₆ (aq.); (iii) RNH₂, NMP, 110 °C, 2 h then NH₄PF₆ (aq.).

Figure 1

Unusual fluorescence lifetime properties of DAOTA‐M2 depicts the effect of double stranded DNA (CT‐DNA, 10 base pair equivalents) on the emission of TOTA, ADOTA‐M and DAOTA‐M2. A) The spectra before (black) and after (green) addition of CT‐DNA (10 mm lithium cacodylate buffer, pH 7.3, 100 mm KCl) are normalised to compound only intensity maximum to highlight the fluorescence quenching and enhancement observed (the quantum yields of the free dyes under this conditions are: Φ=0.100 for TOTA; Φ=0.042 for ADOTA‐M ¹¹; Φ=0.032 for DAOTA‐M2 ¹¹). B) Fluorescence lifetime decay of TOTA (yellow, τ ₁=8.6 ns) and DAOTA‐M2 (grey, τ ₁/f ₁=1.3 ns/93 %, τ ₂/f ₂=3.7 ns/8 %) at physiological conditions (10 mm lithium cacodylate buffer, pH 7.3, 100 mm KCl), as was previously reported. C) Dependence of DAOTA‐M2 fluorescence lifetime on the nucleic acid topology (duplex CT‐DNA in green and G‐quadruplex myc2345 in red).

Figure 2

Electrochemical properties of TOTA, ADOTA‐M and DAOTA‐M2. Representative cyclic voltammograms were recorded in degassed acetonitrile containing 0.1 m nBu₄NPF₆where ferrocene (Fc) is used as an internal standard (see the Supporting Information, Figure S1 for the scan‐rate dependence of various cyclic voltammogram parameters and Tables S1 and S2 for computational modelling energies used to determine theoretical electrochemical properties). [a] Versus NHE; [b] IEFPCM acetonitrile solvation; [c] Using the equation ΔG _0,0=e[E(0⁺/0)−E(R/R ⁻)]−ΔG _0,0−e ²/ɛd, in which E(0⁺/0) is the oxidation potential of guanine (1.3 V vs. NHE),23 E(R/R ⁻) is the experimentally determined reduction potential, ΔG _0,0=(λ _abs,max+λ _em,max) at pH 7.3 and the negligible charge separation (e ²/ɛd) factor is omitted.24 ΔG values below zero indicate that quenching is thermodynamically favourable.

Figure 3

X‐ray crystal structure of the cation present in the crystals of ADOTA‐M.

Figure 4

View in parallel projection perpendicular to the triangulenium ring plane, showing the head‐to‐tail overlap of the triangulenium units in adjacent, centro‐symmetrically related, cations in the structure of ADOTA‐M. The C22⋅⋅⋅C22 separation between the neighbouring cations is approximately 3.67 Å, with the mean interplanar separation being approximately 3.40 Å.

Figure 5

TD‐DFT studies of the effect of the excited (S₁) and ground (S₀) state geometries on the ordering of molecular orbitals in DAOTA‐M2 and its impact upon emission. A) Superimposed optimised ground and excited state geometries. C−H bonds are omitted for clarity, and the main structural differences observed are highlighted in green (ground) and red (excited). B) Molecular orbital energies for both conformations. The dashed arrows indicate the first excited state transition as predicted by TD‐DFT (>90 % orbital contribution). C) HOMO−2 to LUMO ordering of the molecular orbitals for each geometry (see the Supporting Information, Figure S2 for ADOTA‐M data and Table S3 for oscillator strength, energies and configuration interaction (CI) coefficients).

Figure 6

Top: DAOTA‐M2 and bottom: DAOTA‐Pr2 fluorescence enhancements (left), lifetimes (middle) and decay traces (right) in the presence of various DNA topologies (colour coded). The average lifetime ($\bar{\tau }$ /ns, σ=±5 %, n=3) and fluorescence enhancement were calculated from the end point of the corresponding DNA titrations. The decay traces shown are representative of the end point of titrations for the specific DNA topology. Error bars in the left graphs and horizontal black lines in the middle graphs indicate two standard deviations from three independent repeats. For the DAOTA‐M² lifetime graph (top middle), ellipses indicate accuracy of τ ₂ and τ ₃, was as determined by F _χ statistic (F _χ=1.028/1.029, P=0.05, p=33/29, v=1600/1400 for CT‐DNA/other DNA models, respectively) from the systematic variation of τ over the lifetime range. For the DAOTA‐Pr2 lifetime graph (bottom middle), parameter accuracy was not determined, because only one lifetime component was being determined in addition to the free dye. For complete DNA titration decay fittings, numerical τ and plots of the relative contributions of lifetime components at local χ ² _R minima, see the Supporting Information, Figures S6 and S7.

Figure 7

Schematic representation of the proposed fluorescence response of DAOTA‐Pr2 (top) and DAOTA‐M2 (bottom) to double stranded DNA (left) and G‐quadruplexes (right). DAOTA‐Pr2 fluorescence is quenched by exposure to the local aqueous solvent environment. This effect is alleviated to a greater extent upon intercalation between base pairs in duplex DNA as opposed to end‐on π–π stacking with G‐quadruplexes. DAOTA‐M2 fluorescence is predominately controlled by intramolecular photo‐induced electron transfer (PET). This conformation‐dependent quenching process is alleviated to a greater extent upon binding G‐quadruplexes, opposed to duplex DNA.