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.

NMR Structure of a Triangulenium‐Based Long‐Lived Fluorescence Probe Bound to a G‐Quadruplex

“…Relative conformation of morpholino groups with respect to the triangulenium core of the free and CMA-bound ligand could reduce the intramolecular quenching to differed extents and may explain the enhancement in fluorescence intensity when DAOTA-M2 is bound to G-quadruplex. [6] Our current study provides structural rationale for hypothesis [6,7] that DAOTA-M2 has two different excited state lifetimes. Favorable geometric feature of DAOTA-M2 offer extensive stacking in comparison to most reported G-quadruplex ligands that cover only one or two guanines in G-quartet.…”

“…[6] Thekey feature of this probe is that it shows significantly longer fluorescence lifetimes when bound to G-quadruplex DNAt han when it binds to other topologies such as duplex or single-stranded DNA. [7] With the aim of further rationalizing the behavior of DAOTA-M2 and to aid us in the development of even better and more selective triangulenium-based probes,w eh erein present detailed NMR studies to characterize the interaction between DAOTA-M2 and an intramolecular G-quadruplex DNAs tructure.W ef ocused on the modified 19-nucleotide sequence d[TAGGGAGGGTAGGGAGGGT]o riginating from the NHE III 1 region upstream of the P1 promoter of the oncogene c-myc,t he overexpression of which has been implicated in aw ide variety of human cancers. [8] This sequence contains four guanine tracts and has been shown to form aw ell-defined parallel-stranded G-quadruplex (named CMA) under physiologically relevant K + ion concentrations ( Figure 1b).…”

“…Ther elative conformation of the morpholino groups with respect to the triangulenium core of the free and CMA-bound ligand could reduce the intramolecular quenching to differed extents and may explain the enhancement in fluorescence intensity when DAOTA-M2 is bound to G-quadruplexes. [6] Our current study provides as tructural rationale for the hypothesis [6,7] that DAOTA-M2 has two different excitedstate lifetimes.The favorable geometric features of DAOTA-M2 offer extensive stacking in comparison to most reported G-quadruplex ligands that cover only one or two guanines in the G-quartet. [10] Thea pparent differences between the binding affinities and dynamics of interaction of DAOTA-M2 could be attributed to factors such as rearrangement of extra-quartet residues that influence the position of the ligand on outer G-quartets.W eo bserve similar rearrangement of terminal residues to those reported for complexes of Gquadruplexes with acridine BSU6039 and quindoline.…”

Fluorescence pH Probes Based on Photoinduced Electron Transfer Quenching of Long Fluorescence Lifetime Triangulenium Dyes

“…The reduction potential of DAOTA is 350 mV higher than that of ADOTA, and this chromophore should therefore be less prone to PET quenching from the phenol units, as well as it is less quenched by amino acids and DNA bases ,. To investigate this, measurements similar to those described for 3 a – 3 cPF 6 were performed on the DAOTA compounds 5 a – 5 c .…”

Pyridoacridines in the 21st Century

“…Electrophilic substitution of diazaoxatriangulenes such as 162 , for example nitration and Vilsmeier formylation, occurs on the benzene ring with two nitrogens attached, and ortho to a nitrogen . A study of the interaction of trianguleniums as optical probes for G‐quadruplexes, showed 163 to be a unique fluorescence probe . Interaction with a G‐quadruplex from the promoter region of the c‐myc oncogene revealed that they interact at 1:2 binding stoichiometry.…”