Computational understanding and experimental characterization of twice-as-smart quadruplex ligands as chemical sensors of bacterial nucleotide second messengers.

Jie Zhou,David Monchaud,Benjamin T Roembke,Herman O Sintim,Aurélien Laguerre,Gabor Paragi,Célia Fonseca Guerra

doi:10.1038/srep33888

Abstract

A twice-as-smart ligand is a small molecule that experiences a structural switch upon interaction with its target (i.e., smart ligand) that concomitantly triggers its fluorescence (i.e., smart probe). Prototypes of twice-as-smart ligands were recently developed to track and label G-quadruplexes: these higher-order nucleic acid structures originate in the assembly of four guanine(G)-rich DNA or RNA strands, whose stability is imparted by the formation and the self-assembly of G-quartets. The first prototypes of twice-as-smart quadruplex ligands were designed to exploit the self-association of quartets, being themselves synthetic G-quartets. While their quadruplex recognition capability has been thoroughly documented, some doubts remain about the precise photophysical mechanism that underlies their peculiar spectroscopic properties. Here, we uncovered this mechanism via complete theoretical calculations. Collected information was then used to develop a novel application of twice-as-smart ligands, as efficient chemical sensors of bacterial signaling pathways via the fluorescent detection of naturally occurring extracellular quadruplexes formed by cyclic dimeric guanosine monophosphate (c-di-GMP).

Highlights

Flexible and their quadruplex affinic conformation is triggered by the interaction with targets only: the like-likes-like recognition that takes place between native and synthetic G-quartets makes TASQ pioneering smart quadruplex ligands[17,18]
The ability of PyroTASQ to interact with nucleic acids was firstly studied at two levels, i- its affinity for both DNA- and RNA-quadruplexes and its selectivity over duplexes, and ii- its capability of fluorescently label quadruplexes and its selectivity over duplexes
The results collected through FRET-melting[21,22], an in vitro assay routinely used for quantifying the ligand/DNA interactions in a convenient manner, indicate that PyroTASQ displays a high affinity for a series of quadruplex-forming oligonucleotides (QFO) including DNA sequences found in the human telomeres[23] and in the promoter region of myc and kit genes[3] (F21T, F-myc-T and F-kit-T, respectively), and RNA sequences found in telomeric transcripts and in the untranslated region of mRNA of the telomeric repeat binding factor 2 and vascular endothelial growth factor genes (L-TERRA, L-TRF2 and L-VEGF, respectively)[24,25]

Summary

Results

PyroTASQ efficiently labels DNA and RNA quadruplexes. The ability of PyroTASQ to interact with nucleic acids was firstly studied at two levels, i- its affinity for both DNA- and RNA-quadruplexes and its selectivity over duplexes, and ii- its capability of fluorescently label quadruplexes and its selectivity over duplexes. Ground and excited state optimizations show that, while the ‘closed’ conformation models do not structurally change upon excitation (i.e., the quartet and pyrene remain coplanar), the ‘semi-closed’ model experiences deep structural reorganizations, with the isolated guanine that tries to move from a planar to a perpendicular position with respect to the template plane These data allow for proposing a new mechanism (Fig. 3C,D): alone in solution, PyroTASQ adopts an ‘semi-closed’ conformation in which at least one but probably two guanines stack on the pyrene template. These results highlight that PyroTASQ is a promising sensor, capable of disrupting dinucleotide signaling pathways (by promoting the aggregation of the c-di-GMP messenger) and offering a convenient way to detect therapeutically useful biomarkers (by fluorescently labeling the resulting c-di-GMP/TASQ aggregates)

Discussion

Computational part

Additional Information