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      Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes

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          Abstract

          RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV–Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.

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          The application of DNA and RNA G-quadruplexes to therapeutic medicines.

          The intriguing structural diversity in folded topologies available to guanine-rich nucleic acid repeat sequences have made four-stranded G-quadruplex structures the focus of both basic and applied research, from cancer biology and novel therapeutics through to nanoelectronics. Distributed widely in the human genome as targets for regulating gene expression and chromosomal maintenance, they offer unique avenues for future cancer drug development. In particular, the recent advances in chemical and structural biology have enabled the construction of bespoke selective DNA based aptamers to be used as novel therapeutic agents and access to detailed structural models for structure based drug discovery. In this critical review, we will explore the important underlying characteristics of G-quadruplexes that make them functional, stable, and predictable nanoscaffolds. We will review the current structural database of folding topologies, molecular interfaces and novel interaction surfaces, with a consideration to their future exploitation in drug discovery, molecular biology, supermolecular assembly and aptamer design. In recent years the number of potential applications for G-quadruplex motifs has rapidly grown, so in this review we aim to explore the many future challenges and highlight where possible successes may lie. We will highlight the similarities and differences between DNA and RNA folded G-quadruplexes in terms of stability, distribution, and exploitability as small molecule targets. Finally, we will provide a detailed review of basic G-quadruplex geometry, experimental tools used, and a critical evaluation of the application of high-resolution structural biology and its ability to provide meaningful and valid models for future applications (255 references).
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            Structural basis for activity of highly efficient RNA mimics of green fluorescent protein.

            GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
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              Thioflavin T as an efficient inducer and selective fluorescent sensor for the human telomeric G-quadruplex DNA.

              The quest for a G-quadruplex specific fluorescent sensor among other DNA forms under physiological salt conditions has been addressed in this article. We demonstrate for the first time the application of a water-soluble fluorogenic dye, Thioflavin T (ThT), in a dual role of exclusively inducing quadruplex folding in the 22AG human telomeric DNA, both in the presence and absence of Tris buffer/salt, and sensing the same through its fluorescence light-up having emission enhancement of the order of 2100-fold in the visible region. Appropriate conditions allow an apparent switch over of the parallel quadruplex structure in 22AG-ThT (50 mM Tris, pH 7.2) solution to the antiparallel form just by the addition of K(+) ions in the range 10-50 mM. Moreover, addition of ThT cooperatively stabilizes the K(+) induced antiparallel quadruplexes by a ΔT(m) ∼11 °C. The distinction of ThT as a quadruplex inducer has been contrasted with the erstwhile used structurally related dye, Thiazole Orange (TO), which did not induce any quadruplex folding in the 22AG strand in the absence of salt. The striking fluorescence light-up in ThT on binding to the human telomeric G-quadruplex is shown to be highly specific compared to the less than 250-fold enhancement observed with other single/double strand DNA forms. This work has implication in designing new generation dyes based on the ThT scaffold, which are highly selective for telomeric DNA, for potential diagnostic, therapeutic, and ion-sensing applications.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                21 April 2016
                2016
                : 6
                : 24793
                Affiliations
                [1 ]National Laboratory for Molecular Sciences, Centre for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry Chinese Academy of Sciences , Beijing, 100190, P. R. China
                [2 ]University of the Chinese Academy of Sciences , Beijing, 100049, P. R. China
                Author notes
                Article
                srep24793
                10.1038/srep24793
                4838840
                27098781
                86ce2a28-0b5e-428d-a02a-f98b6fcc64cf
                Copyright © 2016, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 07 January 2016
                : 05 April 2016
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