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

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.One of the most important types of nucleic acid structure is the G-quadruplex (G4), which is formed from four guanine bases by stacking of Hoogsteen bonded G-quartets 1 . The G-quadruplexes play a vital role in the human genome and transcriptome 2,3 . The DNA or RNA G-quadruplexes that are formed in cells are associated with many important cellular processes 4-8 . G-quadruplexes have been viewed as emerging therapeutic targets due to their correlation with human diseases [8][9][10][11][12] . Most of the early studies of G-quadruplexes focused on DNA strands, and few studies focused on RNA G-quadruplexes. Recently, RNA G-quadruplexes have been associated with many biological processes, such as telomere maintenance, pre-mRNA splicing and polyadenylation, RNA turnover, mRNA targeting and translation 13 . Therefore, the development of techniques that could efficiently recognize RNA G-quadruplexes structures and investigate their biological functions and impacts is highly desirable. Many techniques including X-ray crystallography and NMR experiments have been utilized to identify high-resolution structures of RNA G-quadruplexes 14,15 . However, these techniques are more suitable for comprehensively studying targeted RNA G-quadruplex structures. Additionally, circular dichroism (CD) has been extensively used to monitor G-quadruplex formation because the positive band at 264 nm and the negative band at 240 nm indicate the formation of parallel-type G-quadruplex structures 16 . However, it is difficult to interpret the G-quadruplex type in the presence of different forms of nucleic acids. Because the probe recognition sites in RNA G-quadruplexes are different from those in other RNA motifs, small molecules that selectively bind to RNA G-quadruplexes and emit fluorescence may be used as RNA G-quadruplex detectors. Our previous study revealed a cyanine dye (CyT) that was able to selectively recognize RNA G-quadruplex structures with ~2000-fold fluorescence enhancement 17 . Due to the important biological functions of RNA G-quadruplex, the development of a multiband probe that can selectively recognize RNA G-quadruplex structures is needed to further expand the application of RNA G-quadruplexes, which have been recognized as significant molecular...

Role of Achiral Nucleobases in Multicomponent Chiral Self‐Assembly: Purine‐Triggered Helix and Chirality Transfer

“…First, there are two negative Cotton effects located at 301 and 311 nm, which show red-shift relative to Fmoc-Glu/A. [11] It is reported that the fluorescence of ThTi ncreases remarkably when it is bound to amyloid fibers or other hydrophobic patches because its freely rotating aromatic rings become fixed. Although Fmoc-Glu/T showed CD signals,i td id not exhibit obvious helical nanostructures.T his indicates that Aa nd Gi nduced more effective expression of helicity in the assemblies than Tand C. Thef luorescence spectra of Fmoc-Glu with four nucleobases are shown in Figure 2D.I nc omparison with plain Fmoc-Glu, Aa nd Gc ause ar ed-shift in the fluorescence emission, while Ta nd Ca lmost maintain the Fmoc-Glu emission, indicating that in the Fmoc-Glu/A (G) system, p-p stacking is stronger than that of the T( C) system.…”

“…Thioflavin T(ThT), as the most popular fluorescent probe,is used for the detection of amyloid fibers and also to study gel formation, due to its environment-dependent emission behavior. [11] It is reported that the fluorescence of ThTi ncreases remarkably when it is bound to amyloid fibers or other hydrophobic patches because its freely rotating aromatic rings become fixed. [12] In this context, ThTw as selected as an indicator to investigate whether the chiral sense of the helix in Fmoc-Glu/A (G) transferred to the achiral moiety.…”

Identification of Compounds that Selectively Stabilize Specific G‐Quadruplex Structures by Using a Thioflavin T‐Displacement Assay as a Tool

“…Such chemical scaffolds are interesting as potentialt herapeutics, for example, the G4-binding compound quarfloxin (CX-3543) reached phase II clinical trials, [12] and as chemicalr esearch tools to furthers tudy the biological functions of G4 structures. [16] We first confirmed that ThT emits af luorescent signal when bound to differentG4structures and then demonstrated that the fluorescent signal was significantly reduced upon displacement of ThT by the G4 ligand PhenDC3. [14] Herein, we have developedacost-effective HTS to find selective G4-binding compounds without utilizing labeled oligonucleotides.…”

“…The HTS process wasb ased on the fluorescent lightup probe Thioflavin T( ThT), [15] which shows selectivity for G4 DNA and G4 RNA over single-or double-stranded DNA (dsDNA). [16] We first confirmed that ThT emits af luorescent signal when bound to differentG4structures and then demonstrated that the fluorescent signal was significantly reduced upon displacement of ThT by the G4 ligand PhenDC3. Therefore, this assay [17] was developed to be used in aH TS format.…”

An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21

“…Recombinant purified DDX21 showed marginally higher affinity for TV2 vs TV2m (100nm vs 300nm) TV2 and TV2m were assayed for rG4 formation by ThT, a fluorescent probe for RNA rG4 formation 35,36 . When ThT is bound to rG4 it exhibits enhanced fluorescence.…”