A Light-Up Strategy with Aggregation-Induced Emission for Identification of HIV-I RNA-Binding Small Molecules.

Fluorescence methods are important tools to identify RNA-binding small molecules and further employed to study RNA-protein interactions. Most reported fluorescence strategies are based on covalent labeling of ligand or RNA, which can impede the binding between them to some extent, or light-off fluorescent indicator displacement methods, which ask for particular indicators. Herein, a label-free fluorescence strategy based on the light-on aggregation-induced emission (AIE) feature of tetraphenylethene (TPE) derivative to screen RNA-binding small molecules is presented. As a result of electrostatic interaction, the selected peptides can induce self-assembly of the TPE derivative to produce strong fluorescent emission; when the peptides are bound to RNA molecules, the TPE derivative is in the deaggregated form and shows no or minimum fluorescence. Based on the phenomenon, a competitive displacement assay combined with the TPE reporter was employed to characterize selected small molecules for their binding abilities to HIV-I RNAs. This AIE feature enables the fluorescence-off state of the TPE derivative in the presence of RNA-peptide complex to be "lightened up" quickly as the RNA-binding molecule is introduced and the peptide is competitively released. This strategy was carried out to test several small molecule binders, and the results are consistent with previous reports. This report gives an inspiring example of AIE-based fluorescent assay for HIV-I RNA-binding molecule screening, which may further be explored to build a drug screening platform for RNA-protein interference.

Chimeric Aptamers-Based and MoS2 Nanosheet-Enhanced Label-Free Fluorescence Polarization Strategy for Adenosine Triphosphate Detection.

Adenosine triphosphate (ATP) as a primary energy source plays a unique role in the regulation of all cellular events. The necessity to detect ATP requires sensitive and accurate quantitative analytical strategies. Herein, we present our study of developing a MoS2 nanosheet-enhanced aptasensor for fluorescence polarization-based ATP detection. A bifunctional DNA strand was designed to consist of chimeric aptamers that recognize and capture ATP and berberine, a fluorescence enhancer. In the absence of ATP, the DNA strand bound to berberine will be hydrolyzed when Exonuclease I (Exo I) is introduced, releasing berberine as a result. In contrast, when ATP is present, ATP aptamer folds into a G-quadruplex structure; thus, the complex can resist degradation by Exo I to maintain berberine for fluorescent detection purpose. In addition, to magnify the fluorescence polarization (FP) signal, MoS2 nanosheets were also adopted in the system. This nanosheets-enhanced FP strategy is simple and facile which does not require traditional dye-labeled DNA strands and complex operation steps. The developed fluorescence polarization aptasensor showed high sensitivity for the quantification of ATP with a detection limit of 34.4 nM, performing well both in buffer solution and in biological samples.

Fluorescent DNA-Protected Silver Nanoclusters for Ligand-HIV RNA Interaction Assay.

Studying ligand-biomacromolecule interactions provides opportunities for creating new compounds that can efficiently regulate specific biological processes. Ribonucleic acid (RNA) molecules have become attractive drug targets since the discovery of their roles in modulating gene expression, while only a limited number of studies have investigated interactions between ligands and functional RNA molecules, especially those based on nanotechnology. DNA-protected silver nanoclusters (AgNCs) were used to investigate ligand-RNA interactions for the first time in this study. The anthracycline anticancer drug mitoxantrone (MTX) was found to quench the fluorescence of AgNCs. After adding human immunodeficiency virus trans-activation responsive region (TAR) RNA or Rev-response element (RRE) RNA to AgNCs-MTX mixtures, the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs. The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations. MTX-RNA interactions were further confirmed in fluorescence polarization and mass spectrometry experiments. The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored. This study provides a new strategy for ligand-RNA binding interaction assay.

A fluorescence aptasensor based on two-dimensional sheet metal-organic frameworks for monitoring adenosine triphosphate.

In the present study, a facile fluorescence aptasensor based on two-dimensional sheet metal-organic frameworks of N,N-bis(2-hydroxyethyl)dithiooxamidato copper(II) (H2dtoaCu) was developed for the sensitive detection of adenosine triphosphate (ATP). The sensing mechanism was based on the noncovalent interaction between FAM-labeled (fluorescein amidite) ATP aptamers and H2dtoaCu. In the absence of ATP, the FAM-labeled aptamer readily adsorbs onto H2dtoaCu, mainly via π-π stacking and hydrogen bond interactions between the nucleotide bases and the H2dtoaCu surface, leading to the reduction of fluorescence intensity of the FAM by photoinduced electron transfer (PET). In the presence of ATP, the FAM-labeled aptamer specifically forms ATP-binding aptamer complexes which exhibit only weak adsorption on the H2dtoaCu surface. Thus, the fluorescence of the FAM-labeled ATP aptamer remained largely unchanged. The fluorescence aptasensor exhibited a good linear relationship between the fluorescence intensity and the logarithm concentration of ATP over a range of 25-400 nM, with a detection limit of 8.19 nM (3S/N). ATP analogs such as guanosine triphosphate, uridine triphosphate, and cytidine triphosphate have negligible effect on the aptasensor performance due to the high selectivity of the ATP aptamer to its target, showing promising potential in real sample analysis.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 ß-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.

Biomass-derived highly porous functional carbon fabricated by using a free-standing template for efficient removal of methylene blue.

Banana peel (BP), a biomass waste, was converted into a valuable highly porous functional carbon material (HPFCM) by a general chelate-assisted co-assembly process. The HPFCMs were fabricated by using Al(III)-based metal-organic framework-like as a free-standing template and commercial Pluronic F127 as a microstructure-directing agent. Several critical variables for fabrication including doses of Al(III) and F127, carbonization temperature had been optimized and the adsorption behavior of HPFCMs was examined by using methylene blue as dye model compound. The optimal adsorbent was validated as HPFCMs-5-1-800, and its equilibrium data were well fitted to the Langmuir isotherm model with a monolayer adsorption capacity of 385.12 mg g(-1) at ambient temperature. The surface physical properties of HPFCMs-5-1-800 were also exemplarily characterized. The findings revealed that the free-standing template is a potential route for preparation of HPFCM from waste BP.