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Cell‐specific aptamers offer a powerful tool to study membrane receptors at the single‐molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling‐based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer‐based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer‐receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer‐based dynamics analysis of two‐ and three‐dimensional motion and fluctuation of highly expressed receptors on the live cell membrane. [ABSTRACT FROM AUTHOR]

The use of aptamers in bioanalytical and biomedical applications exploits their ability to recognize cell surface protein receptors. Targeted therapeutics and theranostics come to mind in this regard. However, protein receptors occur on both cancer and normal cells; as such, aptamers are now taxed with identifying high vs. low levels of protein expression. Inspired by the flexible template mechanism and elegant control of natural nucleic acid-based structures, we report an allosteric regulation strategy for constructing a structure-switching aptamer for enhanced target cell recognition by engineering aptamers with DNA intercalated motifs (i-motifs) responsive to the microenvironment, such as pH. Structure-switching sensitivity can be readily tuned by manipulating i-motif sequences. However, structure-switching sensitivity is difficult to estimate, making it equally difficult to effectively screen modified aptamers with the desired sensitivity. To address this problem, we selected a fluorescent probe capable of detecting G-quadruplex in complicated biological media. [ABSTRACT FROM AUTHOR]

Aptamer binding characterization, including receptor density calculation, single-molecule colocalization, binding site determination, motion and fluctuation analysis, was performed with minimal probe and instrument requirements. Aptamers, Binding Site, Dilution, Fluorescent Probes, Motion Analysis Keywords: Aptamers; Binding Site; Dilution; Fluorescent Probes; Motion Analysis EN Aptamers Binding Site Dilution Fluorescent Probes Motion Analysis 1 1 1 02/22/23 20230301 NES 230301 B A membrane-receptor-analysis method b termed ligand dilution analysis, which combines the concepts of single-molecule localization and random sampling, is reported by Yifan Lyu, Jianhui Jiang, Weihong Tan et al. in their Research Article (e202215387). [Extracted from the article]

Aptamer binding characterization, including receptor density calculation, single-molecule colocalization, binding site determination, motion and fluctuation analysis, was performed with minimal probe and instrument requirements. Back Cover: Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single-Molecule Level (Angew. Keywords: Aptamers; Binding Site; Dilution; Fluorescent Probes; Motion Analysis EN Aptamers Binding Site Dilution Fluorescent Probes Motion Analysis 1 1 1 02/22/23 20230301 NES 230301 B A membrane-receptor-analysis method b termed ligand dilution analysis, which combines the concepts of single-molecule localization and random sampling, is reported by Yifan Lyu, Jianhui Jiang, Weihong Tan et al. in their Research Article (e202215387). [Extracted from the article]

A novel label-free fluorescent strategy for the detection of glutathione (GSH) and cysteine (Cys) is presented. The system consists of two single stranded DNA (ssDNA) with thymine–thymine (T–T) mismatches and used Hg2+ as a mediator, and N-methyl mesoporphyrin IX (NMM) as the signal reporter. The assay is based on the competitive reaction of Hg2+ with GSH/Cys and T–T mismatched double stranded DNA (dsDNA). In the absence of the target, two ssDNA containing T–T mismatches react with Hg2+ to form a T–Hg2+–T dsDNA structure in the solution, which hampers the formation of a G-quadruplex structure. However, in the presence of the target, GSH/Cys reacts with Hg2+ to keep DNA probes in a free single state, resulting in the effective formation of a G-quadruplex structure of the DNA probe (GP). Subsequently, due to the strong interaction between the G-quadruplex structure and NMM, fluorescence was greatly enhanced. This fluorescence strategy does not require any chemical modification, making the assay convenient and cost-effective. This method exhibited a linear relationship between peak fluorescence intensity and concentration of GSH in the range of 10–400 nM with a limit of detection (LOD) of 9.6 nM. A linear range for Cys detection was obtained in the concentration range of 10–500 nM with an LOD of 10 nM. Moreover, the proposed method worked well for the analysis of complex biological samples. [ABSTRACT FROM AUTHOR]

Abstract: Phosphorylation of DNA by polynucleotide kinase (PNK) takes an important role in DNA damage repair, replication and recombination. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, we present a label-free fluorescent method for PNK activity assay using double strand DNA (dsDNA) -templated copper nanoparticles (CuNPs) as a fluorescent indicator. Upon the PNK reaction, the dsDNA template is phosphorylated and then digested by λ exonuclease immediately, prohibiting the formation of fluorescent CuNPs due to the lack of dsDNA template. This homogeneous PNK activity assay does not require any other additional modifications of DNA substrate or complex design, making the proposed strategy simple, cost-effective and high throughput. The proposed strategy is sensitive, selective and exhibits a good assay performance in complex biological samples. The strategy presented here opens a new avenue for PNK assay and nucleic acid phosphorylation related research. [Copyright &y& Elsevier]