DNAzyme-Based Microscale Thermophoresis Sensor

Abstract

Microscale thermophoresis (MST) technology has emerged as a powerful growing method in a molecular interaction study by measuring fluorescence responses of molecules inside a capillary to infrared (IR) laser heating with the benefits of rapid ratiometric measurement, separation-free, no immobilization, and low sample consumption. Combining the advantages of RNA-cleaving DNAzymes in target recognition and enzymatic catalysis and the strength of MST technology for fluorescence signaling, here, we reported a DNAzyme-based MST method for sensitive target detection. We introduced a fluorescein terminal label at the RNA-cleaving DNAzyme, and the substrate was linked to DNAzyme together with a poly-T sequence in a unimolecular design or not conjugated with DNAzyme in a bimolecular design. The presence of the cofactor activated DNAzyme to catalytically cleave the substrate, causing molecular structure alteration and significant changes in MST signals. This DNAzyme MST sensor enabled sensitively detecting activator targets Pb2+ and l-histidine, with a detection limit of 49 pM Pb2+ and 3.9 μM l-histidine. This biosensing strategy is universal and promising for wide applications.

Aptamer–Protein Interactions: From Regulation to Biomolecular Detection

“…The intrinsic properties of molecules, including their size, charge, and hydration shell, determine their thermophoretic movements. , In general, when the binding of aptamers to target proteins occurs, the above-mentioned properties of aptamers change and thus alter their movement . Information on aptamer–protein interactions can be obtained by monitoring the distribution and intensity changes in fluorescence signals of fluorescent dye-labeled aptamers. , …”

“…306 Information on aptamer−protein interactions can be obtained by monitoring the distribution and intensity changes in fluorescence signals of fluorescent dye-labeled aptamers. 307,308 QCM is a microgravimetric sensing strategy for determining aptamer−protein interactions. 309−311 Utilizing the piezoelectric effect of quartz crystals, QCM converts the surface mass change of the quartz crystal electrode into frequency shift of the quartz crystal resonator.…”

Non-covalent dyes in microscale thermophoresis for studying RNA ligand interactions and modifications

“…MST offers the potential to evaluate turnover rates of enzymatic reactions, especially in cases in which no direct detection via uorescence reporter exists. 38,39 Therefore, we sought to determine the intrinsic difference of the substrate's m 6 A modication in thermophoresis behaviour. For this purpose, we simulated different m 6 A methylation levels from 0-100% by mixing the synthetic substrate oligo with its methylated analogue at a total RNA concentration of 100 nM (Fig.…”

Profiling Additional Effects of Aptamer Fluorophore Modification on Microscale Thermophoresis Characterization of Aptamer–Target Binding

“…TRIC reflects fluorescence response of the fluorophores to heating, which is governed by the intrinsic property of fluorophores, local environment of fluorophores, and conformation of fluorescently labeled molecules . Studies have shown that TRIC can be the main contributor for MST signal. , Compared with other methods for the molecular interaction study, − MST has strengths in trace sample consumption, free separation, free immobilization, high throughput, easy sample handling, high speed, and ratiometric analysis. − MST can analyze various types of biomolecular interactions, such as protein–protein interactions, nucleic acid–protein interactions, small molecule–protein interactions, etc. − , Specially, MST is not only useful in molecular interaction studies but also promising for target quantification. ,− …”