Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study

Abstract

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein–small molecule interaction, a newly developed protein–protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

The molecular basis of FimT-mediated DNA uptake during bacterial natural transformation

“…Microscale thermophoresis (MST) experiments were conducted measuring the temperature-related intensity change (TRIC) of the fluorescence signal 66 . A 12 bp fluorescently labelled dsDNA probe was generated by annealing a 5′ fluorescein (FAM)-labelled and an unlabelled strand (Microsynth; Supplementary Table 4 ) and used in all MST/TRIC experiments.…”

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins

“…FA measurements (Figure d) provided dissociation constants of 1.99–4.91 nM, which were similar to previously obtained preliminary K D values (Table S3). The results of the FA assay were confirmed by MST/TRIC experiments (Figure e, Table S6), yielding dissociation constants of 0.76–5.72 nM, respectively, with further information being provided in the Supporting Information (Figure S9). Probes 17–20 were identified as high-affinity ligands for Gαi1, which are superior to both BODIPY FL- and MANT-labeled guanine nucleotides by one to three orders of magnitude.…”

Structural Studies of Inhibitors with Clinically Relevant Influenza Endonuclease Variants

“…In the studies performed here, fluorescence was recorded at two wavelengths (650 and 670 nm), and the change in emission intensity between the 670/650 nm fluorescence signal was monitored as a function of ligand concentration (see the SI for experimental details). This method allows for the determination of binding constants ( K d ) over a large dynamic range from low nanomolar (nM) to high millimolar (mM) values. , In this study, purified PA N endonuclease was used (see the SI for experimental details) . Purified PA N is known to bind substrates and inhibitors with poorer affinity than the complete PA subunit or heterotrimer polymerase complex, which may explain why the K d values reported here (see below) are weaker than reported elsewhere .…”