Binding affinities on all scales from fragment screening to intrinsically disordered proteins with isothermal spectral shift technology

The precise characterisation of biomolecular interactions, their binding constants and stoichiometries, lie at the heart of understanding events in cells and disease. Furthermore, the discovery and development of new drugs often requires the screening of chemical libraries containing hundreds if not many thousands of chemical compounds. After a small number of hit compounds are discovered, the optimisation towards a promising lead compound is greatly aided by a precise knowledge of their binding to their biological targets. Among the many different screening technologies, biophysical techniques that precisely measure the strength of the binding (binding constant Kd) and the composition of the complex (stoichiometry) are often considered the gold standard. However, many therapeutic targets are challenging and some are still entirely within the reach of current biophysical techniques. These include many membrane proteins that cannot be produced at the required amount or concentration. The Dianthus by NanoTemper Technologies, based on a novel spectral shift technology, represents a huge leap in sensitivity which enables the precise measurement of binding constants in very small volumes (less than five microliter) and at very low (nanomolar) concentrations. Hence, analyses of the most challenging protein targets, such as membrane proteins that often require detergents and intrinsically disordered proteins that tend to precipitate even at moderate concentrations, become feasible. Furthermore, combining this technology with high-throughput methods allows the measurements of 384 wells in parallel in less than one hour, which allows the screening of entire chemical libraries with thousands of compounds. Importantly, the Dianthus system will be the centre piece of the biophysical screening platform at the Biophysical Sciences Institute at Durham University which includes a wide range of complementary instrumentation. Taken together with the technical and scientific expertise, the platform offers a unique set of instruments to the academic and industrial user community.

The Dianthus features the latest innovation for measuring binding affinities, an isothermal spectral shift measurement, coupled with the proven TRIC (Temperature Related Intensity Change) technology. The isothermal spectral shift method pioneered in the Dianthus system is based on the well-known phenomenon that organic fluorophores can report changes in their chemical environment by very small modifications of their emission spectrum, e.g. changes in their fluorescence intensity or wavelength. This principle is used to quantify biomolecular interactions in solution with the Dianthus, whereby spectral shift measurements enable precise characterization of interactions for a variety of biomolecules including proteins, antibodies and nucleic acids with each other and with chemical compounds. For this the instrument uses a newly developed dual-emission optical system where, instead of measuring the full emission spectrum, the fluorescence is recorded simultaneously and precisely at two pre-selected wavelengths. (670 nm and 650 nm). A novel ratiometric approach allows the resolution of sub-nanometer changes in the emission spectrum of the fluorescently labelled molecule, making this methodology highly sensitive towards ligand binding events. In addition, the Dianthus instrument can still quantify binding events using the well-established TRIC technology, affording researchers two independent techniques in one platform. Moreover, both methods do not require conformational changes of the target molecule upon binding and use only a few microliters of sample at low nanomolar concentration per data point. The instrument acquires data from standard 384-well plates allowing flexible layouts for both single-dose screening as well as affinity screening. Moreover, it only takes 33 minutes to measure a full plate using spectral shift. Both spectral shift and TRIC measurements require the target molecule to be fluorescently labelled using well-established kits from NanoTemper.