Nano Temper Dianthus

Description

Spectral Shift
Spectral Shift detection quantifies molecular interactions between one binding partner (target) that is labeled with a specific fluorophore optimized to report subtle environmental changes, and another unlabeled binding partner (ligand). Spectral shift detects ligand induced changes in the hydrophobicity of the target biomolecule’s surface by measuring picometer shifts in the fluorescence emission at two wavelengths, 650nm and 670nm. A ratio metric signal of the labeled target as a function of ligand concentration is measured to obtain a dose-response curve.

As a rapid, isothermal measurement, it is non-destructive permitting sample re-analysis, temperature-sensitive studies and studies of sensitive molecules that destabilize quickly. The highly sensitive and robust detection allows work with small amounts of labeled target and suboptimal samples containing aggregation or precipitation.

Temperature-related intensity change (TRIC)

TRIC also measures the interaction between the fluorescently labeled target and unlabeled ligand, using the same samples as Spectral Shift. TRIC detects ligand induced changes in the target’s shape and structural flexibility by measuring the fluorophore response to a small temperature perturbation. compound induced precipitation, providing additional insights into sample quality.

System components
The Dianthus system comprises a benchtop instrument and software for control and analysis. A control computer operates the system, and samples are
introduced through a plate gate at the front. No fluidics, pumps or valves are Beyond Kd determination, TRIC is sensitive to aggregation or
required minimizing daily system maintenance. Dianthus measures at a stable temperature, with active temperature control between 20 – 25°C.
Dianthus is operated as a standalone instrument, or it can be integrated into automation systems (via gRPC framework) enabling walk away operation and hands-free drug discovery workflow processing.

Software

The Dianthus software consists of a control module and an analysis module. The modules have intuitive user interfaces that minimize the time required for experimental setup, assay development, and single dose or dose-response experimental analysis. System installation and introductory training are performed in just one day. Extensive supporting information is provided online at support.nanotempertech.com.

Consumables:

384-well microplates Dianthus uses dedicated 384-well, low profile, black walled, sealable, optically clear, flat-bottomed microplates. This plate design enables direct bottom reading of fluorescence signal avoiding well to well crosstalk. The plates are polymer coated to prevent analyte
adsorption and are uniquely barcoded for identification. The microplates have standard SBS format for compatibility with manual or automated liquid handlers, as well as automated plate handling devices.

Dianthus Screening Analysis
Dianthus Screening Analysis Software provides easy analysis, interpretation & visualization of data from single-dose screening or does response curves using algorithms that also flags outliers, such as precipitation or aggregation. Data can be exported as either .csv or .xlsx for external analyses or formatted for report filing.

Labeling Kits
NanoTemper provides a range of kits specifically optimized for highest sensitivity and optimal signal to noise ratios. The kits
are designed for simple and quick labeling of the target binding partner, permitting rapid assay development, optimization and time to data. The kits contain labeling reagents that attach fluorophores to a specific functional group or fusion tag via covalent or affinity binding. Labeling kits come in multiple size options to accommodate throughput requirements.

Applications
Dianthus is a powerful tool for early-stage drug discovery. It provides single point screening data for Hit ID, dose response data for lead verification, and valuable binding (Kd or EC50) information for Structure Activity Relationships (SAR) studies and hit-to-lead optimization. With the in-solution measurement, Dianthus can address a range of both targets and therapeutic modalities. Examples of use cases are shown below. In-depth application data can be found online in the resource center at resources.nanotempertech.com/application-notes, under the Dianthus tab.

1. Primary Screening of fragment or target-focused compound libraries Adopt Dianthus’ biophysical technology for affinity-based screening of libraries numbering hundreds to thousands of compounds, such as, targeted and focused libraries or fragment libraries. Confirm hits with dose-response curves prior to orthogonal assay analysis.
• Generate reproducible and reliable screening results, with assays regularly achieving Z’ scores > 0.8, and easily >0.5 once assay development is completed.
• Measure binding directly for targets lacking functional or biochemical read-outs e.g., transcription factors or scaffold proteins.
• Perform direct target engagement irrespective of ligand binding site where FRET based approaches are unsuitable.
• Investigate very low affinity (mM) interactions and assess low Dalton sized compounds, e.g., fragments or ions, problematic for other biophysical methods.

• Analyze bivalent analytes, such as heterobifunctional degraders (PROTACs, molecular glues) for binary/ternary complex affinities,cooperativity, and hook effects. Use binary complex assay conditions to progress to ternary complex measurement.
• Investigate covalent inhibitor binding directly with a biophysical readout: no surface = no regeneration.
• Maintain the conformational plasticity of intrinsically disordered proteins (IDPs) by direct in-solution measurement to provide binding studies with data integrity.
• Study membrane protein interactions when solubilized in detergents or within synthetic membrane models such as nanodiscs.
• Directly measure interactions between nucleic acids & small molecules. Cy5 can be used for RNA and DNA labeling.

2. Hit confirmation & validation
Measure binding affinity curves for a range of biomolecular interactions to help determine Structure-Activity-Relationships (SAR). Enable progression of screening hits to leads for lead optimization.
• Understand large multi-complex protein-protein, protein-nucleic interactions.

3. Additional applications – Research and Development
Dianthus is an ideal addition to other biophysical instrumentation within core facilities offering services to internal and external users.
• Address the diverse range of target & ligand molecules, including interactions within complex matrices or samples containing aggregation, which can occur within the research environment.
• Provide simple assay development, and rapid buffer condition testing, to swiftly enable both novice or experienced users obtain data with minimum of fuss and avoiding issues with sensor maintenance.

Specifications

General specifications

Environmental and Dimensions
Size: Width: 61cm (24.0”), Height: 42cm (16.5”), Depth (closed tray): 57cm (22.4”)
Depth (open tray): 69cm (27.2”) , Weight: 70kg (154.3 lbs) net
Operating temperature: 20 – 30 °C (indoor only)
Pollution Degree: 2

Detection technology	:	Spectral Shift, Temperature-related intensity change (TRIC)
Information obtained	:	Single dose ligand categories: binder, nonbinder, outlier; Dose-response: Kd, EC50;
Data presentation	:	Graphics and tables.  Data export format: Excel or CSV (manual mode), JSON (automation mode)
Sample type1	:	Small molecules, fragments, ions, heterobifunctional degraders2 , peptides, proteins3, biologics, RNA/DNA
Sample volume	:	20 µL / well
Sample format	:	Black, flat clear bottom, sealable, polystyrene 384-well barcoded plates
Instrument sample capacity	:	384-well plate
Run time per plate	:	33 min (Spectral Shift), 79 min (Spectral shift + TRIC) Time to obtain a Kd (12-point dilution series): 60 sec (Spectral Shift); 132 sec (Spectral Shift + TRIC)
Time to obtain a Kd (12-point dilution series	:	): 60 sec (Spectral Shift); 132 sec (Spectral Shift + TRIC)
Reproducibility of affinity measurement (32 biological replicates)	:	mean EC50 = 29 µM 95% CI [23.1 µM to 36.6 µM]
Automation	:	Integration with liquid handling systems and automation platforms via gRPC framework
Electric	:	Input Voltage: Single phase AC 100-240 V ± 10% Input Current: AC 6-3.2 A Mains frequency: 50/60Hz
Typical working ranges Temperature control range	:	20 – 25 °C
Maximum difference to room temperature	:	± 5 °C
Temperature control accuracy	:	± 0.25 °C
Affinity range	:	270 pM to mM (Dianthus Pico), 5 nM to mM (Dianthus Nano)
Molecular Weight range	:	101 – 107 Da