nanoTAK (nano Temperature Alteration Kinetics) is a measurement mode available with Monolith Omni that allows you to measure binding kinetics, specifically the association rate constant (k_on) and dissociation rate constant (k_off), directly in solution.

Measurement Principle

Binding kinetics is a fundamental biophysical property that describes how fast two molecules associate and how long their complex remains stable. While affinity (K_d) reflects the equilibrium between bound and unbound states, kinetic rate constants reveal the dynamics of an interaction over time.

nanoTAK is built on Spectral Shift detection and uses the Monolith Omni's integrated infrared (IR) laser to apply a controlled temperature perturbation to pre-equilibrated samples held in capillaries. Because biomolecular interactions are temperature-dependent, a rapid temperature jump shifts the equilibrium between bound and unbound states. The system then relaxes toward its new equilibrium, and this relaxation is monitored in real time via the Spectral Shift signal (fluorescence ratio at 670 nm and 650 nm).

The nanoTAK measurement proceeds in four steps:

• Step 1 - Isothermal Baseline: Spectral Shift is measured isothermally at the starting temperature, establishing the equilibrium signal before any perturbation is applied.

• Step 2 - Temperature Jump: The IR laser rapidly heats the detection volume, creating a defined temperature jump (default: 5°C, e.g., from 20°C to 25°C) in a controlled and reproducible manner. This shifts the equilibrium between the bound and unbound states of the interaction.

• Step 3 - Kinetic Trace Recording: Spectral Shift is recorded over time as the system relaxes toward its new equilibrium at the higher temperature. This produces a kinetic trace for each capillary, reflecting the changing proportion of bound versus unbound labeled target molecules in real time.

• Step 4 - Capillary Cycling and Global Fitting: The next capillary moves into focus and the measurement cycle repeats across the full ligand concentration series. A global fitting algorithm is then applied simultaneously to all traces to extract k_on, k_off, and K_d.

Because multiple concentrations are measured in parallel and fitted globally, the extracted parameters are robust and well-constrained.

Assay Conditions

Labeling: One binding partner (the target) must be fluorescently labeled using a Spectral Shift-compatible labeling kit. The unlabeled binding partner (the ligand) is titrated across a concentration series in standard capillaries.

Pre-equilibration: Samples must be pre-equilibrated at the initial temperature before the measurement begins to ensure that the observed signal change reflects the kinetics of re-equilibration rather than initial binding.

Temperature Jump Parameters: The standard temperature jump is 5°C (e.g., 20°C to 25°C). The kinetics jump temperature range on Monolith Omni is 25 to 40°C (± 0.5°C).

Measurable Kinetic and Affinity Ranges

Measurement Time

Measurement time scales with the dissociation rate of the interaction, as the system must be monitored long enough to capture the full relaxation to the new equilibrium.

Correlation with Affinity Measurements

The equilibrium dissociation constant (K_d) is derived directly from the kinetic rate constants as:

K_d = k_off / k_on

This means nanoTAK provides an independent, kinetics-derived affinity estimate that can be compared with K_d values obtained from Spectral Shift or other affinity methods. However, the primary value of nanoTAK lies not in the K_d alone, but in the resolution of k_on and k_off separately, as two compounds with identical K_d values can have very different kinetic profiles with distinct implications for drug efficacy.

The residence time of a complex can also be estimated from k_off:

Residence Time = 1 / k_off

This is particularly relevant in lead optimization, where a longer residence time may translate to a more sustained pharmacological effect.

Applications for nanoTAK

Kinetic Profiling of Drug Candidates

nanoTAK provides both k_on and k_off, giving a more complete and mechanistically informative picture of an interaction than affinity alone. Two compounds with identical K_d values can have very different kinetic profiles:

Compound A associates rapidly but dissociates relatively quickly. Compound B associates slowly but forms a highly stable, long-lived complex. In a drug discovery context, these differences have significant implications for target residence time, selectivity, and in vivo efficacy.

Lead Optimization

By combining k_on, k_off, and residence time data, nanoTAK supports informed compound prioritization based on mechanism, not just potency. Compounds with favorable kinetic profiles can be identified even when K_d values appear equivalent.

Characterizing Multi-Component and Challenging Systems

nanoTAK is suitable for a broad range of biomolecular interactions, including:

• Small molecule to protein - kinetic profiling of drug candidates against enzymes, kinases, and transcription factors

• Degrader-induced complex formation - binary and ternary complex kinetics for molecular glues and PROTACs

• DNA-DNA hybridization - interactions on different timescales (e.g., k_on ~6.4 x 10⁵ M⁻¹s⁻¹, k_off ~4.8 x 10⁻⁵ s⁻¹)

• Membrane proteins - measured in detergent or nanodiscs without the need for surface tethering

• Intrinsically disordered proteins (IDPs) - compatible due to the solution-based, surface-free format

Data Analysis

nanoTAK data is analyzed using the dedicated nanoTAK app on the NanoTemper App Hub, a server-based analysis platform. The app:

• Evaluates temperature-induced fluorescence changes across the ligand concentration series

• Applies global fitting to extract k_on and k_off

• Presents time traces and fitted curves for quality assessment

Results are exported and can be merged with affinity data from MO.Control for a consolidated interaction profile.