What is nanoLISA?

nanoLISA is a measurement mode available with Monolith Omni that allows you to assess protein thermal stability using extrinsic fluorescence. It provides information about the folding state and stability of a protein under the assay conditions and complements affinity and kinetic measurements by showing whether a ligand stabilizes or destabilizes the target protein.

Measurement Principle

Protein thermal stability is a fundamental biophysical property that reflects the folded state of a protein. When proteins are heated, they undergo a transition from their native folded state to an unfolded state. This transition can be monitored using fluorescence-based methods.
nanoLISA uses extrinsic fluorescent dyes that exhibit different fluorescence properties depending on the protein's folding state. As the protein unfolds, hydrophobic regions become exposed, altering the dye's local environment and changing its fluorescence emission spectrum.

The nanoLISA measurement involves:

Sample heating: An infrared laser heats the sample from ambient temperature up to 95°C over 60 seconds
Fluorescence monitoring: The Spectral Shift ratio (fluorescence at two emission wavelengths) is recorded continuously during heating
Melting curve generation: The ratio change over time produces a sigmoidal curve representing the unfolding transition

**Figure 1.** nanoLISA monitored by fluorescence ratio measurement. Schematic (top) shows fluorescently labelled protein transitioning from folded to unfolded state upon heating in a capillary over 30-60 seconds. Graph (bottom) displays the fluorescence ratio (670 nm / 650 nm) versus time, showing the characteristic sigmoidal unfolding curve with the inflection point (IP) marking the midpoint of the thermal transition.

The inflection point (IP) is the time at which 50% of the protein population has unfolded. This parameter is analogous to the melting temperature (Tm) used in other thermal stability methods, but is reported as a time rather than a temperature due to the rapid heating approach.

Assay Conditions

Concentration: The concentration range is similar to Spectral Shift assays, with a typical range between 5 nM and 20 nM. Fluorescence will decrease upon temperature increase, therefore sub-nanomolar concentrations should be tested first.

Labelling: For affinity-based labelling, the protein-dye ratio should be adjusted closer to 5:1 to ensure that upon temperature increase the dye stays bound to the target protein.

Measurement time: Laser time can be selected between 30 seconds and 60 seconds per capillary.

Applications for Optical Unfolding

Protein Quality Assessment

Before starting binding experiments, verify that your protein is properly folded and stable. A well-behaved protein should show:

A clear sigmoidal unfolding transition
Reproducible inflection point across replicates
No evidence of aggregation (irregular curve shapes)

Binding Validation

Ligand binding often stabilises proteins, shifting the unfolding transition to higher temperatures (later inflection times). By comparing the unfolding behaviour of protein alone versus protein with ligand, you can:

Confirm that binding has occurred
Obtain an independent measure of binding (orthogonal to affinity measurements)
Distinguish true binders from non-binders or artefacts

**Figure 2.** nanoLISA curves of a protein with different ligands. Different inflection points between DMSO (green) and Hit (purple) confirm binding, whilst the noise in the red curve suggests a ligand which induces protein aggregation.

Identifying Problematic Compounds

Aggregators

Compounds that induce protein aggregation typically cause destabilisation or irregular unfolding curves. The melting curve may show:

Decreased inflection point (earlier unfolding)
Loss of sigmoidal shape
Increased baseline noise

Fluorescent Compounds

Auto-fluorescent compounds can interfere with the measurement by contributing to the fluorescence signal. This may result in:

Abnormal baseline fluorescence
Irregular curve shapes
Concentration-dependent artefacts

Characterising Multi-Component Systems

nanoLISA is particularly useful for studying complex systems such as ternary complexes.

For molecules like PROTACs that bring together two proteins, a possible experimental setup can be:

1. E3 ligase alone: Baseline stability
2. Binary complex (E3 ligase + PROTAC): Stabilisation from PROTAC binding
3. Ternary complex (E3 ligase + PROTAC + target protein): Additional stabilisation from
protein-protein interactions

**Figure 3.** nanoLISA curves for the binary complex VCB-MZ1 (top left) and ternary complex VCB-MZ1-BRD2-BD2 (top right). The colour gradient shows increasing concentrations of MZ1, shifting the equilibrium further towards a bound state and increasing the detected inflection point. VCB alone, as well as the saturated binary and ternary complex optical unfolding curves, are displayed in the bottom figure.

The ternary complex typically shows greater stabilisation than the binary complex due to the additional protein-protein interface, providing evidence of ternary complex formation.

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