Kinetic Förster Resonance Energy Transfer (kinFRET) enables the kinetic measurement of analyte-induced ternary complex formation, providing invaluable insights into ternary protein complexes, PROTACs and molecular glues. By leveraging kinFRET on the heliX platform, researchers can gain a deeper understanding of these complex interactions, facilitating drug discovery, therapeutic development, and fundamental biological research.
At 2bind, our team of experienced scientists harnesses the full potential of heliX kinFRET to deliver comprehensive solutions for your research needs. We combine state-of-the-art instrumentation with expert data analysis and interpretation to ensure accurate, reliable, and actionable results.
kinFRET operates by measuring the energy transfer between two fluorescent probes attached to the arms of a Y-shaped nanolever, which are functionalized with two different biomolecules. When the biomolecules – and consequently the two fluorescence probes – come into FRET-enabling proximity due to a binding event, energy is transferred from the donor probe to the acceptor probe. This results in a measurable decrease in donor emission and a corresponding increase in acceptor emission. The energy transfer signal serves as a direct readout of the interaction and is monitored in real time allowing the calculation of association and dissociation rates.
The heliX platform’s kinFRET mode therefore boasts a key advantages for ternary complex formation induced by molecular glues or PROTACS: All binding events are detectable in a single experiment. There is no need for step-wise, multi-experiment approaches that involve complex data deconvolution.
Association rate constant. Provides information on how fast complexes form; can be used for KD determination.
Dissociation rate constant. Provides information on how fast complexes dissociate; can be used for KD determination.
Equilibrium dissociation constant. Can be obtained by kinetic or classical equilibrium binding analysis. Provides information about the strength but not the dynamics of an interaction.
Dose-response data. Ligand concentration that gives half-maximal response or half-maximal activity.
Drug Discovery: Characterize the binding and mechanism of action of PROTACs, molecular glues, and other targeted protein degradation therapies.
Biotherapeutic Development: Optimize the design of PROTACS and other multi-target therapeutics.
Basic Research: Elucidate the molecular mechanisms underlying complex biological processes, such as signaling pathways and protein complex assembly.
Diagnostics: Develop novel assays for detecting and quantifying biomarkers of disease.
The helix sensor chips comprise a gold microelectrode to which DNA nanolevers are tethered. Due to the inherent rigidity of DNA double helices and their negative charge, their orientation on the chip surface can be precisely controlled by applying an electrical potential to the electrode. Depending on the electrical potential, two distinct measurement modes are possible: dynamic and static measurement mode.
Kinetic FRET is measured the static measurement mode, where the nanolever is in an upright position and target molecules as well as fluorophores are in a maximal distance from the gold surface. If two fluorophores form a donor/acceptor FRET pair and are within a maximal distance of 10 nm, energy is transferred from the donor to the acceptor fluorophore. Consequently, in a FRET-enabling situation the fluorescence of the acceptor can be measured upon excitation of the donor.
For kinFRET special Y-shaped DNA structures are use, together with two FRET-pair forming fluorophores. When a bi-specific ligand (e.g. bi-specific antibody, PROTAC, or molecular glue) binds to two neighboring but differently colored DNA levers, it brings those levers together and induces a FRET (foerster resonance energy transfer) from one dye to the other. The microfluidic system and the single photon counter allow for a real-time monitoring of the complex formation induced by PROTACs, or molecular glues and removes any need for complicated, step-wise approaches.
The kinFRET measurement mode of the heliX® platform can be used for measuring the kinetics of ternary complex formation induced by PROTACs or molecular glues using the Y-shaped DNA nanolevers specifically optimized for these applications.
Here is a list of typical applications of kinFRET with the heliX® platform:
Ternary complex formation induced by PROTACs or molecular glues
Key questions of assay design typically include the immobilization strategy. heliX® biosensor chips are equipped with single stranded anchor sequences and the most convenient way is the immobilization of target molecules via DNA hybridization. Different anchor sequences attached to electrode 1 and electrode 2 enable targeted immobilization to each spot. Typically, adapter strands carrying the fluorophore and ligand strands are prehybridized. Each electrode is equipped with a two-color detector and interactions of one analyte with up to four target molecules can be measured in parallel.
Immobilization via DNA hybridization is usually the method of choice to immobilize target DNA or RNA sequences. However, convenient, gentle and controlled conjugation methods as well as the proFIRE® purifier streamline the production of protein-DNA conjugates with high quality and purity, which can be easily implemented into the heliX® workflow. Ready-to-use kits for coupling to primary amines exist for molecules with usual pIs as well as for low pI molecules. If the target of interest contains a His-tag, coupling can be directed to amines in proximity of the tag. Alternatively, DNA strands can be coupled to thiol groups of free cysteines with high efficiencies and recovery rates.
As an alternative to chemical modifications of the targets of interest, tag-based capturing can be used. Methods and tools for capturing via His tags, biotin, twin-strep-tag, Fc parts and GFP have been developed and are ready to use.
Ligand strands are compatible with standard adapters as well as with DNA nanostructures. Rigid DNA-origami nanolevers are recommended for sizing experiments with large molecules. Antibody-like Y-structures with flexible arms reduce the distance between attached ligands and enable bivalent binding of smaller bispecific molecules like PROTACS. Ternary complex formation and dimerization can be followed by time-resolved FRET.
kinFRET requires a maximal distance of 10 nm between the two fluorophores upon complex formation. Antibodies can reach larger distances with the paratopes located on the two arms of the antibody molecule. Therefore, FRET is not the most sensitive method to measure the simultaneous binding to two targets in the case of classical knob-into-hole bispecific antibodies. However, bispecific formats with shorter distance between the paratopes can induce FRET upon binding to their two target molecules. In any case, using the Y-structure in the FPS mode can be useful in bispecific antibody projects, because it enables the immobilization of the two target molecules in a defined distance.
