The static measurement mode/fluorescence proximity sensing can be used for high-performance kinetics of any molecular interaction. The broad detection range enables the investigation of very weak as well as very tight binders. The two colors allow for the differentiation of monovalent affinities and multivalent avidity to investigate for example bispecific antibodies.

Another unique feature of the static measurement mode is its ability to depict the catalytic activity of nucleic acid-modifying enzymes. It is possible to measure the activity of polymerases, transcriptases, helicases, etc in real-time and to separate their activity from their binding affinity.

The dynamic measurement mode can be used to detect conformational changes because these will change the hydrodynamic friction of the attached molecule, which affects the switching speed.

A third option how fluorescence can be quenched is by Förster resonance energy transfer (FRET). By combining two different fluorochromes on one nanolever, FRET can be induced and changes in FRET intensity can be related to binding events in more complex set-ups, e.g. for multi-domain proteins or to examine nucleic acid tertiary structure formation (e.g. aptamers). FRET technology is also the basis for investigations of proximity induced binding (e.g. PROTACs, molecular glues) with the Y-shaped DNA nanolevers specifically optimized for these applications.

The biochips offer endless opportunities for customized functionalizations

Kinetics applications

• Protein-protein interactions
• Receptor-ligand interactions
• Virus-like particle-peptide interactions
• Protein-small molecule interactions
• RNA/DNA-protein interactions
• RNA/DNA-small molecule interactions
• Transcription factors
• Splicing factors

Conformations applications

• Small molecules or ions inducing conformational changes
• Formation of multimeric protein complexes
• Posttranslational modifications
• Structure-function relationship
• Efficacy and mode of action of drugs

Multispecific Binders

• Multispecific antibodies binding mechanisms
• Optimization of antibody formats
• PROTACs
• Molecular glues

Nucleic acid targeting applications

• Screening of RNA targeting small molecules
• Polymerase/transcripase/helicase/etc inhibitor screening
• Small molecule – aptamer binding
• Riboswitches
• DNA damage repair
• Viral/bacterial transcription machinery
• CRISPR-Cas9

The heliX® fluidic system has been designed as best-in-class to enable the quantitation of fast and slow kinetics (10⁺¹ – 10^-6 s^-1) with confidence. Typically, kinetics are measured with nanolevers in an upright position (static mode). Binding events are detected by fluorescence proximity sensing.

Single-photon-counting detection for highest fluorescence sensitivity and data collection at 10 ms resolve even the fastest kinetics in real-time. High flow rates up to 500 µL/min eliminate mass-transport limitations and rebinding artefacts and thereby improve the measurement of rapid kinetics. Signals are stable and dissociations can be measured for up to 5000 min to quantify slowest dissociation rates.

Each of the two electrodes is equipped with a dual color detector and interactions of one analyte with four ligands can be measured in parallel.

Any constant temperature from 15°C to 40°C can be chosen for real time kinetic measurements.

The measured instantaneous velocity of the nanolever depends on the hydrodynamic friction of the analyte.

Conformational changes are detectable by comparing to the initial state. Compaction results in reduced friction, whereas extension due to analyte binding or multimerization leads to increased friction.

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.

Actually, one chip type allows multiple assay designs. The chips are equipped with unique anchoring sequences in electrode 1 and electrode 2.

Dependent on the requirements of a specific experiment, the anchoring sequences can be hybridized with adapter strands of different shape in a functionalization step (see section “What types of adapter strands have been designed up to now?”). The DNA can be conjugated with the ligand, or with a capture molecule.

Via the ligand strand sequence, two different target molecules can be applied in the same functionalization step and directed to the two different electrodes. This is useful for measurement of a real-time reference.

However, DNA-targeting also enables full control of immobilization densities and spatial distribution of immobilized binding partners on each electrode. Anchoring DNA sequences are fixed on the electrodes in a defined distance. By diluting the fluorescently labeled ligand strand with ligand-free strand hybridized with adapter strand without fluorophore, the density of target on the surface can be reduced and the distance between target molecules can be increased in a highly controlled and reproducible manner.

Obviously, it is also possible to load two antigens on the surface. Prehybridization with the alternative fluorophore enables the measurement of interaction of an analyte with two target molecules on the surface simultaneously. The same is true for electrode 2, leading to a total number of four interactions that can be measured in parallel.

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 real-time FRET.

The two standard fluorescent probes for heliX® are the red and green dye Ra and Ga, respectively.

Fluorescence proximity sensing is based on the change in the local environment of the dye upon binding of an analyte to the ligand, which in turn results in a change of the fluorescence signal. This effect depends on the chemical nature of the dye and the interaction partner.

Therefore, a different dye may yield a higher signal response depending on the type of interaction. Dye scouting enables to screen for the most sensitive fluorophore for the respective application.

Three red and three green fluorophores with different chemical properties are available for dye scouting. The dyes differ in net charge and hydrophobicity as depicted below.

heliX® is equipped with LEDs and lasers to excite at 485 to 515 nm, or 600 to 630 nm, and to detect emissions at 520 to 580 nm, or 650 to 690 nm. Any dye respecting those characteristic can be used in heliX® applications.