Bruker Biosensors helix+ Y-Structure Kit 1: User Manual for Proximity Binding Assays

Key Features

Ideal for studying ternary complex formation upon binding of bispecific small molecules (e.g., PROTACs, molecular glues), homo-dimerization, and bispecific antibodies with weak affinities.
The new optimized Y-structure design allows higher FRET sensitivity.
Compatible with helix+ Adapter Chip.
The Y-structure Red Adapter strand carries a moderately hydrophilic red dye (Ra) with a single positive net charge.
The Y-structure Green Adapter strand carries a hydrophilic green dye (Ga) with a single negative net charge.
Green and red dyes can detect binding on each arm via Fluorescence Proximity Sensing (FPS), or report if the structure is OPEN or CLOSED via sensitive FRET.
Homo-/hetero-proteins can be coupled easily to the arms via exchangeable ligand strands.
The flexible hinge region confines two proteins to a small volume and defined distance.

Product Description

Order Number: HK-NYS-1

Table 1. Contents and Storage Information

Material	Cap	Concentration	Amount	Buffer	Storage
New Y-structure Red Adapter strand with Ra	Red	400 nM	5 x 50 μL	TE40 [1]	-20°C
New Y-structure Green Adapter strand with Ga	Green	400 nM	5 x 50 μL	TE40 [1]	-20°C
New Y-structure Ligand Free Strand (lfs) - Red	Yellow	500 nM	3 x 100 μL	TE40 [1]	-20°C
New Y-structure Ligand Free Strand (lfs) - Green	Yellow	500 nM	3 x 100 μL	TE40 [1]	-20°C
cAnchor strand 2	Black	100 nM	5 x 200 μL	TE40 [1]	-20°C

For research use only.

This product has a limited shelf life, please see expiry date on label.

To avoid many freeze-thaw cycles, please aliquot the nanolever.

heliX+ Adapter Chip Overview

Ternary binding

This diagram illustrates the setup for ternary binding assays. It shows two anchor strands immobilized on the chip surface at distinct spots. At Spot 1, a Red adapter strand, conjugated with a Red dye (Ra), is attached to an anchor strand and a ligand. At Spot 2, a Green adapter strand, conjugated with a Green dye (Ga), is attached to another anchor strand and a second ligand. This configuration allows for the study of interactions involving three components.

Binary binding in green

This diagram illustrates the configuration for binary binding assays detected in the green channel. It shows an anchor strand at Spot 1 with a Red adapter strand and a Red dye, linked to a ligand. At Spot 2, another anchor strand holds a Green adapter strand linked to a Green dye and a second ligand. This setup is used when the interaction is monitored via the green dye.

Binary binding in red

This diagram shows the setup for binary binding assays detected in the red channel. Similar to the green binding setup, but the ligand at Spot 2 is linked to a Ligand-Free strand (Green) and the Red dye at Spot 1 is linked to the primary ligand. This configuration monitors interactions via the red dye.

Preparation

The following steps outline the hands-on sample preparation for the helix+ Adapter Chip functionalization.

Step 1: Couple Ligand

The Ligand strands can be extended at either the 5' (Ligand strand 1 - Red arm) or 3' (Ligand strand 2 - Green arm) end with any DNA/RNA sequence. Additionally, they can be crosslinked to a protein of interest through amine coupling using the specialized helix+ Amine Coupling Kits (HK-NYS-NHS-1, which hybridizes to the red arm, and HK-NYS-NHS-2, which hybridizes to the green arm). It is highly recommended to purify the conjugation product with proFIRE® before conducting kinetic studies.

TIP: For higher FRET quality, the covalent coupling of the ligands is recommended. Alternatively, the protein can be captured via His-tag using the HK-NYS-NTA kit. For any questions, please contact the support team at support.dbs@bruker.com.

Step 2: Select Adapter

For surface functionalization, the new Y-structure Red Adapter strand harboring the red dye Ra and the new Y-structure Green Adapter strand harboring the green dye Ga need to be pre-hybridized with the Ligand-strands in order to build the Y-structure. In solution hybridization of Y-structure strands:

Mix the new Y-structure Red Adapter strand with Ra (400 nM), the new Y-structure Green Adapter strand with Ga (400 nM), conjugated Y-structure Ligand strand 1 (500 nM), and conjugated Y-structure Ligand strand 2 (500 nM) at a 1:1 ratio (volume by volume).
Incubate the solution of step i) at room temperature for 2 hours to ensure complete hybridization. Overnight incubation at 4°C is also possible, but it depends on the stability of the conjugated protein.

Step 3: Mix

Mix solution of step ii) and cAnchor strand 2 (100 nM) at 1:1 ratio (volume by volume).

Step 4: Functionalization

Solution is ready to use for helix+ Adapter Chip functionalization.

The preparation process is supported by a fully automated workflow on the helix+ instrument, where the functionalized chip is made ready for use.

Example

Required volume for 1 functionalization: 35 μL with a final concentration of 50 nM.

Vial 1				Vial 2
Y-structure Red Adapter strand (400 nM)	Y-structure Green Adapter strand (400 nM)	Conjugated Ligand strand 1 (500 nM)	Conjugated Ligand strand 2 (500 nM)	cAnchor strand 2 (100 nM)
4.5 μL	4.5 μL	4.5 μL	4.5 μL	18 μL

Assay Setup in heliOS

For studying ternary complex formation upon binding of bispecific small molecules (e.g., PROTACs, molecular glues), go to heliOS > create a New Assay Workflow > add Custom Assay > load Y-Structure FRET Kinetics > modify the parameters based on your needs and run the assay.

Suggested assay parameters (e.g., flow rates, functionalization time, LED power, etc.) are within the heliOS assay.

IMPORTANT: For binary interaction in red, please set LED red >= 1. However, do not forget to set it back to 0 when FRET interactions are under investigation. For more details, please refer to the helix+ guide available at this link.

Alternatively, Y-Structure FRET Kinetics - auto LED assay can be utilized, where the helix+ system automatically adjusts the LED power to optimize the fluorescence signal for a better signal-to-noise ratio. This approach is highly recommended for weak binders or screening applications.

For studying bispecific antibodies with weak affinities (e.g., Hemlibra binding to Factor X and IX), go to heliOS > create a New Assay Workflow > add simply Kinetics with Functionalization from the Custom Assay list > modify the parameters based on your needs and run the assay.

TIP: Antibodies are big proteins which do not allow to bring the two dyes in close proximity, therefore FRET cannot be recorded. This is the reason why classic kinetics workflow and Fluorescence Proximity Sensing (FPS) is used for detecting binding.

For further questions, please contact the support team at support.dbs@bruker.com.

Useful Order Numbers

Table 2. Order Numbers

Product Name	Amount	Order No.
Y-structure Amine coupling kit 1 - Red	3 conjugations	HK-NYS-NHS-1
Y-structure Amine coupling kit 2 - Green	3 conjugations	HK-NYS-NHS-2
New Y-structure Kit 2: for proximity binding assay Spot 2	400 nM x 250 μL	HK-NYS-2
New Y-structure His Capture Kit	500 nM x 200 μL	HK-NYS-NTA

Contact

Dynamic Biosensors GmbH
Perchtinger Str. 8/10
81379 Munich
Germany

Bruker Scientific LLC
40 Manning Road, Manning Park
Billerica, MA 01821
USA

Order Information: order.biosensors@bruker.com

Technical Support: support.dbs@bruker.com

www.dynamic-biosensors.com

Instruments and chips are engineered and manufactured in Germany.

For Research Use Only. Not for use in clinical diagnostic procedures.

[1] TE40: 10 mM Tris, 40 mM NaCl, pH 7.4, 0.05 % Tween20, 50 μM EDTA, 50 μM EGTA

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Bruker Biosensors helix+ Y-Structure Kit 1

User Manual for Proximity Binding Assay - Spot 1