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switchSENSE

switchSENSEunderstanding interactions

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Understanding Interactions

Our proprietary switchSENSE technology enables scientists investigating bio­molecules and their interactions to take their research to the next level.

switchSENSE combines the advantages of a molecular dynamics measurement with the benefits of a chip­based biosensor system. In addition to standard inter­action parameters such as affinity and binding kinetics, our dynamic measure­ment technology gives you the opportunity to analyze a multitude of additional parameters such as molecular size, aggregation, conformational changes and much more. All in one measurement, label­free, and with superior sensitivity.

The DRX 2400 instrument in conjunction with the versatile 4 × 6 biochip is designed to combine outstanding flexibility and straightforward usability, helping you to obtain more meaningful data with unprecedented analytical content. The propri­etary time­resolved measurement, the finetunable molecule density, and the powerful parallel chip functionalization scheme do not only allow you to better describe molecular interactions, but to truly understand them.

Employ switchSENSE for a wide range of applications including antibody develop­ment, analysis of DNA­binding proteins, thermodynamic characterization of com­pounds, and many more.

Transform your understanding of interactions and unravel complex mechanisms with switchSENSE.

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Unrivaled Information Content Parallel and Label­free

Interaction Analysis

KineticsAffinity

Cooperativity

Biophysical Analysis

Size & AggregationShape & Conformation

Thermodynamics

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Electrically Switchable Nanolevers – the switchSENSE Principle

DNA nanolevers are electrically actuated at high­ frequency on microelectrodes, while their orientation is monitored by time­resolved single photon counting. The binding of analyte molecules slows the switching dynamics in a characteristic way, providing an unprecedented level of information about the target.

2μs 4μs 6μs

1

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High Sensitivity, High PerformanceMeasure analyte concentrations from fM to mM and capture ultra­fast and ultra­slow kinetics.

Kinetics and AffinityDetermine binding rate constants kON, kOFF in real­time and dissociation constants KD from kinetic as well as from titration measurements.

Size and ConformationAnalyse protein diameters on chip with 0.2 nm accuracy, and monitor conformational changes.

Cooperativity and AvidityIdentify multiple binding sites in a single measurement using variable capture molecule densities.

Variable Temperature, 4 °C – 75 °CCharacterize melting transitions, thermal stability or perform thermodynamic analyses.

More Information from Molecular Dynamics

The DRX 2400 instrument is the first system to measure the motion of biomolecules on a chip. switchSENSE allows four different measure­ment modes in order to extract the maximum of information from your sample.

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Parallel and Versatile24 microelectrodes, individuallyaddressable through DNA encoding.4 separate flow channels.

One Chip – Unlimited Possibilities

Versatile functionalization with proteins, DNA, and small molecules. Reusable and disposable with integrated flow channels for maintenance­free operation.

DNA­Encoded AddressingswitchSENSE allows for the parallel functionalization of all electrodes within a single flow channel by hybridization of specifically designed sequences with negligible cross­reactivity. Encode up to six different capture molecules with dedicated complementary nanolever sequences using standard kits, and load them onto the chip in a single step.

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S1 S2 S3 S4 S5 S6

2 3 4 5 6

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Less is MoreWhen it comes to sensor performance, a low number of capture molecules means higher sensitivity and better control over binding stoichiometry. switchSENSE features microelectrodes which are only 120 μm in diameter, and enables the user to adjust the distance between capture molecules. Through a proprietary desorption method, the density of molecules on the detection spot can be tuned for optimal assay conditions.

Easy RegenerationThe switchSENSE chip is functionalized by DNA–DNA hybridization. While being a reliable method to “zip” capture molecules onto the chip, they can be removed as easily by splitting the DNA duplex using an automated routine. No need to establish unknown regeneration conditions, no need to worry about the activity of capture molecules.

Although switchSENSE uses a fluorescence readout, the actual measurement parameter derives from the molecular dynamics of the DNA switching – which is hardly affected by changes in the absolute fluorescence signal. Thus, Dynamic Response readings remain stable over many regeneration cycles.

Straightforward FunctionalizationswitchSENSE chips can be functionalized in two alternative ways for maximal practicability: on-chip or in-vitro. DNA­encoded addressing enables 6 × parallelity for each flow channel.

Diagram shows in­vitro conjugation workflow

parallel funct. by DNA encoded

addressing

PREPARECONJUGATE

use providedkits & protocols

real­timelabel­free target

parallel

kineticsaffinity

sizecharge

HYBRIDIZECHIP

MEASUREINTERACTION

ANALYZEDATA

Dyn

amic

Res

pons

e (D

RU

)

Fluo

resc

ence

Am

plit

ude

(kco

unts

)

00 5 10 15 20

Regeneration Cycle

DRU

Fluo.

50

100

150

200

0.0

0.5

1.0

1.5

2.0

Elec. 1 Elec. 2 Elec. 3

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Interaction Data for In­Depth AnalysisProteins, Small Molecules, Nucleic Acids – One Platform.

Equilibrium titration analysis of small molecule – protein interactions (biotin antibiotin). The full IgG features a low apparent KD = 4.2 ± 0.3 pM and a steep transition due to multivalent binding, while the monovalent antigen binding IgG fragment (Fab) can be well described by a 1:1 interaction model with a KD = 103 ± 11 pM.

Capture Molecule on Chip

Analyte in Solution

Proteins W W W W

DNA, RNA W W W W

Small Molecules W W W

0

0 1p 10p 100p

Concentration (M)

Fraction Bound (%)

1n 10n

20

40

60

80

100

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Protein–ProteinCollect real­time data for kinetic analyses or conduct equilibrium binding studies. Utilize the exceptional performance of switchSENSE to characterize even complex and ultra­strong binders.

Real­time measurement of the association and dissociation kinetics of a high­affinity monoclonal antibody interacting with human carbonic anhydrase 1 (CA; DNA­tethered). The Dynamic Response decreases during antibody binding, as the higher hydrodynamic drag of the mAb­CA complex slows the switching of the nanolever. A monophasic (single exponential) association is followed by a complex, biphasic dissociation, with apparent KD values in the pico­ and femto­molar con­centration range, respectively.

Association

0.2 nM

1 nM2 nM

Dyn

amic

Res

pons

e(n

orm

aliz

ed)

0 min

0

1

15 min 0 h 6 h 12 h Time

Dissociation

Rate constants of association (kON) and dissocation (kOFF), and dissociation constant (KD)

kON kOFF KD

3.28 ± 0.06 ×106 M–1s–1 5.1 ± 1.0 ×10–4 s–1

2.3 ± 0.3 ×10–6 s–1160 ± 30 pM700 ±100 fM

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Kinetic analysis of the association and dissociation of phi29 polymerase to double stranded DNA.

Sequence screening by modular exchange. The switchSENSE Anchor Chip features six short sequences to which target sequences of interest can be bound. Subsequently, a titration analysis or binding kinetics measurement can be performed with a DNA­binding protein. After an automatic regeneration step, the next sextuple of sequences can be investigated.

dye­labeledstrand

user­definedexchangeable

target sequencesurface­tetheredanchor strand

0

5 nM phi29 buffer

kON = (3.9 ± 0.1) × 106 M–1 s–1

kOFF = (2.2 ± 0.1) × 10–4 s–1

KD = 0.56 nM

50 100 150

Time (min)

Dyn

amic

Res

pons

e(D

RU)

Association Dissociation

DNA­Binding ProteinsAs DNA sequences are an integral component of the 4 × 6 chip, switchSENSE is the ideal platform for the analysis of trans­cription factors, polymerases or nucleases. Response elements in the sequence of interest may be exchanged swiftly through a modular anchor system, which facilitates screening applications. Simultaneously, the enzymatic activity of polymerases or nucleases can be directly evaluated.

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DNA detection by low frequency switching. The switching modulation amplitude increases as single stranded 48nt targets bind to surface­tethered capture strands, because mechanically rigid DNA duplexes can be manipulated more efficiently by the applied electric field.

Optimization of the conditions for the detection of a carcinogenic SNP in a 51 bp fragment of the p53 gene with a 15mer PNA probe. The false­positive signal for a wt sequence with a single mismatch (GT) could be reduced to ~0% at elevated temperatures of 60°C and at a low NaCl concentration of only 1 mM, while the sensitivity for the fully complementary target sequence was maintained. Standard methods were used for sample preparation.

Nucleic AcidsswitchSENSE features best­in­class sensitivity for the detection of label­free sequences. The chip can be configured with natural (DNA, RNA) and synthetic (PNA, LNA) nucleic acids and can be used for the detection and quantification of target sequences. Make use of the exceptional specificity to identify single mismatches (SNP detection) or record melting curves on chip in temperature dependent experiments.

0

0

50

100

Time (s)

Fluoresence (kcps)

50 100

perfect matchmm0

one mismatchDNA probe

25 °C, high salt

one mismatchPNA probe

25 °C, high salt

one mismatchPNA probe

60 °C, low salt

one mismatchPNA probe

60 °C, low saltafter washing

100%89%

40%

12%0%

True Positive False Positive, Single Mismatch

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Unrivaled Information ContentswitchSENSE enables you to investigate a wide range of biophysical parameters. One platform. In­depth analysis.

Measurement Modes• Nanolever Actuation | high­frequency (time­resolved), low­frequency, variable voltage• Variable Temperature | 4 – 70 °C• Variable Capture Molecule Density | proprietary electrical desorption process

Analyzable Parameters• Affinity | avidity, cooperativity, bispecific binders • Kinetics | rate constants, dissociation constant• Size | hydrodynamic diameter, aggregation, co­factor binding• Shape, Conformation | denaturation, unfolding, ligand­induced shape change• Chemical Modifications | phosphorylation, glycosylation, …• Charge | charging state, isoelectric point• Thermodynamics | melting point, enthalpy, entropy

0

0

DNA

IgGFab

1

2 4 6 8

Time (μs)

DNA Fab IgG

10 12 14 16

Fluo

resc

ence

(nor

mal

ized

)

Time­resolved upward motion of a DNA nanolever before and after binding an IgG antibody (150 kDa) and an antibody fragment (50 kDa), respectively. Proteins slow the nanolever motion according to their hydrodynamic friction, which leaves a fingerprint in the switching dynamics trace that is characteristic for their size and shape.

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The switchSENSE analysis software includes a theoretical model for the calculation of the motion of the nanolever–protein complex. By comparison with experimental data, it yields the effective hydrodynamic diameter of the protein, i.e. the diameter of a sphere with the same hydrodynamic friction coefficient as the protein under investigation.

The figure compares diameters analyzed with switchSENSE to literature data obtained by dynamic light scattering (DLS, circles) and x­ray diffraction (squares), showing a linear correlation.

Sizing – Protein DiameterswitchSENSE is the only biosensor capable of sizing molecules on a chip – with minimal sample consumption. Time­resolved switchSENSE data can be analyzed with a model that yields the hydrodynamic diameter DH of a protein sphere with an accuracy of 0.2 nm.

2

Ub

Cyt­C

INFα

CAT

ERK2

CA

G

Fab

L

DHFR

2

3

4

5

6

7

Diameter, literature (nm)

Diameter, switchSENSE (nm)

3 4 5 6 7

A

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Conformation & Posttranslational ModificationsThe switching dynamics measurement is sensitive to slightest changes in a molecule’s conformation, revealing even post­translational modifications or ligand­induced shape changes.

Thermal & Denaturation AnalysesswitchSENSE can be used to investigate the compaction, expansion or unfolding of a protein’s tertiary structure. Measure the switching dynamics of proteins as a function of temperature for the determination of thermodynamic parameters; or evaluate protein stability with chaotropic agents.

Detection of post­translational modification states of human chorionic gonadotropin (hCGβ) and extracellular signal­regulated protein kinase 2 (ERK2). The glycosylation of hCGβ leads to a compaction of the protein, while the phosphorylation of ERK2 increases the protein’s negative charge – both effects result in a faster switching motion, i. e. a higher Dynamic Response.

The calmodulin (CaM) measurement is exemplary for the detection of ion­induced conformational changes in proteins. When Ca2+ binds to CaM, its conformation changes from a globular to a dumb­bell shape with higher friction, slowing the switching dynamics. All data are normalized to the DR of a bare nanolever.

[left] The temperature­ dependent unfolding of proteins increases the Dynamic Response (lower friction), while the expansion of protein domains decreases the DR.

[right] The urea­induced unfolding of tertiary structure is seen in the switchSENSE data, whereas a conventional CD measurement indicates the loss of secondary structure.

Dynamic Response (%)

hCGβ ERK2 Calmodulin

80

100

glycosylate

d

not glyc

osylated

phosphorylate

d

not phosphory

lated

without C

a2+

with Ca

2+

Protein AS. aureus

switchSENSE

Circular Dichroism220 nm

Loss of Tertiary StructureLoss of Secondary Structure

5 D

RU

2 D

RU

2 D

RU

2 m

deg

Unfold

ing

Unf

oldi

ngExpansion

Human SerumAlbumin

30 35 40 45 50 55 60 65Temperature (°C)

1 2 3 4 5 6 7Urea (M)

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Multivalent and Cooperative BindersThe characterization of multivalent interactions (avidity) requires a careful control of binding stoichiometry on the sensor surface. switchSENSE interfaces are specifically designed for this purpose: the number of nanolevers on a switchSENSE chip is variable, and can be adjusted to exceptionally low densities (less than 1010 capture molecules/cm²). This unique feature allows the unequivocal identification of multivalent interactions.

Equilibrium titration analysis for proteins with one and two binding sites, respectively: proteinG­His6 (circles) and His6­proteinA­His6 (squares).

The density of NTA3­modified DNA nanolevers was adjusted to allow for simultaneous 1:1 and 1:2 binding.

Dissociation kinetics of a high­affinity IgG (antibiotin) were simultaneously measured on three electrodes with different nanolever densities. Analysis with a double­ exponential function yielded two different time constants: the amplitude of the long time constant increases with nanolever surface density, providing clear evidence of a multivalent interaction (nanolever interlinking through the antibody).

10 p 100p 1 n 10 n 100 n

0.0

0.5

1.0

Concentration (M)

Fraction bound

35

400 800 1200

Fr

acti

on o

f 2:1

inte

ract

ions

(%)

Fluorescence Intensity (kcps)

40

45

50

KD2 = 91 pM

KD1 = 1.5 nM

KD = 6.1 nM

fast dissociation slow dissociation

1:1 2:1

Nanolever density

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Analytical PerformanceLimit of detection LOD = 10 fM (MW > 10 kDa)Dissociation constant KD = 100 fM – 1 mMAffinity constant KA = 103 M–1 – 1013 M–1

Association rate constant kON = 103 – 109 M–1 s–1

Dissociation rate constant kOFF = 10–6 – 1 s–1

Size Hydrodynamic protein diameter DH quantification for 2 … 9 nm (8 kDa – 70 kDa), ΔDH = ± 0.2 nm; relative sizing up to 300 kDa

Shape Conformational changes, ΔDH > 0.2 nmCharge, pI value Estimation of effective molecular charge and charge ranking,

5.5 < pH < 10, ΔpI = ± 0.5Temperature 4 °C – 75 °C ChipDetection spots 24 electrodes, diameter = 120 μmIntegrated flow channels 4 channels × 6 electrodesRegeneration by oligonucleotide denaturation and hybridization,

cycle duration ~ 3 min Sample HandlingAutosampler Capacity 36 × 1.5 ml + 12 × 10 ml vials,

adjustable temperature 4 °C – 45 °CPump max. pump rate 4000 μl/min for fast kinetics, capacity 5 ml

Technical DataDRX 2400

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Dynamic Biosensors GmbHLochhamer Str. 1582152 Martinsried/PlaneggGermanywww.dynamic­biosensors.com