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Monitoring intracellular protein interactions using NanoLuc® Binary Technology (NanoBiT™) Brock Binkowski, Christopher Eggers, Braeden Butler, Marie Schwinn, Michael Slater, Thomas Machleidt, Mei Cong, Keith Wood & Frank Fan

Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399

1. Introduction 4. GPCR interaction with β-arrestin-2 7. Agonist-induced RTK dimerization

2. NanoBiT overview 5. RAF dimerization using stable expression 7. Quantifying agonist binding via dimerization

3. Real time kinetics of PPI interaction dynamics 6. Nuclear hormone receptor dimerization 9. Summary

NanoBiT is extremely bright

• Fusion partners can be expressed at very low levels, minimizing potential artifacts

• 100-1,000 fold brighter than split firefly luciferase

NanoBiT components are small & stable

• LgBiT, 18 kDa; SmBiT, 11 amino acids

• LgBiT evolved for increased structural stability, providing a stable fusion partner

NanoBiT is reversible

• Monitor both protein association and dissociation events in real time

NanoBiT offers experimental flexibility

• Monitor protein interaction dynamics at a single time point or continuously for 1-2 hours

• Room temperature or 37 °C measurements

• Validated in 96-, 384- & 1536-well formats

Check http://www.promega.com/nanobit for new NanoBiT PPI expression vectors

For more information on NanoBiT, please see ACS Chemical Biology, 11(2), p. 400-408

www.promega.com 5/2016 Corresponding author: brock.binkowski@promega.com

• LgBiT and SmBiT are fused to proteins A & B

• A:B interaction facilitates LgBiT:SmBiT interaction, generating a bright luminescent enzyme

• LgBiT:SmBiT with low affinity (KD = 190 µM), limiting non-specific association and reducing

assay background

• LgBiT:SmBiT interaction is reversible (kon = 500 M-1sec-1; koff = 0.2 sec-1)

• LgBiT evolved for increased structural stability making it a better fusion partner

• Non-lytic assay format allows real-time measurements of protein interaction dynamics for 1-2

hrs

0 2 0 4 0

0

2 5

5 0

7 5

1 0 0

0 .1

1

1 0

1 0 0

1 ,0 0 0

T im e (m in )

Na

no

BiT

, n

orm

Glo

Se

ns

or, n

orm

N a n o B iT G lo S e n s o r

IS O P R O F S K

• Transient expression of SmBiT-PRKACA & LgBiT-PRKAR2A in HEK293

• Modulators of intracellular cAMP added sequentially at indicated time points

• Inverse correlation for NanoBiT vs. GloSensor cAMP 22F (cAMP biosensor)

• NanoBiT can monitor reversible PPIs in real time

0 2 0 4 0

0

5

1 0

1 5

2 0

T im e (m in )

No

rm

ali

ze

d r

es

po

ns

e

A D R B 2 :A R R B 2 A V P R 2 :A R R B 2

2

2

Inactive

PKA

ATP

Agonist

2 1

CA R2A

CA R2A

1 2

R2A

R2A cAMP

1 = SmBiT

2 = LgBiT

1

CA

CA

1

Active

PKA

LgBiT

SmBiT

PM

ARRB2 P

P

PM

ARRB2

Agonist

LgBiT SmBiT

Internalization

Transient or stable association for GPCR:ARRB2

ARRB2 P

P

SmBiT

LgBiT

• Transient expression of ADRB2-LgBiT/SmBiT-

ARRB2 or AVPR2-SmBiT/LgBiT-ARRB2 in

HEK293 cells

• Saturating ISO or AVP added at time zero

• Expected transient interaction seen for

ADRB2:ARRB2 (class A receptor)

• Expected stable interaction seen for

AVPR2:ARRB2 (class B receptor)

• NanoBiT does not interfere with endogenous

biology

+

LgBiT

18 kDa

SmBiT

11 amino

acids

Protein A Protein B A:B interaction

Structural complementation

gives a bright, luminescent

enzyme

• Stable expression of BRAF-LgBiT & CRAF-SmBiT

via bi-directional CMV promoter

• Recombinase-mediated, single copy integration

into HEK293 genome

• Expected rank order potency observed for BRAF

inhibitors that promote dimerization

• Z’ values >0.5 in 384-well format using manual

dispensing

• NanoBiT can be miniaturized to 384- & 1536-

well formats

Protein:protein interactions (PPIs) are essential to the cellular signal transduction pathways

that contribute to cancer. Although numerous approaches exist to monitor PPIs in vitro,

methods for intracellular detection have been more limited. We developed NanoLuc® Binary

Technology (NanoBiT), a two-subunit system based on NanoLuc® luciferase that can be

applied to the intracellular detection of PPIs. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT;

11 amino acid peptide) subunits are expressed as fusions to proteins of interest, where PPI

facilitates subunit complementation to give a bright, luminescent enzyme. Unlike related

approaches where an enzyme or protein is simply split, LgBiT was independently optimized for

structural stability and SmBiT was selected from a peptide library specifically for the PPI

application. The result is a subunit pair that weakly associates (KD = 190 µM) yet still maintains

30% of the activity of full-length NanoLuc at saturation. In contrast to many split systems, the

LgBiT:SmBiT interaction is reversible, allowing the detection of rapidly dissociating proteins.

PPI dynamics can be followed in real-time in living cells using the Nano-Glo® Live Cell

Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate.

Advantages over split systems include better sensitivity, reversibility, fusion to a peptide or a

small, structurally stable protein domain, real-time measurements using a non-lytic assay

format, and subunits with reduced affinity for self-association.

0 1 0 2 0 3 0

0

2

4

6

8

T im e (m in )

No

rm

ali

ze

d R

es

po

ns

e

H E R 1 :H E R 1

N R G 1E G F V e h ic le

0 1 0 2 0 3 0

0

2

4

6

T im e (m in )

No

rm

ali

ze

d R

es

po

ns

e

H E R 1 :H E R 2

N R G 1E G F V e h ic le

0 1 0 2 0 3 0

0

2

4

6

8

T im e (m in )

No

rm

ali

ze

d R

es

po

ns

e

H E R 1 :H E R 3

N R G 1E G F V e h ic le

0 1 0 2 0 3 0

0

2

4

6

8

T im e (m in )

No

rm

ali

ze

d R

es

po

ns

e

H E R 2 :H E R 3

N R G 1E G F V e h ic le

• The cytoplasmic domain of HER1,

2 & 3 was replaced with linker-BiT

• Transient expression in U2OS cells

• Saturating EGF or NRG1 added at

time zero

• The expected selectivity profile was

seen for EGF and NRG1

• NanoBiT can monitor RTK

homo- or heterodimerization in

real time

HER

1

EGF

HER

1

EGF

0 1 0 2 0 3 0

0

1

2

3

4

T im e (m in )

No

rm

ali

ze

d R

es

po

ns

e

1 E -6

3 .3 3 E -7

1 E -7

3 .3 3 E -8

1 E -8

3 .3 3 E -9

1 E -9

3 .3 3 E -1 0

1 E -1 0

V e h ic le

[E G F ] (g /m l)H E R 1 :H E R 1

-1 1 -1 0 -9 -8 -7 -6 -5

0

1

2

3

4

lo g [E G F ] (g /m l)

No

rm

ali

ze

d R

es

po

ns

e

E C 5 0 = 4 .0 n g /m l

H E R 1 :H E R 1

0 1 0 2 0 3 0

0

2

4

6

T im e (m in )

No

rm

ali

ze

d R

es

po

ns

e

1 E -6

3 .3 3 E -7

1 E -7

3 .3 3 E -8

1 E -8

3 .3 3 E -9

1 E -9

3 .3 3 E -1 0

1 E -1 0

V e h ic le

[N R G 1 ] (g /m l)H E R 2 :H E R 3

-1 1 -1 0 -9 -8 -7 -6 -5

0

2

4

6

lo g [N R G 1 ] (g /m l)

No

rm

ali

ze

d R

es

po

ns

e

E C 5 0 = 9 .3 n g /m l

H E R 2 :H E R 3

• HER truncations used as

described above

• Transient expression in

U2OS cells

• Varying EGF or NRG1

added at time zero

• CRC data at 7 minutes

• Quantify agonist

interaction with RTK via

dimerization

0 2 0 4 0 6 0 8 0

1 0 3

1 0 4

1 0 5

1 0 6

T im e (m in )

Lu

min

es

ce

nc

e (

RL

U)

R 1 8 8 1 (1 0 0 n M )

V e h ic le

R 1 8 8 1

• Transient expression of LgBiT-AR & AR-SmBiT in HEK293 cells

• R1881 agonist added at time zero, which induces AR dimerization

• CRC data plotted at 30 minutes

• Validated for GR homodimerization as well

• NanoBiT can monitor nuclear hormone receptor dimerization in real

time

1 0 0 1 0 1 1 0 2 1 0 3 1 0 4

0

21 0 5

41 0 5

61 0 5

81 0 5

[ In h ib ito r ] (n M )

Lu

min

es

ce

nc

e (

RL

U)

G D C 0 8 7 9A Z 628P L X 4 7 2 0

S o ra fe n ibM L 7 8 6

1 2 3

1 0 3

1 0 4

1 0 5

1 0 6

P la te

Lu

min

es

ce

nc

e (

RL

U)

G D C 0 8 7 9

V e h ic le

Ras Ras

P

B-Raf C-Raf

B-Raf C-Raf

Active

Inhibitor

HER

1

EGF

HER

1

EGF

N D L

N D L

N D L Ag

N D L

-1 2 -1 1 -1 0 -9 -8 -7 -6

0

2 0

4 0

6 0

8 0

lo g [R 1 8 8 1 ] (M )

No

rm

ali

ze

d R

es

po

ns

e