NanoBiT® Technology Platform –

Dr. Erik Bonke

Application Specialist

Innovative Tools to Study Cellular Protein Biology

2

A Continuously Grown Expertise in

Luciferase-based Technologies

• Reporter Gene Assays

• GloSensorTM (cAMP, Protease Assays)

• GloResponseTM (Signaling Pathways)

• Rapid ResponseTM

• Cell-Health Assays

• Bioassays (ADCC, PDL1)

• NanoBRETTM / NanoBiT®

• HiBiT Protein Tagging System

Keith V. Wood, Ph.D.

Detection Modes – The Advantage of Bioluminescence

3

Matching Plate Type and Detection Mode

4

ABSORPTION

FLUORESCENCE

LUMINESCENCE

Yes

No !!

“clear” “black” “white”

“solid or clear bottom” “solid or clear bottom”

TC treated

sterile

with lid

• 400 – 800 nm (VIS)

• 200 – 400 nm (UV)

➢ quarz glass

➢ cyclic olefin copolymer

(COC)

Yes

(Yes)

• plastic autofluorescence (↓)

• background (↓)

• crosstalk (↓)

(Yes)

Yes

• maximal reflection

• (!) phosphorescence

• crosstalk (↓)

Clear differences in absolute signal intensity and crosstalk

White Plate ≠ White Plate

5

281500000 207800 29240 9741 4974 2556 1626 1063 676 446 456 396 495600000 2070 76 43 43 50 50 36 96 83 96 96

82710000 145700 25640 8537 4364 2523 1526 966 593 500 403 376 123900000 1033 63 46 66 40 60 60 83 100 116 120

10130000 59820 18130 6518 3391 2153 1196 910 556 476 370 456 13190000 326 60 50 40 56 46 43 93 66 90 86

1281000 28090 12580 4014 2376 1563 1020 750 546 440 326 263 1676000 120 60 73 36 50 43 86 76 80 110 86

157000 14680 8287 2360 1736 1366 840 640 416 393 280 316 1186000 56 46 53 60 30 46 46 73 83 83 143

41940 8530 5518 1460 1156 933 660 510 356 303 286 233 90620 33 36 66 46 46 40 26 56 73 63 86

19840 5578 3554 1170 940 746 643 493 370 313 296 306 40010 40 36 43 36 43 66 66 60 63 70 76

281500000 207800 29240 9741 4974 2556 1626 1063 676 446 456 396 495600000 2070 76 43 43 50 50 36 96 83 96 96

82710000 145700 25640 8537 4364 2523 1526 966 593 500 403 376 123900000 1033 63 46 66 40 60 60 83 100 116 120

10130000 59820 18130 6518 3391 2153 1196 910 556 476 370 456 13190000 326 60 50 40 56 46 43 93 66 90 86

1281000 28090 12580 4014 2376 1563 1020 750 546 440 326 263 1676000 120 60 73 36 50 43 86 76 80 110 86

157000 14680 8287 2360 1736 1366 840 640 416 393 280 316 1186000 56 46 53 60 30 46 46 73 83 83 143

41940 8530 5518 1460 1156 933 660 510 356 303 286 233 90620 33 36 66 46 46 40 26 56 73 63 86

19840 5578 3554 1170 940 746 643 493 370 313 296 306 40010 40 36 43 36 43 66 66 60 63 70 76

PBSLuciferase

DIL

TU

ION

Plate A

Plate B

PBSLuciferase

DIL

UT

ION

Chemistry of Luciferase Reactions

Photinus pyralis

Renilla reniformis

Sea Pansy

Firefly Reaction

Renilla Reaction

6

7

NanoLuc® Luciferase – A New Experimental Reporter

Wang et al. 2015

Oplophorus gracilirostris

8

Live Cells Expressing Nluc 384-well plate –

Smartphone picture

9

Key charateristics of NanoLuc® Luciferase

• Enables the setup of highly sensitive assays

• No overexpression of reporter required

✓ ~100-fold fewer molecules of NanoLuc® than Firefly to get measurable signal

✓ Work at physiologically relevant expression levels

Bright, brighter, NanoLuc®

150-fold higher specific activity

10

NanoLuc® Compared to Firefly/Renilla Luciferase

FireflyRenillaNanoLuc®

Amino acids MW [kDa] MV [Å3]

Nluc 171 19.1 14

Rluc 312 36.0 32

Fluc 550 60.6 44

Small, smaller, NanoLuc®

< <

11

Additional Key Charateristics of NanoLuc® Luciferase

• Thermal stable enzyme

• Active over broad pH range

• Monomeric enzyme

• No PTM detected in mammalian cells

• No disulfide bonds

• Uniform distribution in cells

Nluc-H3

Nucleus

Calreticulin-Nluc-KDEL

ER

Nluc-b2 AR

Cell Membrane

12

Application Portfolio of NanoLuc® Luciferase

Transcriptional Regulation

Compound Screening

Target Engagement

Endocytosis

Imaging

Target ID

RNA Interference

Protein Interaction

Biosensors

Cell SignalingProtein Stability

Novel Technologies for Research based on NanoLuc®

13

HiBiT Protein Tagging

System

Autophagy LC3

HiBiT Reporter Assay

NanoBiT® Protein:Protein

Interaction System

RealTime-GloTM Annexin V

Apoptosis Assay

Target Cell

Killing Assay

NanoBiT® Cell-based

Immunoassays

Development of the Nano Binary Technology(NanoBiT®)

14

Small Tag Size (11 aa)

• Reduced risk to influence the function of

protein of interest

HiBiT Protein Tagging System – Principle & Features

7 logs

Sensitive & Quantitative

• Sub-attomole levels can be detected

• High linear range of >7 logs

Facilitates Endogenous Protein

Assays with CRISPR/Cas9

• Maintain native expression level

• No cloning required

• Optimized protocol available

Simple & Short Detection Protocol

• Homogenous 1-step assay („add only“)

• No antibodies and no washing steps

required

15

Measure Protein Regulation at the Translational & Post-Translational Level

1

Protein Expression

4

Protein

Secretion / Release

5

Receptor

Biology

3Viral

Infection

Protein

Degradation &

Stabilization

2

16

Many Options to Detect HiBiT-Tagged Proteins

1

3

2

All systems resemble homogenous, add-mix-measure (plate-based) assays.

4LIVE-CELL REAL-TIME QUANTIFICATION

!≤ 2 h Nano-Glo® Live Cell Assay System

Nano-Glo® Endurazine/VivazineLive Cell Substrates2 h - days

17

Transient Expression from a Vector

Promega‘s HiBiT entry vectors

Use own vector and append HiBiT

via PCR amplification (e.g. internal

placement of tag)

1

2

Naturally occurring secretion signals shall be removed

*

*

Two Options

18

TRANSIENT TRANSFECTION &

ECTOPIC EXPRESSION

Studying Cellular Protein Biology – Protein Tagging

19

• Constitutive promoter

• Gene dosage effects

• No transcriptional

regulation

• Aberrant expression levels

☺

GENOMIC ENGINEERING &

ENDOGENOUS EXPRESSION

vs

• Native promoter

• No gene dosage effects

• Transcriptional regulation

maintained

• Native expression levels

Stable Expression from an Endogenous Locus

Cloning-free 10-Day Protocol Available!

www.promega.com/protocol-crispr-hibit

1 gRNA (crRNA + tracrRNA)

2 Cas9 endonuclease

3 ssDonor DNA

Three components required:

20

Transfer

Validate after 24 - 48 h

gRNA: guide RNA; crRNA: CRISPR RNA; tracr RNA: transactivating crRNA

2018

2017

Stable Expression from an Endogenous Locus

Cloning-free 10-Day Protocol Available!

www.promega.com/protocol-crispr-hibit

1 gRNA (crRNA + tracrRNA)

2 Cas9 endonuclease

3 ssDonor DNA

Three components required:

21

Transfer

Validate after 24 - 48 h

gRNA: guide RNA; crRNA: CRISPR RNA; tracr RNA: transactivating crRNA

2018

2017

• PAM as close as possible to integration site (knock-in↑)

• Choose PAM so that the coding region is not cut

• Mutate PAM within the donor DNA

• Ideally crRNA should span the insertion site

POINTS TO CONSIDER

A Rapid Cloning-free CRISPR/Cas9 Workflow

• Design & order crRNA

• Design & order Donor DNA

• Order tracrRNA & Cas9

22

Donor ssDNA

A Rapid Cloning-free CRISPR/Cas9 Workflow

• Design & order crRNA

• Design & order Donor DNA

• Order tracrRNA & Cas9

• Anneal crRNA & tracrRNA

to form gRNA

• Add Cas9 to form RNP complex

23

• Electroporate/Transfect

• Validate tagging

• Perform experiments

Donor ssDNA

Validation of Genomic Editing

#1

#2

#3

#4

#5

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

1 0 8

RL

U

g u id e R N A

M o c k

• 24 – 48 h post-electroporation, lytic assay and blotting can be performed

• Differing levels of luminescence observed for different guides

Nano-Glo® HiBiT Blotting System

GAPDH-HiBiT Edited Pools

Nano-Glo® HiBiT Lytic Detection System

or Nano-Glo® Live-Cell Assay Substrates

Validation of Genomic Editing – Imaging

25

Olympus

LV200 Imaging System

nuclearCDK1-HiBiT

Guide RNA 1 Guide RNA 2 Guide RNA 3

Expression & Editing Efficiency

Subcellular Localization

Live cell bioluminescence imaging confirms …

Validation of Genomic Editing – Stability of Edit

26

Expression of GAPDH-HiBiT in cell pools remains steady without selective pressure

P10 P18

P22 P25

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

1 0 2

1 0 3

1 0 4

1 0 5

1 0 6

1 0 7

1 0 8

d a y s p o s t-e d it

RL

U

A 549

H e L a

H E K 2 9 3

GAPDH-HiBiT expression over time

Measure Protein Regulation at the Translational & Post-Translational Level

1

Protein Expression

4

Protein

Secretion / Release

5

Receptor

Biology

3Viral

Infection

Protein

Degradation &

Stabilization

2

27

• Transcription factors

• PROTACs

• Autophagy

The HIF1α Pathway – Schematic Overview

28

Study Intracellular Signaling – Stabilization of HIF1α

• High expression levels mute

the biological response

• endogenous expression with

CRISPR/Cas9 yields highest

assay window

Exp

ress

ion

Le

vel

Re

spo

nse

29

BNIP3-HiBiT ANKRD37-HiBiT HILPDA-HiBiT KLF10-HiBiT

Induction of HIF1α Downstream Targets

3030

• Hypoxia leads to induction of HIF1α downstream

targets BNIP3, ANKRD37, and HILPDA

• The putative target KLF10 shows only weak

induction (potentially no regulation by HIF1α)

Schwinn, M.K., et al. (2017) CRISPR-mediated tagging of endogenous proteins with a luminescent peptide. ACS Chem. Biol. DOI: 10.1021/acschembio.7b00549

CONCLUSION

Nano-Glo® Live Cell Substrate phenanthroline

Live-Cell Monitoring of Protein Abundance in Real-Time

31

Schwinn, M.K., et al. (2017)

+

32

Study Protein Degradation – PROTACs

PROTAC = Proteolysis targeting chimera

Exp

ress

ion

Le

vel

Re

spo

nse

JQ1 Thalidomide

33

Time-dependent Degradation/Recovery – PROTACs

0 1 0 2 0 3 0

0 .0

0 .5

1 .0

D e g ra d a t io n o f B E T F a m ily b y M Z 1

T im e (h o u rs )

Fra

cti

on

al

RL

U

B R D 4

B R D 3

B R D 2

0 1 0 2 0 3 0

0 .0

0 .5

1 .0

D e g ra d a t io n o f B E T F a m ily b y M Z 1

T im e (h o u rs )F

ra

cti

on

al

RL

U

B R D 4

B R D 3

B R D 2

BET family degradation by MZ1

Time (min) 0 15 30 60 120

HiBiT-BRD4

adapted from Riching KM et al. 2018, ACS Chem Biol., 13(9):2758-2770

HiBiT-BRD4

+JQ1

Time (hrs) Time (hrs)

34

The Autophagy LC3 HiBiT Reporter Assay

• Stable ready-to-use cell lines

available (U2OS and HEK293)

• Vector system for stable

transfection of other cell lines

35

The Autophagy LC3 HiBiT Reporter Assay

• Stable ready-to-use cell lines

available (U2OS and HEK293)

• Vector system for stable

transfection of other cell lines

HaloTag Domain Enables Optional Imaging Modality

36

VEHICLE

Basal autophagy

Lipidated LC3 formation

PP242

• U2OS Autophagy LC3 HiBiT Reporter Cells treated with compounds for 4 h

• No-wash Janelia Fluor® 646 HaloTag Ligand added at time of compound addition

WORTMANNIN

Cytosolic LC3 accumulation

PP242 + WORTMANNIN

early stage inhibitor

Cytosolic LC3 accumulation

BAFILOMYCIN A1

Lipidated LC3 accumulation

PP242 + BAFILOMYCIN A1

late stage inhibitor

Lipidated LC3 formation/accumulation

Measure Protein Regulation at the Translational & Post-Translational Level

1

Protein Expression

4

Protein

Secretion / Release

5

Receptor

Biology

3Viral

Infection

Protein

Degradation &

Stabilization

2

37

• Receptor tagged on extracellular side

with HiBiT

• Non-lytic detection with cell

impermeable LgBiT protein

• Measure both ligand potency and extent

of internalization within minutes

Receptor Endocytosis – ADRB2 & EGFR

38

ADRB2 EGFR

Development of the Nano Binary Technology(NanoBiT®)

39

Small tag size

minimal influence on fusion partner

Bright signal upon

complementation

Enables low expression levels

Low intrinsic affinity

Reversible:

Investigation of PPI dynamics

NanoBiT® PPI – Key Characteristics

+

SmBiTLgBiT

1.3 kDa18 kDa

DISSOCIATION

ASSOCIATION

Transfection-based

High dynamic range

Live-cell assay

40

NanoBiT® PPI Workflow

41

NanoBiT® PPI – The Protein Kinase A Model

42

NanoBiT® PPI – The Protein Kinase A Model

Isoproterenol (ISO)

ADRB agonist (cAMP ↑)

Propranolol (PRO)

ADRB antagonist (cAMP ↓)

Forskolin (FSK)

activator of adenylate cyclase (cAMP ↑)

ISO PRO FSK

• Endogenous biology is maintained with the NanoBiT® PPI System

• The NanoBiT® PPI System functions in a reversible manner

Conclusions

43

In-House Established NanoBiT® PPI Assays

44

AR/AR (NR3C4/NR3C4) AR/SRC1 BRD4/Histone 3.3 L3MBTL3/BCLAF1

MYC/MAX KRAS 2A G12D:CRAF EPOR(1-273)/EPOR(1-273) PDGFRA(1-549)/PDGFRA(1-549)

FKBP/FRB PD-1/SHP1 GR/GR (NR3C1/NR3C1) PDGFRA(1-549)/PDGFRB(1-553)

p53/MDM2 PD-L2/PD-L2 EGFR/GRB2 PDGFRB(1-553)/PDGFRB(1-553)

PRKACA/PRKAR2A CX3CR1:ARRB2 HER1(1-668)/HER2(1-675) RELA/NFKBIA

BRAF/CRAF ADRB2:ARRB2 HER1(1-668)/HER3(1-664)

HER2(1-675)/HER3(1-664) AVPR2:ARRB2 HER3(1-664)/HER3(1-664)

VEGFR1(1-785)/VEGFR1(1-785) CRAF/CRAF KRAS (G12C)/CRAF

EGFR(1-673)/EGFR(1-673) BRAF/BRAF KRAS (G12V)/CRAF

PD-L1/PD-L1 BCL2/BAK KRAS/CRAF

• Pre-optimized ready-to-use protein:protein interaction pairs

• Ready-to-use stable cell lines

• BiBiT System: Generate your own stable cell line

• Available through Custom Assay Service (CAS)

Annexin V & Apoptosis

• Phospholipid asymmetry is maintained by flippases, floppases and scramblases

• Alteration of their activities during apoptosis leads to PS „flip-out“ and recruitment

of macrophages

• Annexin V binding to PS blocks phagocytosis by macrophages

45

RealTime-Glo® Annexin V Apoptosis Assay

46

How does the Data look like?

HEALTHY EARLY APOPTOSIS SECONDARY NECROSIS

47

How does the Data look like?

3.75 h 6 h

9 h 12 h

U2OS cells treated with 1 µM staurosporin at 0 h

Data provided by Olympus48

Multiplexing with Caspase-Glo® 3/7

• Caspase activation precedes PS exposure

• Multiplexing data supports Annexin V data

Conclusions

49

NanoBiT® Homogenous Cell-based Immunoassays

50

Total Protein Detection

Phosphoprotein Detection

• Fast and convenient plate-based assay

• No aspiration steps required

• High sensitivity, low cell number, no IP enrichment of PTM needed

• HTS compatible

• Any cell type including primary cells

FACTS

NanoBiT® Homogenous Cell-based Immunoassays

51

• Fast and convenient plate-based assay

• No aspiration steps required

• High sensitivity, low cell number, no IP enrichment of PTM needed

• HTS compatible

• Any cell type including primary cells

FACTS

0 1 0 2 0 3 0

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

T i m e ( m i n )

Lu

min

es

ce

nc

e (

RL

U)

T o t a l I B

P - I B

0 1 0 2 0 3 0

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

T i m e ( m i n )

Lu

min

es

ce

nc

e (

RL

U)

T o t a l I B

P - I B

+ TNFα

Signaling Pathway and Kinase Activity Analysis

52

• IκBα phosphorylation (pS32)

• Immediately followed by rapid degradation

CONCLUSION

pS32-IκBα

total IκBα

0 1 0 2 0 3 0

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

T i m e ( m i n )L

um

ine

sc

en

ce

(R

LU

)

T o t a l I B

P - I B

Signaling Pathway and Kinase Activity Analysis

53

MG132

+ TNFα / MG132

• Decrease in IκBα degradation

• Accumulation of phosphorylated IκBα

CONCLUSION

pS32-IκBα

total IκBα

Validation / Identification of Kinase Inhibitors

54

IKK16

• Assay reveals expected pharmacology

CONCLUSION

+ TNFα

+ TNFα

Available NanoBiT® Cell-based Immunoassays

Disease or cellular process Pathway Kinase(s) Kinase substrates

Cancer mTOR / Akt / Pi3K pathway mTOR Akt pS473 and total

Inflammation and Immunity

NF-B pathway IKK IBα pS32 and total

JAK / STAT pathway JAKs STAT3 pY705 and total

• DIY kit for targets / PTMs of interest

• NanoBiT® Labeling Kit

• NanoBiT® Lysis and Detection Module

ADDITIONAL MATERIAL

Pathway-specific ready-to-use assays

NanoBiT® Universal Target Cell Killing Assay

56

Measure death of a specific cell population within a mixed population of cells

• Cell-mediated cytotoxicity (CMC)

• Antibody-dependent cell-mediated cytotoxicity (ADCC)

• Cell death on co-cultures; applicable to primary cells

• No cell engineering required

• Endpoint or kinetic detection possible

FACTS

ADCC with NanoBiT® Universal Target Cell Killing Assay

57

Prepare effector cells (PBMC)

Label target cells

Mix PBMC and

labeled target cells

Plate cells in media

Add antibody

Incubate

Add detection reagents

Detect luminescence

Raji ADCC

Daudi ADCC

Raji or Daudi

Rutiximab

58Promega CorporationPromega Corporation©2015 Promega Corporation. Proprietary Information. Not for further distribution.

Thank You For Your Attention!

THANK YOU!

QUESTIONS?58