Applications of a smaller, brighter, more versatile luciferase:

Applications of a smaller, brighter, more versatile luciferase: NanoLuc™ Luciferase Technology

Kyle Hooper, Ph.D. Fall 2012

Today, I will speak about…

Origins of NanoLuc Luciferase

NanoLuc Luciferase as a reporter

• Full-Length Nluc

•Destabilized, full-length NlucP

• Secreted, full-length secNluc

NanoLuc Luciferase as a fusion partner

• Protein Translocation

• Protein Stability

• Protein:Protein Interactions

• Receptor Interactions

• Biosensors

NanoLuc™ Luciferase Technology

2

What is NanoLuc™ Luciferase?

NanoLuc™ (Nluc) is a 19.1 kDa, ATP-independent luciferase that utilizes a novel coelenterazine analog (furimazine) to produce high intensity, glow-type luminescence.

3

2-furanylmethyl-deoxy-coelenterazine

Furimazine

Furimamide

Light

NanoLuc™ Luciferase

Coelenterazine

Light

19kOluc

Promega Advanced Technologies Group Hall, M.P., et al. (2012) ACS Chem Biol in press

Light

Coelenterazine


81,000X

enzyme evolution

Evolution of NanoLuc from ocean to lab bench

130kDa Oplophorus luciferase

7X brighter than native Renilla Luciferase

Shimomura, O., et al. (1978)

19kOluc 19kDa subunit is catalytic.

Light output & stability compromised.

Inouye, S., et al. (2000)

Coelenterazine

Light Coelenterazine

Oplophorus gracilirostris first cataloged in 1881

Light

Light

Furimazine


2,500,000X

substrate evolution

4

NanoLuc™ is very small

5

Firefly (Fluc) Renilla (Rluc) NanoLuc™ (Nluc)

Amino acids

M.W. Mol.

Vol. Å3

Nluc 171 19.1 14

Rluc 312 36.0 32

Fluc 550 60.6 44

NanoLuc is bright

Transfected DNA (pg)

Lu

min

escen

ce (

RL

U)

100 102 104 106

102

104

106

108

101 0

HepG2 / NLuc

HepG2 / FLuc

Hela / NLuc

Hela / FLuc

Purified NanoLuc™ Luciferase is ~100-fold brighter

NanoLuc™ Luciferase is ~100-fold brighter in cells

6

CMV-driven NLuc/Nano-Glo™ Assay CMV-driven FLuc/ONE-Glo™ Assay

Recombinant NLuc/Nano-Glo™ Assay Recombinant FLuc/ONE-Glo™ Assay Recombinant RLuc/Renilla-Glo™ Assay

7

NanoLuc™ has excellent physical properties

Thermal stable enzyme

• Retains activity following 30 min incubation at 55 °C

• Melting temps: Nluc, 58 °C; Fluc, 31 °C

Active over broad pH range

• Fully active between pH 7-9

• Retains significant activity at pH 5-7

• Fluc: sharp decrease in activity below pH = 8

Monomeric enzyme

• Facilitates use as transcriptional reporter or fusion partner

No post-translational modifications detected in mammalian cells

No disulfide bonds

• Supports high levels of activity inside living cells

Uniform distribution in cells

• No apparent compartmental bias in the absence of targeting sequences

400 500 600 7000

20

40

60

80

100

NanoLucRenilla

(nm)

Re

lati

ve e

mis

sio

n

Firefly

565 nm480 nm460 nm

unfused NLuc Immunofluorescence

U2OS cells

Nano-Glo™ Luciferase Assay Reagent

8

Nano-Glo™ Luciferase Assay Reagent: Furimazine

• Provides maximal brightness

Glow kinetics (no flash reaction)

• Half-life routinely >2 hour at room temperature

Low autoluminescence background

• Enhances assay sensitivity

Stable reconstituted reagent:

• ~10% decrease in activity over 8 hrs at RT

Time (min)%

sig

nal d

ecay

0 50 100 150 2000

25

50

75

100

Nluc (Nano-Glo)

FLuc (One-Glo)

% Signal Decay

Add-Mix-Measure format like • ONE-Glo™ Luciferase Assay System • Bright-Glo™ Luciferase Assay System • Steady-Glo® Luciferase Assay System • Renilla-Glo™ Luciferase Assay System

3 Varieties of NanoLuc™ Luciferase for you

Intracellular Formats

Secretion signal

Protein destabilization domain


NanoLuc™ Luciferase PEST

NanoLuc™ Luciferase IL6

Nluc (513 bp)

NlucP (636 bp)

secNluc (597 bp)

Secretion Format

pNL1.1

pNL1.2

pNL1.3

9

Intracellular stability of NanoLuc™ & Firefly

Cell line FLuc FLucP NLuc NLucP

HEK-293 >6 h 2.0 ± 0.4 h >6 h 18 ± 11 min

HeLa 3.8 ± 1.3 h 1.4 ± 0.2 h >6 h 20 ± 6 min

U2OS (n=1) >6 h 2.8 h >6 h 36 min

Relative protein stability in cells: NlucP < FlucP < Fluc < Nluc 10

NLuc

NLucP

NLuc

NLucP

NlucP gives the greatest dynamic response

11

NLuc 13-236 fold brighter than Fluc (79 fold avg.)

NlucP 2-27 fold brighter than FLucP (10 fold avg.)

Nluc 10-78 fold brighter than NlucP (34 fold avg.)

→Very similar pharmacology/EC50s

Experimental details: transient transfection of HEK293 cells with NF-kB inducible constructs. rhTNFa treatment for 5 hours.

0.001 0.01 0.1 1 10 100104

105

106

107

108

109

101 0

FlucP

FlucNlucP

NLuc

TNFa ng/mL

Lu

min

escen

ce (

RL

U)

Brightness

Nluc > NlucP > Fluc > FlucP (18 experiments)

NlucP responds earliest to stimuli

12

0 1 2 3

1

10

100

1000

Time (hours)

Fo

ld r

esp

on

se

NFkB/TNFa

NlucP FlucP

FlucNluc

Experimental details: transient transfection of HEK293 cells with NFκB inducible constructs; addition of 100 ng/ml rhTNFα at time zero.

Relative Response NlucP > FlucP > Fluc > Nluc

NLucP allows study of weakly induced responses

13

Time (hours)

Fo

ld in

du

cti

on

0 1 2 3 4 5 60

2

4

6

8

10

NLuc

NLucP

FLuc

FLucP

HSE/17-AAG

Experimental details: transient transfection of Hela cells w/ Hsf1 inducible constructs; addition of 500 nM 17-AAG at time zero.

Relative Response NlucP > FlucP > Fluc,Nluc

NanoLuc Luciferase as an intracellular reporter

NlucP for a faster response

NlucP for greatest dynamic range

NlucP for measuring weak responses

Nluc where maximum brightness is needed.

14

Should I switch from Firefly to NanoLuc™ Luciferase?

Firefly (Fluc) NanoLuc™ (Nluc)

Does it allow you to do your work? Do you plan to do work in vivo? Firefly is a great reporter Excellent signal:background Excellent dynamic range

We just released new response element signaling pathway detection pGL4 vectors:

ARE p53 ATF6 MRE

HSE HRE XRE AP1

ISRE SIE SBE

TCF-LEF

STAT5 NFAT CRE

NF-kB

SRE SRF

Not necessarily

15

Should I switch from Firefly to NanoLuc™ Luciferase?

Firefly (Fluc) NanoLuc™ (Nluc)

Yes, if …

Transfection efficiency limits you to easy-to-transfect cell lines

Signals are too weak to move to 96-well plates

FLuc is just too big

• The increased brightness could allow a subtle signal become a reliable signal.

• The small size could allow gene replacement with minimal impact, especially in viral constructs

16

3 Varieties of NanoLuc™ Luciferase for you

Intracellular Formats

Secretion signal

Protein destabilization domain


NanoLuc™ Luciferase PEST

NanoLuc™ Luciferase IL6

Nluc (513 bp)

NlucP (636 bp)

secNluc (597 bp)

Secretion Format

pNL1.1

pNL1.2

pNL1.3

17

Secretion based format using secNluc

18

Experimental details: transient transfection of HEK293 cells with CREB inducible construct; addition of 10 µM forskolin at time zero.

Time (hrs)

Lu

min

escen

ce (

RL

U)

0 1 2 3 4 5 6103

104

105

106

107

DMSO

Forskolin (FSK)

Medium exchange+

10 M FSK

• Sample medium at multiple time points without cell lysis

• Kinetic studies from the same set of wells

• Half-life of secNluc protein > 4 days at 37°C in medium

• Response dynamics similar to unfused Nluc

• Similar pharmacology vs. Nluc/NlucP

Time (hours)

S/B

0 2 4 60.1

1

10

100

1000

NLuc

NLucP

secNLuc

Gluc kits: bright, but high autoluminescence background

19

Experimental details: HepG2 cells (DMEM +10% FBS) transiently transfected w/CMV promoter constructs; removal of aliquots after 22 hrs; n = 12 per treatment.

Sec

rete

d

Auto

Sec

rete

d

Auto

Sec

rete

d

Auto

101

102

103

104

105

106

107

108

Lu

min

escen

ce (

RL

U)

secNluc/NanoGlo

Gluc/Flash

Gluc-Dura/Glow

secN

luc/

Nan

oGlo

Glu

c/Fla

sh

Glu

c-Dura

/Glo

w

1

10

100

1,000

10,000

100,000

Sig

nal/au

to b

kg

d

Signal over autoluminescencebackground

Gluc kits: high background limits sensitivity & dynamic range

Handling the bright signal

R&D used GloMax® Instruments for NanoLuc™ Development

GloMax®-Multi+ Microplate Multimode Reader

GloMax®-Multi Microplate Multimode Reader

GloMax® 96 Microplate Luminometer

Largest dynamic range

available

21

NanoLuc Assays should be

designed for this range.

Getting NanoLuc™ Luciferase Signals in range

Transfect fewer cells

Transfect less DNA

Switch to NlucP

Use a weaker constitutive promoter

0.001 0.01 0.1 1 10 100104

105

106

107

108

109

101 0

FlucP

FlucNlucP

NLuc

TNFa ng/mL

Lu

min

escen

ce (

RL

U)

22

More details on NanoLuc™ Development

Webinar recording by lead R&D scientist for development of the NanoLuc™ Vectors and Nano-Glo™ Assay System

Broadcast from June 2012 ~30 minutes long

www.promega.com/webinars

Click Previous Webinars in the grey box

Brock F. Binkowski, Ph.D. Sr. Research Scientist II

23

NanoLuc™ Luciferase as a protein function probe Applications of full-length NanoLuc™ Luciferase.

NanoLuc™ Luciferase as a fusion partner: Proof of concept experiments

25

Prot X

NLuc Prot Y

HT

BRET

HT NLuc DEVD

BRET Caspase 3

Ligand

Re

c

Re

c

BRET

NLuc

Protein Stability

Protein-Protein Interactions

Biosensors

Receptor Interactions

Protein Translocation

NanoLuc™ Luciferase excels in bioluminescent imaging applications

Nluc brightness leads to short exposure times:

• Fluc/Rluc: 1-5min/exposure

• Nluc: 1-5sec/exposure

Why bother? Fluorescence works.

• Fluors are susceptible to photobleaching.

• Excitation can cause autofluorescence of other fluors

• Luciferases will generate light as long as substrate is available

Olympus LV200 Bioluminescence Imager

Unfused NLuc

26

NanoLuc™ Fusions can go anywhere…

NLuc-Nrf2 NLuc-b2 AR NLuc-MLS Calreticulin- NLuc-KDEL

Mitochondria ER Anchored Nucleus Cell-Surface

NanoLuc™ Luciferase fusions could be a useful tool to investigate cell biology

27

NanoLuc fusion to Glucocorticoid Receptor

Time lapse: 13 minutes

HeLa cells; 500nM dexamethasone treatment

cytoplasm → nucleus

Bioluminescence imaging of protein translocation

28


30

Prot X

NLuc Prot Y

HT

BRET

HT NLuc DEVD

BRET Caspase 3

Ligand

Re

c

Re

c

BRET


Biosensors



NLuc

Protein Stability

Monitoring Protein Stability with NanoLuc™ Luciferase

31

Can NanoLuc™ Luciferase be added to a protein as a probe for protein stability?

Ub

Ub Ub p53

p53

p53

Ub

Degradation

+ p53

Ub

mdm2

Unstressed Cell

mdm2

mdm2

DNA Damage

NLuc

NLuc

NLuc

NLuc

p53 model

The fusion can be used as a probe of stability


32

Prot X

NLuc Prot Y

HT

BRET

NLuc

Protein Stability



Ligand

Re

c

Re

c

BRET


HT NLuc DEVD

BRET Caspase 3

Biosensors

Bioluminescence Resonance Energy Transfer (BRET)

Luciferase Donor

Fluor Acceptor

Fluorescent Signal

luciferin oxyluciferin

350 400 450 500 550 600 650 700

Important characteristics for BRET applications in research:

• Donor emission must overlap with acceptor excitation spectra

• Donor & Acceptor must be close (<10nm)

• Acceptor emission must be discernable from Donor emission

• Output intensity is dependent upon donor intensity

donor emission

acceptor excitation

Acceptor emission

Occurs Naturally Renilla Luciferase → GFP in Renilla reniformis

Based on publications from S.S. Gambhir at Stanford

Cross-over Donor emission 33

Could NanoLuc™ Luciferase work better as the luminescent donor?

Donor brightness is a key limiter to current BRET technologies. NLuc

Fluor Acceptor

BRET

More spectral overlap needed to get sufficient signal

BRET-beneficial aspects of NanoLuc Luciferase:

~100-fold brighter than Rluc need less spectral overlap with fluor gain greater spectral separation

34

RLuc → GFP

We have a potential acceptor fusion protein:

HaloTag® Fusion Protein 34.1kDa protein engineered from halophilic bacterial hydrolase.

• Engineered to lock into enzyme: substrate intermediate for covalent attachment .

• No homolog in mammalian cells.

Coumarin

Alexa Fluor® 488 Oregon Green®

diAcFAM Rhodamine 110

TMR Alexa Fluor® 660

Goes anywhere in the cell Variety of fluors ready-to-use 35

Testing the concept: NanoLuc™ & HaloTag® Fusion can perform BRET

BRET Emission spectrum BRET Ratio (= Emacc /Emdon )

OR TMR0.00

0.25

0.50

0.75

1.00

1.25

1.50

untr + HT ligand

0

5

10

15

20

S/B

HT-Ligand

BR

ET

Ra

tio S

/B R

atio

0

10000

20000

30000

40000

50000

60000

400 450 500 550 600 650 700

Re

lati

ve L

igh

t U

nit

s

wavelength

TMR NL OR

Oregon Green® BRET spectra

Tetramethyl- rhodamine

BRET Spectra

NanoLuc™ emission

NLuc HT BRET

36

Donor Only

Donor + Acceptor

S/B

HT NLuc DEVD

BRET Caspase 3

Biosensors

Ligand

Re

c

Re

c

BRET



37

NLuc

Protein Stability


Prot X

NLuc Prot Y

HT

BRET


Can NLuc:HT Pair be used for Protein-Protein BRET?

FKBP

NLuc FRB

HT

FKBP

NLuc FRB

HT

BRET

+ Rapamycin

HT-TMR

590 nm

Same model system used with BRET 6 System RLuc8.6 → TurboFP

Dragulescu-Andrasi, A., et al (2011) PNAS 108, 12060-5.

38

Bright Future for NanoLuc™ Luciferase fusions

Brightness improves bioluminescent imaging

Versatility to go anywhere in cell

Versatility to allow stability measurements

Brightness allows BRET with HaloTag® Fusions • Biosensors • Protein:Protein Interactions

Brightness allows BRET with fluorescent ligands

• Ligand binding assays 39

Get a FREE Sample of the NanoLuc™ Vectors.

Ask for the < Source Code >

www.promega.com/nanoluc

pNL 1.1 (NLuc)

pNL 1.2 (NLucP)

pNL 1.3 (secNLuc)

40

Plus more information about all the pNL vectors

Technical Services Scientists ready to help

By phone: 800-356-9526

•Available 7am-6pm Central M-F

Online Chat @ promega.com

•Available 7am-6pm Central M-F

•Global Chat with Branch office tech serv scientists, too, after hours (language dependent)

[email protected]

•Guaranteed answers within 24hr

•Most responses within 2hrs

41

mailto:[email protected]

Questions?

Documents

Applications of a smaller, brighter, more versatile luciferase: