Applications of a smaller, brighter, more versatile luciferase:
Transcript of 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
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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.
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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
NanoLuc™ Luciferase
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
NanoLuc™ Luciferase
2,500,000X
substrate evolution
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NanoLuc™ is very small
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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
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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
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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
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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
NanoLuc™ Luciferase PEST
NanoLuc™ Luciferase IL6
Nluc (513 bp)
NlucP (636 bp)
secNluc (597 bp)
Secretion Format
pNL1.1
pNL1.2
pNL1.3
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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
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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
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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
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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.
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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
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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
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3 Varieties of NanoLuc™ Luciferase for you
Intracellular Formats
Secretion signal
Protein destabilization domain
NanoLuc™ Luciferase
NanoLuc™ Luciferase PEST
NanoLuc™ Luciferase IL6
Nluc (513 bp)
NlucP (636 bp)
secNluc (597 bp)
Secretion Format
pNL1.1
pNL1.2
pNL1.3
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Secretion based format using secNluc
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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
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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
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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)
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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
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NanoLuc™ Luciferase as a protein function probe Applications of full-length NanoLuc™ Luciferase.
NanoLuc™ Luciferase as a fusion partner: Proof of concept experiments
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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
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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
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NanoLuc fusion to Glucocorticoid Receptor
Time lapse: 13 minutes
HeLa cells; 500nM dexamethasone treatment
cytoplasm → nucleus
Bioluminescence imaging of protein translocation
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NanoLuc™ Luciferase as a fusion partner: Proof of concept experiments
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Prot X
NLuc Prot Y
HT
BRET
HT NLuc DEVD
BRET Caspase 3
Ligand
Re
c
Re
c
BRET
Protein-Protein Interactions
Biosensors
Receptor Interactions
Protein Translocation
NLuc
Protein Stability
Monitoring Protein Stability with NanoLuc™ Luciferase
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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
NanoLuc™ Luciferase as a fusion partner: Proof of concept experiments
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Prot X
NLuc Prot Y
HT
BRET
NLuc
Protein Stability
Protein-Protein Interactions
Protein Translocation
Ligand
Re
c
Re
c
BRET
Receptor Interactions
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
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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
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Donor Only
Donor + Acceptor
S/B
HT NLuc DEVD
BRET Caspase 3
Biosensors
Ligand
Re
c
Re
c
BRET
Receptor Interactions
NanoLuc™ Luciferase as a fusion partner: Proof of concept experiments
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NLuc
Protein Stability
Protein Translocation
Prot X
NLuc Prot Y
HT
BRET
Protein-Protein Interactions
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.
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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
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www.promega.com/nanoluc
pNL 1.1 (NLuc)
pNL 1.2 (NLucP)
pNL 1.3 (secNLuc)
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