NanoLuc®: A Smaller, Brighter, and More Versatile Luciferase ...
Transcript of NanoLuc®: A Smaller, Brighter, and More Versatile Luciferase ...
©2010, Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.
NanoLuc®: A Smaller, Brighter, and More Versatile Luciferase Reporter
Terry L. Riss, Ph.D. Senior Product Specialist, Cell Health Promega Corporation
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 7:1848-1857.
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
4
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
Confidential and Proprietary. Not for Further Disclosure.
NanoLuc is very bright
Purified enzyme
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
Living HEK293 cells in 96-well plate (50,000 cells per well). Imaged by a hand-held iPhone
Expression in mammalian cells
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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
Reduced false hits with NanoLuc® Luciferase in HTS
Level of inhibition
≥ 10% ≥ 20% ≥ 30% ≥ 50%
NanoLuc 1.2% 0.5% - -
Firefly 1.9% 0.7% 0.5% 0.3%
% of library compounds
Experimental details: LOPAC library members at 10 µM final concentration; incubation with purified NanoLuc or firefly luciferase for 2 min.; Fluc detection using ONE-Glo™ Luciferase Assay..
LOPAC library (Sigma)
• Library of Pharmaceutically Active Compounds
• 1280 compounds
• Small organic ligands w/ well documented pharmacological activities
• Used to screen for non-specific luciferase activity modulators
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FLuc NLuc
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 12
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
Summary: 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
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 is well behaved in mammalian cells
19714
19714
Expression in HEK cells
Expression in E. coli
Uniform intracellular distribution
Immunofluorescence Luminescence
No post-translational modifications
U2OS cells U2OS cells
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NanoLuc™ Fusions can be designed to 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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NanoLuc™ Luciferase can 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
100%
Bioluminescence Resonance Energy Transfer (BRET)
Luciferase Donor
Fluor Acceptor
BRET signal
luciferin oxyluciferin
...in Nature
first described by C.H. Johnson & colleagues in 1999
ProtX -Luc
TRANSFECT FUSION
CONSTRUCTS
ProtY -Fluor
BRET signal
...in the Lab
Luc Donor
luciferin oxyluciferin
X
Fluor Acceptor
Y
BRET signal
350 400 450 500 550 600 650 700
Donor Em
Acceptor Ex
Acceptor Em
Requirements for BRET: • Donor & Acceptor must be close (10-100 Å) • Donor emission spectrum must overlap with
Acceptor excitation spectrum
Aequorea victoria Renilla reniformis
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Could NanoLuc™ work better as a BRET donor?
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Nluc Fluor Acceptor
BRET signal
BRET-beneficial properties of NanoLuc Luciferase:
100x brighter than Rluc… less spectral overlap of Donor & Acceptor needed Better S:B and dynamic range
Blue-shifted emission… Lots of space on the spectrum for the Fluor Acceptor
Nluc 460nm; Rluc 480nm; Fluc 560nm
We have a potential acceptor fusion protein:
HaloTag® 34.1kDa protein engineered from halophilic bacterial hydrolase
• Forms covalent attachment to functional ligand
• Add ligand to cells expressing HaloTag fusion
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
Confidential and Proprietary. Not for Further Disclosure.
HT Protein of
Interest
HaloTag technology – affinity tag alternative
Protein-mediated covalent attachment:
• Strong binding – covalent bond is essentially irreversible
• High specificity – modified catalytic mechanism
• High binding rate – optimized protein structure
+ HT Protein of
Interest
Reactive linker Functional group
Confidential and Proprietary. Not for Further Disclosure.
Multiple uses with one genetic construct
Cellular imaging
HaloTag Vector
Protein
HT
Expression of HaloTag fusion
Covalent capture
Interaction
s
Protein labeling
Purification Detection
HaloTag fluorescent ligands HaloTag surfaces
Protein display
O O Cl
O O Cl
Ligand
Re
c
Re
c
BRET
Receptor Interactions
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
HT NLuc DEVD
BRET Caspase 3
Biosensors
Biosensor: Disrupting BRET with a cleavable bridging peptide sequence
Staurosporine
0.1 1 10 100 1000 100000.000
0.002
0.004
0.006
0.008
nM [Staurosporine]
BR
ET
ra
tio
TRAIL
0.01 0.1 1 10 100 1000100000.000
0.002
0.004
0.006
0.008
ng/ml [TRAIL]
BR
ET
rati
o
HT NLuc DEVD
BRET BRET
HT-TMR
590 nm
Caspase 3 Activation
HT NLuc DEVD HT-TMR
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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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Confidential and Proprietary. Not for Further Disclosure.
BRET assay for detecting bromodomain interactions with histones
HT
H3 or H4
HaloTag fusion NanoLuc
BRD4
NanoLuc fusion
BRET (600/480nm) Measurement
TMR
585 nm
Furimazine
460 nm
0.000
0.001
0.002
0.003
0.004
0.005
0.006
BRD2 BRD3 BRD4 CBP p300 Control
Histone H3
Histone H4
BRET Measurement in HCT116 Cells
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Histone H3
Histone H4
BRET Measurement in HCT116 Cells
BR
ET
Ra
tio
Confidential and Proprietary. Not for Further Disclosure.
Measuring effect of BET inhibitor on histone-bromodomain interactions
IC50 = 65.4 nM
IC50 = 222.8 nM
Histone H4-HaloTag
Histone H3.3-HaloTag
NLuc-BRD4
NLuc-BRD4 NLuc-CBP
NLuc-CBP
Glucorticoid Receptor Dimerization
0.1 1 10 100 10000.0008
0.0009
0.0010
0.0011
0.0012
0.0013
0.0014
0.0015
0.0016
nM [Dexamethasone]
BR
ET
Rati
o
GR
NLuc GR
HT
GR
NLuc GR
HT
BRET + Dexa -
methasone
HT-TRM
590 nm
cytosol nucleus
Translocation into nucleus
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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
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Nano-Glo™ Luciferase Assay System
Cat.# N1150 10x100ml (10,000 assays) • 5 x 4ml Nano-Glo™ Luciferase Assay Substrate
• 10 x 100ml Nano-Glo™ Luciferase Assay Buffer
Cat # N1110: 10ml (100 assays) • 200μl Nano-Glo™ Luciferase Assay Substrate
• 10ml Nano-Glo™ Luciferase Assay Buffer
Cat.# N1120 100ml (1,000 assays) • 2 x 1ml Nano-Glo™ Luciferase Assay Substrate
• 100ml Nano-Glo™ Luciferase Assay Buffer
Cat # N1130 10x10ml (1,000 assays) • 10 x 200μl Nano-Glo™ Luciferase Assay Substrate
• 10 x 10ml Nano-Glo™ Luciferase Assay Buffer
Just make enough 1X Nano-Glo Assay Reagent from the 50X substrate and buffer to meet your needs. No need to make up all the reagent at once.
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Confidential and Proprietary. Not for Further Disclosure.
Currently Available Vectors
Plasmid Reporter Marker Promoter MCS
Catalog #
pNL1.1 Nluc - - Y N1001
pNL1.2 NlucP - - Y N1011
pNL1.3 secNluc - - Y N1021
pNL2.1 Nluc Hygro - Y N1061
pNL2.2 NlucP Hygro - Y N1071
pNL2.3 secNluc Hygro - Y N1081
pNL3.1 Nluc Hygro minP Y N1031
pNL3.2 NlucP - minP Y N1041
pNL3.3 secNluc - minP Y N1051
pNL1.1.CMV Nluc - CMV N N1091
pNL1.3.CMV secNluc - CMV N N1101
pNL3.2. NF-kB-RE
NlucP Hygro NF-kB-
RE/minP N N1111
The pNL vectors are built on the pGL4 Vector backbone allowing transfer of inserts from pGL4 series vectors to the pNL vectors.
49
Confidential and Proprietary. Not for Further Disclosure.
Want to know what vectors are coming?
www.promega.com/cam
Stay on the cutting edge and get research tools before they are available to everyone!
New vectors using Promega exclusive technologies are typically offered as a custom product before they become a catalog product.
Visit the Current Research Materials page at:
You can also request construction of a special vector to meet your exact needs with Custom Assay Services
50
Confidential and Proprietary. Not for Further Disclosure. Confidential and Proprietary. Not for Further Disclosure.
Applications
Brock Binkowski
Braeden Butler
Mike Valley
Matt Robers
Thomas Machleidt
Chris Eggers
Frank Fan
Hui Wang
Substrate Design
Poncho Meisenheimer
James Unch
Ruslan Arbit
Dieter Klaubert
Enzyme Design
Lance Encell
Mary Hall
Monika Wood
Kris Zimmerman
Paul Otto
Hélène Benink
Gedi Vidugiris
Mike Slater
Danette Daniels
Marie Schwinn
Jacqui Mendez
Keith Wood
©2012, Promega Corporation.
Questions Welcome