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.

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 7:1848-1857.

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

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

8

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

9

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

10

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

11

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

13

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

14

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.

16

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

17

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

18

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

19

Secretion based format using secNluc

20

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

21

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

23

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

24

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

25

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

26

NanoLuc fusion to Glucocorticoid Receptor

Time lapse: 13 minutes

HeLa cells; 500nM dexamethasone treatment

cytoplasm → nucleus

Bioluminescence imaging of protein translocation

27


29

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

30

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


31

Prot X

NLuc Prot Y

HT

BRET

NLuc

Protein Stability



Ligand

Re

c

Re

c

BRET


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

32

Could NanoLuc™ work better as a BRET donor?

34

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


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


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



40

Prot X

NLuc Prot Y

HT

BRET

NLuc

Protein Stability



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

41

HT NLuc DEVD

BRET Caspase 3

Biosensors

Ligand

Re

c

Re

c

BRET



42

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.

43


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


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

46

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

47

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.

48


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


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

NanoLuc®: A Smaller, Brighter, and More Versatile Luciferase ...

Documents

Transcript of NanoLuc®: A Smaller, Brighter, and More Versatile Luciferase ...