1. Introduction
2. NanoLuc: a brighter, smaller luciferase for reporter
gene assays and protein fusions
3. The Nano-Glo® Dual-Luciferase® Reporter
(NanoDLR™) Assay System
4. Improved reagent stability and signal stability to
facilitate batch processing of plates
5. Orders of magnitude greater sensitivity for Nluc in
NanoDLR™ assay compared to Renilla luciferase
6. Using two different transcriptional reporters can
greatly improve confidence in HTS results
7. Coincidence reporter to screen for increased
expression from an endogenous promoter
8. Coincidence reporter distinguishes transcriptional
activators from compounds stabilizing reporter
9. Summary
www.promega.com
Add ONE-Glo™ EX
Reagent Measure Fluc
luminescence
Add NanoDLR™ Stop&Glo®
Reagent (containing Fluc
inhibitors)
Measure Nluc
luminescence
• “Add-read-add-read” homogeneous format is conducive to HTS.
• The Fluc signal is quenched over a million-fold upon addition of the NanoDLR™ Stop&Glo®
Reagent, which also supplies the substrate for Nluc activity.
• The increased brightness of Nluc compared to Renilla luciferase and improved inhibition of the
Fluc signal give orders of magnitude greater sensitivity than the existing dual assay.
• ONE-Glo™ EX Reagent utilizes new chemistry that provides a two-hour signal half-life for firefly
luciferase, enhanced room temperature stability, lack of odor from DTT, steadier signal decay
kinetics, and improved performance in the presence of phenol red.
• Both ONE-Glo™ EX and NanoDLR™ Stop&Glo® reagents were designed for improved
room temperature stability once reconstituted (Panel A).
Consistent luminescence values throughout long screening runs.
• Both reagents exhibit a steady rate of signal decay with a half-life of about two hours
(Panels B).
A stable signal facilitates batch processing and decreases plate-to-plate and well-to-
well variability.
Nluc
homology
model
• NanoLuc® luciferase (Nluc) is a 19.1 kDa, ATP-independent luciferase that
utilizes a novel coelenterazine analog (furimazine) to produce high-intensity,
glow-type luminescence (about 150-fold brighter than firefly or Renilla).
• The broad dynamic range and sensitivity of Nluc make it an ideal transcriptional
reporter. Adding a PEST sequence to destabilize the protein (NlucP) gives
maximal temporal dynamics and fold induction (Panel A). Adding a secretion
signal (secNluc) allows transcription to be measured without cell lysis.
• Because of their brightness, Nluc fusions can enable such applications as
1) measurement of protein stability at endogenous levels (Panel B), 2) BRET
studies of protein-protein interactions, and 3) bioluminescent imaging.
0 1 2 3
1
1 0
1 0 0
1 0 0 0
T im e (h o u rs )
Fo
ld r
es
po
ns
e
In d u c tio n o f N F B R E w ith T N F
N lu c P
F lu c P
F lu c
N lu c
• The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System provides robust and
sensitive measurement of firefly luciferase (Fluc) and NanoLuc (Nluc) in a single well.
Homogeneous assay, not requiring cumbersome aspiration, wash, and lysis steps.
“Glow” (not “flash”) kinetics, with two-hour signal half-life, allow for batch processing of plates.
Improved stability means reconstituted reagents can be kept at room temperature for long runs
with little decrease in luminescence.
The increased brightness of Nluc and improved quenching of the Fluc signal give orders of
magnitude greater sensitivity of detection, compared to Renilla luciferase in Dual-Glo.
This increased sensitivity enables both luciferases to be used as dynamic reporters, enabling
additional formats, like coincidence reporting.
• A coincidence reporter system with Fluc and NlucP can dramatically improve the quality of data
coming out of a high-throughput screen.
Luciferase inhibitors can represent a significant fraction of hits in HTS because of their
stabilization of the reporter enzyme in cells or their inhibition of reporter activity.
Fluc and Nluc are non-homologous luciferases utilizing different substrates, and they have
dissimilar profiles of compound interference.
Performing screens with an Fluc-P2A-NlucP coincidence reporter can eliminate assay
artifacts, reducing the number of actives >5-fold and increasing the confidence that hits are
biologically relevant.
• Compounds causing interference with
Fluc (Panels C-E) or Nluc (Panel A) alone
showed an increase in signal only for that
reporter.
• The addition of a PEST sequence to only
Nluc allowed for the identification of
compounds that interfere globally with
protein stability and turnover, such as the
proteasome inhibitor, MG-132, which
caused an increase in the NlucP, but not
Fluc, signal (Panel B).
• Those compounds truly increasing
transcription, such as the histone
deacetylase inhibitor, panobinostat,
showed coincident increases in both
reporters (Panel F).
Luciferase-based reporter-gene assays remain a cornerstone of high-throughput screening of
compounds because of their high sensitivity and dynamic range. However, a substantial number of
non-relevant hits can be generated due to direct interaction of compounds with the luciferase
reporter. To help differentiate compounds modulating the biological pathway of interest from those
affecting the stability or activity of the reporter enzyme, we have developed a second-generation
coincidence reporter system in which transcriptional activation leads to stoichiometric expression of
two orthologous reporters that have dissimilar profiles of compound interference. In this system,
firefly luciferase (Fluc) and PEST-destabilized NanoLuc® luciferase (NlucP) are expressed off the
same promoter using ribosome skipping mediated by the P2A peptide.
To sensitively measure both Fluc and NanoLuc (Nluc) in the same sample, we have developed the
Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System, a homogeneous lytic assay
performed in an “add-read-add-read” format, in which the Fluc signal is quenched over a million-fold
by addition of the Nluc reagent. The increased brightness of Nluc and improved Fluc inhibition
means that Nluc can be detected at over 2-3 orders of magnitude lower molar concentration than
Renilla luciferase in the existing homogenous firefly/Renilla dual-luciferase assay (Dual-Glo),
allowing both luciferases to be dynamic reporters.
Following single-copy integration of the Fluc-2A-NlucP biocircuit into a gene locus relevant to
Parkinson’s disease, HTS using NanoDLR easily distinguished compounds affecting one of the
reporters from those affecting transcription, yielding a >5-fold decrease in the number of hits.
nrf2 NanoLuc
N rf2 -N a n o L u c F u s io n in H C T 1 1 6
C e lls T re a te d w ith D ,L -S u lfo ra p h a n e
1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4
0
2 0 0 0 0 0
4 0 0 0 0 0
6 0 0 0 0 0
8 0 0 0 0 0
1 0 0 0 0 0 0
2 h
4 h
0 h
1 h
0 .5 h
[D ,L -S u lfo ra p h a n e ], M
RL
U
• The Fluc-P2A-NlucP biocircuit was
integrated into a gene locus relevant to
Parkinson’s disease by TALEN-mediated
genome editing in order to screen for
compounds increasing transcription
(Panels A & B).
• P2A-peptide-mediated ribosome skipping
allowed unfused Fluc and NlucP to be
expressed stoichiometrically from the same
endogenous promoter.
• 315,000 compounds were screened in 5-
point qHTS format in 1536-well plates.
• Approximately 6300 compounds (2%)
displayed activation of an individual
reporter, but only ~950 compounds (0.3%)
were positives with coincidence, yielding a
>5-fold reduction in hits.
NanoLuc®
Luciferase
Furimazine
+ O2
+ CO2
+ light
Nluc as transcriptional reporter Nrf2-Nluc fusion to measure oxidative stress response
0 1 2 3 4 5 6
0
5 0
1 0 0
O N E -G lo E X a n d N a n o D L R S to p & G lo
R e a g e n ts D is p la y Im p ro v e d
R o o m T e m p e r a tu re S ta b ility
T im e (d a y s )
% A
cti
vit
y
Re
ma
inin
g
O N E -G lo E X
O N E -G lo
D u a l-G lo
N a n o D L R S to p & G lo
N a n o -G lo
0
1
2
3
4
5
6
7
8
9
1 0
-1 7 -1 6 -1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8
L u c ife ra s e S ig n a l
in th e A b s e n c e o f F lu c
lo g [ lu c ife ra s e ] (M )
log
RL
Us
N a n o D L R S to p & G lo (N lu c )
D u a l-G lo S to p & G lo (R lu c )
0
1
2
3
4
5
6
7
8
9
1 0
-1 7 -1 6 -1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8
L u c ife ra s e S ig n a l
in th e P re s e n c e o f 1 0 0 n M F lu c
lo g [ lu c ife ra s e ] (M )
log
RL
Us
N a n o D L R S to p & G lo (N lu c )
D u a l-G lo S to p & G lo (R lu c )
sensitivity
difference
sensitivity
difference
• The inherent brightness of Nluc compared to Rluc makes NanoDLR more than 100-fold more
sensitive than Dual-Glo in the absence of Fluc (Panel A).
• With very high Fluc concentrations, uninhibited Fluc signal can increase the background for the
second luciferase. The improved Fluc inhibition in NanoDLR yields over 1000-fold greater
sensitivity than Dual-Glo under these conditions (Panel B).
• Compounds directly binding the reporter can inhibit and/or stabilize the enzyme, leading to
positives not related to altered transcription (Auld, DS, et al. ACS Chem. Biol., 2008; 3: 463-470.)
• A coincidence reporter system, in which transcription is linked to two reporters with dissimilar
profiles of compound interference, can dramatically decrease the number of hits arising from
direct interactions with the reporter (Cheng, KC & Inglese, J. Nature Methods, 2012;9:709–736.)
• Firefly luciferase (Fluc) and PEST-destabilized NanoLuc (NlucP) make a particularly attractive
pair of orthologous reporters (Ho, P. et al. ACS Chem. Biol., 2013; 8: 1009-1017.)
Reporter 1 2A Reporter 2
Promoter
• Reporter 1: Fluc
• ATP-dependent
• Firefly luciferin (benzthiazole)
• Adenylate intermediate
• Reporter 2: NlucP
• ATP-independent
• Fz + O2 → Fz + CO2 + h
• No sequence homology to FLuc
GSGATNFSLLKQAGDVEENPGP
linker -helix cis-acting hydrolase
element
Bicistronic
transcript
Activation of
target pathway
Unfused reporters Compound
Compound
Direct binding to
one of the reporters
activity
activity
Stabilization
A B
A B
A B
0 1 2 3 4 5
1 0
1 0 0
F lu c a n d N lu c S ig n a ls D e c a y w ith
a H a lf-L ife o f A b o u t 2 H o u rs
T im e (h o u rs )
Pe
rc
en
t S
ign
al
O N E -G lo E X
N a n o D L R S to p & G lo
50
[email protected] January 2014
A New Dual Luciferase Assay Using NanoLuc Enables a Second-Generation Coincidence Reporter System to Reduce False Hits in HTS
Christopher Eggers1, Samuel Hasson2, Brock Binkowski1, Matt Robers1, James Unch3, Braeden Butler1, Keith Wood1, James Inglese2, and Frank Fan1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399; 2National Center for Advancing Translational Sciences, 9800 Medical Center Dr, Rockville, MD 20850; 3Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401
Abstract #285