Download A Novel Assay for DNA-Dependent DNA Polymerase Activity

A Novel Assay for DNA-Dependent
DNA Polymerase Activity
Anu Mathew,
Renée Benson, Lisa Gazzillo,
Robert Umek, James L. Wilbur,
George B. Sigal, Eli N. Glezer,
Hans A. Biebuyck and Jacob N. Wohlstadter
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TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
Abstract
Human, viral, and bacterial DNA polymerases are attractive targets for drug
interventions, and as such are candidates for HTS screening efforts. We have
developed an assay for DNA-dependent DNA polymerase activity using a new
assay platform developed by Meso Scale Discovery (MSD ). This platform
combines array technologies and electrochemiluminescence detection to achieve
ultra-fast, highly sensitive assays in a no-wash format. Polymerase activity
results in the extension of a primer containing a ruthenium complex that
emits electrochemiluminescence. The assay is exquisitely sensitive to the
processivity of the enzyme and only detects full-length products. Several
distinct polymerases (Klenow and the HIV RT enzymes) were analyzed in the
assay. Several known inhibitors were screened to validate the assay format. We
have demonstrated feasibility of assay automation in a 96-well format, and
developed protocols to enable minimal manipulations.
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
Multi-Array Technology
TM
Unified technology platform with instruments, plates and reagents
for drug discovery.
Combines the power of microarrays with the sensitivity of
electrochemiluminescence.
96-, 384- and 1536 microplate formats.
Multi-Spot plates with high density arrays for multiplexing.
TM
Sector HTS Instrument: High resolution imaging detection and
robotic integration for HTS and large-scale proteomics.
TM
Sector HTS
Sector PR Instrument: Medium throughput benchtop reader for
assay development, cellular and molecular biology, research in
therapeutic areas, secondary screening, QC. Assays developed on
Sector PR port to Sector HTS.
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Sector PR
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
Schematic of the DNA-Dependent DNA Polymerase Assay
Ru
A
Ru
B
pol
NaOH
Ru
NaH2PO4
B
Ru
Ru
a
B
e
C
The substrate is a heteroduplex containing an annealed region (red) for priming
and a template region (blue) for elongation (A). The primer to be elongated has a
ruthenium chelate (Ru) attached to the 5’ end. After elongation by the DDDP
activity of the polymerase (B), the reaction is stopped with NaOH. Subsequently,
the reaction is neutralized by the addition of NaH2PO4 containing a biotinylated
oligonucleotide complementary to the newly synthesized strand. The entire reaction
is conducted in a well containing an avidin-coated (a) carbon electrode (e). The
biotinylated oligonucleotide serves as a capture probe that tethers the elongated,
Ru-containing strand to the electrode. In the standard reaction conditions, a 20ul
reaction is conducted using 1nM HIV-RT (Worthington) and 50nM substrate in
reaction buffer (10mM Tris pH 7.5, 5mM MgCl2, 3mM DTT, 10uM dNTPs). The
reaction is stopped by the addition of 25ul of base to a final concentration of
30mM followed by 25ul of 90mM NaH2PO4 containing 4 pMol of biotinylated
capture probe. The microtiter plate is agitated throughout the 45 minute capture
period. Note that re-annealing of sister strands (C) competes with capture and
immobilization of the Ru-labeled species at the surface of the electrode. The data
shown here were obtained using HIV-RT. However, with slight changes to the
reaction buffer, similar results were obtained with the Klenow polymerase of E. coli
and human DNA polymerase beta. The reaction is distinguished from those that
measure nucleotide incorporation in that only full-length products capable of
hybridizing are detected.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
Kinetic Analysis of the Polymerase Reaction
B
A
300000
160000
250000
120000
Average Signal
Average Signal
200000
150000
100000
50000
0
Total signal
0
1
2
3
4
5
100nM
80000
40000
0
6
50nM
Bkgd
0
40
RT enzyme (nM)
Total
100000
Average Signal
80000
60000
40000
20000
0
0
50
100
150
120
160
200
Substrate (nM)
C
120000
80
200
250
Bkgd
The DNA polymerase reaction was performed under standard
conditions except for variation in enzyme (A) and substrate
(B) concentrations. The reaction is linear with respect to
enzyme concentration over the range studied. The standard
reaction conditions were chosen such that the linear
dependence on enzyme (A) and time (C) are represented
while substrate is present in excess.
Time (min)
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TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
The DNA-Dependent DNA Polymerase Assay Facilitates
EC50 Determinations
Suramin
AurintricarboxylicAcid
60000
90000
75000
Total signal
30000
EC50
7.8 µM
Bkgd
15000
0
Average Signal
Average Signal
45000
60000
0.0
EC50
30000
39 µM
Bkgd
15000
0
-0.5
Total signal
45000
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
2.0
ATA (LOG µM)
Suramin (LOG µM)
The effects of suramin and aurintricarboxylic acid (ATA), two inhibitors known to affect the reverse transcriptase
(RNA-dependent DNA polymerase) activity of the HIV RT enzyme, were tested for effects on the DNA-dependent DNA
polymerase activity of this multifunctional enzyme. Our results generated EC50 values for both inhibitors as indicated.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
A Simplified Protocol that Omits Agitation Retains a High
Signal/Background Ratio
Total Signal
Average Signal
75000
50000
25000
agitation -Total
no agitation -Total
agitation -Bkgd
no agitation -Bkgd
75
agitation
no agitation
Signal/Background Ratio
100000
Signal/Background
50
25
0
0
The standard DNA-dependent DNA polymerase assay was conducted except the protocol eliminated agitation during the
capture step. To compensate for the lack of agitation, the capture time was extended to 3 hours. In the absence of
agitation, there is a significant decrease in total signal obtained, despite the longer capture time allowed. This decreased
signal is likely the result of increased sister strand re-annealing (see schematic). The signal/background ratio also
decreases. However, the signal/background ratio remains high (22) despite the elimination of the agitation step. The
results support the notion that an automated assay without agitation can be executed.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
Validation of Alternative Assay Formats for HTS
+ wash, + agitation
-wash, -agitation
200000
75000
150000
100000
total signal
z factor
0.798
Signal
Signal
50000
total signal
25000
50000
z factor
0.617
20
30
bkgd
0
bkgd
0
0
10
20
30
40
50
0
Sample No.
10
40
50
Sample No.
Two workflow protocols were compared for performance based on Z-factor scores. The standard assay was compared to
an assay in which there were no washes. In the no-wash assay, the agitation during capture was eliminated and the
capture time was extended to 3 hours. In both cases, the assays were executed in an automated fashion in which a
Rapid-PlateTM (Zymark) and a MultidropTM (Labsystems) were used for reagent delivery, aspiration and washing, where
appropriate. The HIV RT enzyme was used, though similar results have been obtained with the Klenow polymerase for
non-washed and washed formats. Acceptable Z-factor values (>0.5) support the notion that both protocols can be used
in HTS.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM
A Novel Assay for DNA-Dependent DNA
Polymerase Activity
Conclusion
A DNA-dependent DNA polymerase assay has been developed on a novel
platform that utilizes disposable carbon electrodes arrayed in microtiter
plates.
A DNA capture probe immobilizes the product that carries an
electrochemiluminescent reporter.
The assay is particularly sensitive to the processivity of the enzyme since
only full-length products are detected.
The assay is compatible with HTS and facilitates the characterization of
EC50 values of inhibitors.
TM
TM
A division of Meso Scale Diagnostics, LLC.
TM