Improved Ligation Specificity with Chemically Modified
Ligation Components
Sabrina Shore, Alexandre Lebedev, Elena Hidalgo Ashrafi, Gerald Zon, Natasha Paul, Richard Hogrefe.
TriLink BioTechnologies, Inc. San Diego, CA 92121.
P
0.4
0.2
0
U
A
B
C
D
2) Acceptor Probe
O
Standard Conditions
O
5'
O P
O
O
Enzyme
A
O
P
O
O
B2
P
O
O
O P N
OH H
O
HO
O
O
-O
OH
P
B3
Lead Modification
Name
O
O
O
O
O P
HO O O
O
O
O
Nicked DNA
3' HO
O
B1
3'
Chemical modifications to the base, sugar, or phosphate of the donor
and acceptor probes were evaluated for their influence on improved
mismatch discrimination
C
D
A
B
C
D
Ligation Product
Template
Acceptor
Donor
P
5’- ... T C G
T G T ... -3’
3’- ... A G C - A C A ... -5’
P
5’- ... T C A
T G T ... -3’
3’- ... A G C - A C A ... -5’
(normalized to unmodified)
B
B No DNA Ligase
C No ATP
D Control Reaction
The strong propensity for T4 DNA ligase to join mismatched ligation
probes necessitates a screening of modified ligation components for
improved stringency of ligation
Ligation conditions: 1X Ligase Buffer (50 mM Tris (pH 7.5), 10 mM DTT, 10 mM MgCl2),
Ligation Probes (1 µM), ssDNA Template (1 µM), ATP analogs (1 mM), T4 DNA Ligase (0.5 Weiss units), 20 µL.
Thermal cycling conditions: 95ºC (3 min), 4ºC (3 min), add Ligase, 22ºC (60 min), 65ºC (10 min).
1.2
7
O
HO P O P O P O
5
N
5'
4'
8
O
6
2
N9
4
X1=O, X02 = CH10
3
-0.3
20
Cycles
,30
Y3 = H 40
-0.3
∆Cq
T4
0.7
0
10
E. coli
20
30
1.7
-0.3
40
∆Cq
0.7
0
10
Cycles
Mismatch G-T
20
30
40
Cycles
Mismatch C-T
Mismatch T-T
Mismatch G-T Mismatch C-T Mismatch T-T
Modifications evaluated:
Y1, Y2, Y3 = H or OCH3
Cq
(dRn)
ΔCq
Cq
(dRn)
ΔCq
Cq
(dRn)
ΔCq
Cq
(dRn)
ΔCq
X1, X2 = O, or CH3 Standard Conditions
20.15
0
20.96
1
21.48
1
21.10
1
Acceptor Modifications 2 22.97
0
24.40
4
29.84
40
34.31
14
Cofactor Modifications 2 22.52
0
28.14
8
28.09
8
30.45
10
Chemically modified ligation components increase ΔCq's between
matched and mismatched targets compared to standard conditions
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), CleanAmp™ Precision Primers (0.1
µM), 0.2 mM dNTPs, 5 µL Ligation product (Diluted 105), 1.25 U IVGN Taq DNA polymerase, SYTO®9 Green (2 µM),
ROX (0.03 µM), 25 µL.
Thermal cycling conditions: 95°C (10 min); [95°C (30 sec), 56°C (30 sec), 72°C (1min)] 40X.
Cystic Fibrosis SNP detection by Ligase Detection
Reaction (LDR)
Ligation Probes
C-T Mismatch
0.8
A-T Match
0.4
WT (+) or Mut (-)
0.4
0
0
ed
fi
di
m
Un
PS
)
)
)
)
)
)
(n n-1 (n n-1 (n n-1
( Me
( Me
(
PS P PMe '-O
Me
O
2
2'
ed
fi
di
o
m
Un
P
PS
)
)
)
)
)
)
(n n-1 (n n-1 (n n-1
( Me
( Me
(
PS P PMe '-O
Me
O
2
2'
+
-
+
-
+
-
+
-
+
-
+
P
5'-... G T T
A G T ... -3'
3'-... C A X - T C A ... -5'
5'-... G A C
C T C ... -3'
3'-... C T X - G A G ... -5'
X = wild type (A) or mutant (C)
X = wild type (G) or Single Nucleotide Polymorphism (SNP) (A)
Different lead acceptor probe modifications identified
depending on the DNA ligase family (ATP or NAD+)
Chemically modified components improve mismatch
discrimination in LDR
Ligation conditions: 1X Ligase Buffer (50 mM Tris (pH 7.5), 10 mM DTT, 10 mM MgCl2, 1 mM ATP),
Ligation Probes (1 µM), ssDNA Template (1 µM), (0.5 Weiss units T4 DNA Ligase or 5 Units E.Coli DNA Ligase), 20 µL.
T4 DNA Ligase Thermal cycling conditions: 95ºC (3 min), 4ºC (3 min), add Ligase, 22ºC (20 min), 65ºC (10 min).
E.Coli DNA Ligase Thermal cycling conditions: 95ºC (3 min), 4ºC (3 min), add Ligase, 16ºC (20 min), 65ºC (20 min).
LDR conditions: 1X Ligase Buffer (20 mM Tris (pH 7.5), 20mM KCl, 1 mM DTT, 10 mM MgCl2, 0.1% IGEPAL®), 0.01
mM Cofactors, 0.50 µM Donor probe, 0.25 µM Acceptor probe, ssDNA template (50 nM), 4 Units Pfu DNA Ligase, 20
µL.
Cycling conditions: 94°C (2 min), Cycle 20x [94°C (30 sec), 65°C (4 min)]
Conclusion
Ligation Product
Template
Ligation Probes
N
3
1) Chemical modifications made to the ATP cofactor, donor probes and
acceptor probes provide improved ligation specificity for T4 DNA ligase.
2) Optimal chemical modifications to acceptor probes vary depending on
the DNA ligase family (ATP or NAD+).
2'-OMe (n-2)
2'-OMe (n-1)
2'-OMe (n)
PMe (n-2)
PMe (n-1)
No Modification
Investigation of chemically modified donor probe
modifications
Match
A-T
N1
PMe (x-1)
Y1, Y2, Y3 = H
1.7
2.7
0.8
Core Structure of ATP Cofactor
H2N
X1=CH0.73, X2 = O
DNA
ligase
Ligation Product
No Ligase
Design of chemically modified ATP Cofactors
PMe (x)
2.7
3.7
ssDNA Template
Figure 7
Figure 3
Modification
on position Y
3.7
1.2
1.6
o
A No Template
2.7
Modification
o
1.7
Cofactor Modifications 2
E.Coli DNA Ligase
(NAD+ dependent)
T4 DNA Ligase
(ATP dependent)
Relative Ligation Yield
A
2' OMe (n-1) Donor
Cofactor Modification 1
Cofactor Modification 2
Figure 10
Comparison of acceptor probe modifications using ATP or
NAD+ ligases
Mismatch
Match
3.7
Match A-T
Figure 6
Limitations of T4 DNA ligase in mismatch discrimination
Acceptor Modifications 2
Match A-T
O
O
Donor
P
Probe 2, Y3 = H, F, or OCH3 -O O
Acceptor
Probe
B1
B2
X1, X2 = O, S, CH3
Joined DNA
Figure 2
D
E
F
Real-time ligation-PCR using chemically modified ligation
components
Fluorescence (dRn)
B3
O
3) Donor Probe
Unmodified
PMe (n) Acceptor
2' OMe (n-1) Acceptor
PMe (n-1) Donor
Mismatch T-T
No Ligase
Three Potential Components to Modify:
1) ATP Cofactor
O
-O
DNA-AMP
Mismatch C-T
F
Figure 9
E-AMP
Donor
Probe
Nicked DNA
E
Ligation conditions: 1X T4 Ligase buffer (50 mM Tris (pH 7.5), 10 mM DTT, 10 mM MgCl2), Ligation probes (1
µM), ATP analogs (1 mM), ssDNA template (1 µM), T4 DNA Ligase (0.5 Weiss units), 20 µL.
Thermal cycling conditions: 22°C (1 hour), 65°C (10 min).
Acceptor and donor modifications evaluated
AMP
P
Mismatch G-T
D
Each of the modified ligation components can improve
discrimination between match and mismatched targets
Ligation conditions: 1X Ligase Buffer (50 mM Tris (pH 7.5), 10 mM DTT, 10 mM MgCl2),
Ligation Probes (1 µM), ssDNA Template (1 µM), ATP analogs (1 mM), T4 DNA Ligase (0.5 Weiss units), 20 µL.
Thermal cycling conditions: 95ºC (3 min), 4ºC (3 min), add Ligase, 22ºC (20 min), 65ºC (10 min).
-O
O
% Ligation Yield
No Template
Modification F
Modification E
Modification D
5'-... G T T
A G T ... -3'
3'-... C A X - T C A ... -5'
Modified Cofactors B and C provide robust ligation yield and
improved match versus mismatch discrimination
O
O
U
A
B
C
C
Cofactor 2
Acceptor
Probe
OH
F
B
Match A-T
P
Lysine
O P
O O
O P
HO O O
O
O P
HO O O
Cofactor
OH
E
A
X = wild type (A) or mutant (C)
NH 2
O
0.2
Cofactor 1
OH
OH
0.4
PMe (n-1) acc.
HO
0.6
Mismatch
T-T
Modification
Lysine
O
0.8
PMe (n) acc.
O
HO
PPi
OH
O
O P N
OH H
1
U
Template
Lig. Probes
Mismatch
C-T
Enzyme
A
1.2
0
Unmodified
A
O
O
O
O P O P O P OH
OH OH OH
OH
Modification C
Match
C-G
0.6
O
Lysine
Relative Ligation Yield to Standard Conditions
Lig. Product
5'
Enzyme
NH 2
O
Mismatch
C-T
Fluorescence (dRn)
Lysine
Ligation Product
Mismatch
G-T
0.8
E-AMP
Enzyme
O
Modification B
1
Modification A
1.2
Unmodified (ATP)
C-T Mismatch
No ATP Cofactor
C-G Match
U A B C D E F
Template
Ligation Probes
Figure 5
E + ATP
HO
Match
A-T
5'-... T C X
T G T ... -3'
3'-... A G C - A C A ... -5'
Proposed approach for improved DNA ligase specificity
O
Evaluation of lead probe and cofactor modifications with
T4 DNA Ligase
Fluorescence (dRn)
Figure 1
Evaluation of chemically modified cofactor analogs
No Ligase
Ligases are gaining utility in molecular biology applications, such as
nucleotide sequence detection, single nucleotide polymorphism
(SNP) detection, protein detection and “next generation”
sequencing by ligation. With the increased demand for DNA ligases in
the field of biotechnology, comes increased demand for ligation
fidelity. Described approaches to improved ligation fidelity include
ligases from different biological sources, point mutations of key
amino acid residues within the ligase, modified reaction conditions
and addition of crowding reagents, such as PEG. Although most
approaches to improved ligation fidelity have focused on the ligase
itself, further improvements are needed and may be attainable by a
different approach. Herein a strategy to improve the discrimination
between matched and mismatched targets is described which
employs chemical modification to the nucleic acid components of
the reaction, such as the donor probe, the acceptor probe and the
ATP cofactor. The results demonstrate that chemically modified
components increase the stringency of DNA ligase-mediated nucleic
acid detection, providing a unique approach for SNP genotyping.
A
Figure 8
Figure 4
Ligation Yield
(normalized to ATP)
Abstract
3) Cofactor modifications provide the greatest improvement to ligation
fidelity of T4 DNA ligase.
4) The lead ATP cofactor demonstrated the most consistent increase in
ΔCq’s in real-time PCR.
1'
3'
2'
HO
OH
Ligation Product
Mismatch
C-T
Adenosine-5'-triphosphate (ATP)
P
Legend
Base modifications
Sugar modifications
Triphosphate modifications
Template
Ligation Probes
Crystal structure suggests several key
amino acid contacts with AMP
Crystal structure of ligase adenylate structure of the
Cholorella virus DNA ligase. Image captured from Tomkinson et. al. Chem Rev. (2006) 106, 687-99.
Chemical modifications to the cofactor may influence
interactions at key ligase contacts to improve ligation fidelity
5'-... G T T
A G T ... -3'
3'-... C A X - T C A ... -5'
X = wild type (A) or mutant (C)
Several candidate modifications to the donor probe decrease
mismatch ligation
5) LDR reactions employing chemically modified ligation components
yield more specific match vs. mismatch discrimination.
Acknowledgements:
Terry Beck, Angela Tenenini, Anton McCaffrey, Julie Powers, Elizabeth Hill
NIH Grants:
1R43GM085860-01, 2R44GM085860-02 & 5R44GM085860-03
For further information, please contact Natasha Paul, npaul@trilinkbiotech.com
Ligation conditions: 1X Ligase Buffer (50 mM Tris (pH 7.5), 10 mM DTT, 10 mM MgCl2),
Ligation probes 1 μM, ssDNA Template 1 μM, ATP 1 mM, T4 DNA Ligase 2 Units, 20 uL
Thermal Cycling Conditions: 95°C (3min), 4°C (3 min), add Ligase, 16°C (20 min), 65°C (10 min)
9955 Mesa Rim Road San Diego, CA 92121
800-863-6801 | www.trilinkbiotech.com | info@trilinkbiotech.com
The Modified Nucleic Acid Experts