DNA-based biosensors for metal detectionDNA-based biosensors for metal detection Juewen Liu...
Transcript of DNA-based biosensors for metal detectionDNA-based biosensors for metal detection Juewen Liu...
DNA-based biosensors for metal detection
Juewen Liu
Department of Chemistry Waterloo Institute for Nanotechnology
University of Waterloo
June 6, 2018
GWF meeting (Hamilton)
Problem to solve: heavy metal contamination
Importance and challenges of metal detection
http://userpage.chemie.fu-berlin.de
• Environmental monitoring
• Food analysis
• Biomedical diagnosis
http://www.nanoscience.imdea.org
• Instrumentation analysis1. Highly accurate, reliable2. Multiplexed detection3. Industrial standard4. Expensive5. Long turnaround time6. Detects total metal
• (Bio)sensors1. Cost effective2. On-site, real-time detection3. Detects bioavailable metal
Various metal ligands
Hg2+ Zn2+
Nat. Chem. Biol. 2008, 4, 168-175
Small molecule fluorescent chelators Peptides, proteins, & antibodies
Genetically engineered cells Nucleic acids
Coord Chem Rev, 256, 2012, 2175 Curr Opin Chem Biol, 12, 2008, 214
http://2010.igem.org/Team:Peking/Project/Biosensor
UO22+
Highly stable, cost effective, easy to modify, combinatorial selection + rational design
DNA for metal binding
Sigel, R. K. O.; Sigel, H. Acc. Chem. Res. 2010, 43, 974.
• Phosphate: electrostatic interaction, binds hard Lewis acids
• Bases: metal coordination
In vitro selection of a cerium-dependent DNAzyme
rAF Ce4+
PCR1
1
2 PAGEN50
P2
P1
PCR2rAFP3
P4
PAGE 345
119 nt 91 nt
P. J. Huang, J. Lin, J. Cao, M. Vazin and J. Liu*, Analytical Chemistry, 86, 1816-1821 (2014)
1013-1014 DNA sequences
Ce(NH4)2(NO3)6
Selection progress
Round 1 Round 2 Round 3
9.3%0.6%
82.1%
99.0%
99.9%
Round 4
10.7%
Round 5
25.2%
74.0%
Round 6
26.8%
71.4%87.8%
Sequence alignment
AAATGATCCACTAATACGAGTCACTATrAGGAAGATG
TTCTACCAGTGAT
GCGA
AA
N50
5’
Cleavage site Start of the N50 region
-ACGAGTCACTATrAGGAAGATGGC
TACCG-TG CAGTGATA
GAAAC
AGGTT
5’
A CCT
TTGAA GT
CAGC
GGTCG
C
TA
TA
TACT
AGGTCAA
GTGGGTGG A
Ce13
GTCACGAGTCACTATrAGGAAGATGGCGAAA
TACCGCTTTCAGTGCTCAGTGATATT AGGTCA
AGTGGGTGG A
Ce13d
5’
A general lanthanide-dependent DNAzyme
• Y3+ shows similar activity like other lanthanides• Pb2+ is the only active divalent metal ion
Left lane: 10uM Right lane: 100uM
La3+ Ce3+ Ce4+ Pr3+ Nd3+ Sm3+ Eu3+ Gd3+ Tb3+ Dy3+ Ho3+ Er3+ Tm3+ Yb3+ Lu3+
Sr3+ Fe3+ Y3+ In3+ Tl3+
Mg2+ Mn2+ Fe2+ Co2+ Cu2+ Zn2+ Hg2+ Pb2+
P. J. Huang, J. Lin, J. Cao, M. Vazin and J. Liu*, Analytical Chemistry, 86, 1816-1821 (2014)
Highly sensitive lanthanide detection
t (min)
0 20 40 60 80 100 120
F (a
.u.)
120
150
180
210
240
270
300
-MCH+MCH
t (min)
0 20 40 60 80
F (a
.u.)
100
130
160
190
220
250
280
0
5 nM10 nM
20 nM50 nM0.1 µM0.5 µM1 µM
[Ce3+] (nM)
0 200 400 600 800 1000
dF/d
t
0
3
6
9
12
15
0 10 20 30 40 50012345
t (min)
0 10 20 30 40 50
F (a
.u.)
90
120
150
180
210
240
270
300
No metalCe3+
Sc3+
Fe3+
In3+
Tl3+
t (min)
0 10 20 30 40 50
F (a
.u.)
90
120
150
180
210
240
270
300
No metalCe4+
Y3+
La3+
Ce3+
Pr3+
Nd3+
Sm3+
Eu3+
Gd3+
Tb3+
Dy3+
Ho3+
Er3+
Tm3+
Yb3+
Lu3+
F Ce3+
Q Q
Ce3+Pb2+
OH
SH
MCH
P. J. Huang, J. Lin, J. Cao, M. Vazin and J. Liu*, Analytical Chemistry, 86, 1816-1821 (2014)
Characterization by phosphorothioate (PS) modifications
rAGGAAG
TT AGGTCA
AGTGGGTGG A
OO
O
O-
P
O
O OH
A
G OO
O
S-
P
O
O OH
A
G
Ce Mg Mn Fe Co Ni Cu Zn Cd Hg PbCe13d
PO
PS
So, it’s just another DNAzyme?
Anything special about this one?
Ce13d with PS substrate can detect thiophilic metals as a group.P Huang and J Liu, Analytical Chemistry, 86, 5999–6005 (2014).
Selection of DNAzymes using a PS-modified library
OO
O
S-
P
O
O OH
A
GFAM-AAATGATCCACTAATACGAGTCACTATrAGGAAGATG
TTCTACCAGTGAT
GCGA
AA
N50
5’
Can we get new DNAzymes that can discriminate different thiophilic metals?
Single modification made at the fixed region.
Perrin, Angew. Chem. Int. Ed. 2008, 47, 4346 –4350
So far, DNAzymes for soft metals are rare.
Solution 1: modified bases Solution 2: modified phosphate backbone
A highly Cd2+-specific DNAzyme
Cd2+ Pb2+ Cu2+ Ce3+
Rat
e (m
in-1
)
0.00
.02
.04
.06
.08
.10
.12
.14
Red bars = Ce13d
GTCACGAGTCACTATrA*GGAAGATGGCGAAA
T-CCTTCTACCGCTTTCAGTGCTCAGTGATA
5’
A TA CA GT ATGAT
5’BN-Cd16
Substrate
Enzyme
Black bars = new enzymeGTCACGAGTCACTATrAGGAAGATGGCGAAA
TACCGCTTTCAGTGCTCAGTGATATT AGGTCA
AGTGGGTGG A
P Huang and J Liu, Nucleic Acids Research, 2015
Highly sensitive Cd2+ detection
RefMg2+
Ca2+Mn2+
Fe2+Co2+
Ni2+Cu2+
Zn2+Ba2+
Hg2+Pb2+
Cd2+
dF/d
t (a.
u.)
0.0
.2
.4
.6
.8
1.0
1.2
1.4
100 nM1 M
t (min)
0 10 20 30
F (a
.u.)
15
20
25
30
35
40t (min)
0 10 20 30 40
F (a
.u.)
10
15
20
25
30
35
40
0 nM 2 nM5 nM10 nM20 nM50 nM100 nM200 nM
A
C
[Cd2+] (nM)
0 100 200 300 400 500
dF/d
t (a.
u.)
0.0
.5
1.0
1.5
2.0
2.5
3.0
0 10 20 30 40 500.0
.3
.6
.9
1.2
B
D
Cd2+
Other metals
FCd2+
F
Q
P Huang and J Liu, Nucleic Acids Research, 2015
Highly sensitive Cu2+ detection
OO
O
S-
P
O
O OH
A
G
SubstrateFAM-AGTCACTATrA*GGAAGATGGCGAAC
CTTCTACCGCTTGQ-CAGTGATA
5’
A AA CG CGAPSCu10
DNAzyme
Cu2+
Dete
LOD = 1.6 nM Cu2+
Huang P and Liu J, Anal. Chem. 88, 3341–3347, 2016
Highly sensitive Ag+ detection (using normal DNA)
GTCACGAGTCACTATrAGGAAGATGGCGAAA
TTCTACCGCTTTCAGTGCTCAGTGATAT
5’
G C G A C TG GG AG
GAG
AC
T
A TT
T
AA
A
GCGAC
G
C
TC
TT AG
Pb2+
No Metal Ag
+ K+ Li+ Tl+
Mg2+
Ca2+
Ni2+
Cu2+
Zn2+
Co2+
Sr2+
Cd2+
Mn2+
Hg2+
Fe2+
Fe3+
Ce3+
Ga3+
Tl3+
Y3+
Cle
avag
e (%
)
0
10
20
30
40
50
60
7010 M Mn+
100 M Mn+
Cle
avag
e(%
)
0
20
40
60
80NaNO3 NaCl
[Na+] (mM)
0
12.5 25 50 100 200 300 400 500
750 1000
Saran R. and Liu J. Anal. Chem. 2016, 88, 4014-4020
Monovalent, thiophilic Ag+, but it works very well!This is a surprise!
Only 52 sequences out of >50,000
Efficient, 0.41 min-1 Time (min)0 20 40 60
Rel
ativ
e Fl
uore
scen
ce
1.0
1.5
2.0
2.5
3.0 500 nM 400 nM 300 nM200 nM160 nM
25 nM
120 nM 100 nM 50 nM
0 nM
Detection limit = 24 nM Ag+
EtNa has the best Ca2+ specificityA
C D
EtNa
E5 10-23
B
8-17 variants
ChemBioChem, 18, 518-522. (2017)
EtNa binds two Ca2+ but only one Mg2+
Slope = 2
Slope = 1
ChemBioChem, 18, 518-522. (2017)
C
Ca2+ detection
Limit of Detection = 17 µM Ca2+
A DNA code for metal ions
• Many new metal-specific DNAzymes isolated.
• Please start to use them.
Wenhu Zhou, Runjhun Saran and Juewen Liu, Chemical Reviews, 117, 8272–8325 (2017)
Acknowledgments
$$: University of Waterloo, NSERC, CIHR, CFI, Ontario MRI, CFREF
•Postdoctoral FellowDr. Xu ZhangDr. Feng Wang
•Graduate StudentsNeeshma DavePo-Jung Jimmy HuangAjfan BaessieAlexander IPImran KhimjiBiwu LiuHoward TsaiZiyi SunMahsa VazinRunjhun SaranWenhu ZhouLingzi MaZhicheng HuangUndergraduates
Rachel Pautler Ahmed Zaki David Lin Jeehae ShinThomas Kennedy Anand Lopez Erin Kelly Firas El-HamedJames MacLean Nathan Lang Marissa WuNish Bhatt Kevin Joseph Murat KiyBrendan Smith Kiyoshi Morishita Youssef HelwaArsalan Beg Menhaj Jenny Lin Zachary Jacobi
Visiting Students/ScholarsSylwia WalczkZenata MatuszekElsa MateronDr. Hui Jiang
Cr3+, Na+, Ca2+
Fe3+, Ln3+, Cu2+, Cd2+
Ag+