Polymerase/Nicking Enzyme Synergetic Isothermal Quadratic DNA
Machine and Its Application for One-Step Amplified Biosensing of
Lead Ion (II) at Femtomole Level and DNA Methyltransferase
Yongxi Zhao1, Feng Chen1, Qing Zhang1, Yue Zhao1, Xiaolei Zuo2 and Chunhai
Fan2
1Key
Laboratory of Biomedical Information Engineering of Education Ministry, School of Life
Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, P. R. China
2Division
of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility,
CAS Key Laboraotory of Interfacial Physics and Technology, Shanghai Institute of Applied
Physics, Chinese Academy of Sciences, Shanghai 201800, P. R. China
Correspondence: Professor Y Zhao, Key Laboratory of Biomedical Information Engineering of
Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an,
Shaanxi 710049, P. R. China. E-mail: yxzhao@mail.xjtu.edu.cn; Professor C Fan, Division of
Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key
Laboraotory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics,
Chinese Academy of Sciences, Shanghai 201800, P. R. China. E-mail: fchh@sinap.ac.cn
Table S1 Sequences of oligonucleotides used in this work
Sequence(5’ to 3’) a
note
DNAzyme
AAT CAT CTC TGA AGT AGC GCC GCC GTA TAG TGA
GTT TTT TTT TTC TCA CTA TrA bG GAA GAG ATG ATT
Dam-probe
GGA AGA GAT GAT CCT GAA GTT TTT ACT TCA GGA
TCA TCT CAA AA
Basic
SDA AAG TGT GTG TGT CCT CGC TGA GGT TAA TCA TCT
template
CTT CC
SDA template
CAC GAG TCA GTG TCC TCG CTG AGG TTA ATC ATC
TCT TCC
SDA primer
GGA AGA GAT GAT T
SDA product
TCA GCG AGG ACA CAC ACA CTT
Hairpin probe 1 GAT GTG AGT GTC CTG CTT GCT GAG GAC ACT
(H1)
Hairpin probe 2 AAG TGT GTG TGT CCT CCG TGC GCT GAG GAC ACA
(H2)
C-P
Hairpin probe 3 GTA AGT GTG TGT GTC CTC CGT GCG CTG AGG ACA
(H3)
CAC AC
Molecular beacon FAM-CCA CGA GTC AGT GTC CTC AGC GTG
(MB)
G-DABCYL
a
The underlined regions in all oligonucleotides repesents the stem sequence, and
italic bold letters is the recognition site for Nt.BbvCI. The 5’ end and 3’ end of the
molecular beacon are labeled with fluorescein (FAM) and quencher (DABCYL),
respectively.
b
rA denotes adenosine ribonucleotide while all others are deoxyribonucleotides.
Before use, hairpin probes, molecular beacon, Dam-probe and DNAzyme were
incubated at 90 oC for 5 min and then slowly cooled to room temperature over 30 min
to make these probes perfectly fold into hairpin structures
Fluorescence Measurement. Fluorescence measurements were carried out by using a
FluoroMax-4 fluorescence spectrometer (Horiba Jobin Yvon, Edison, NJ). The
emission spectra were obtained from 510 nm to 600 nm with an excitation wavelength
of 495 nm at room temperature in steps of 1 nm. Both the excitation and emission slit
widths were set at 5 nm.
Gel Electrophoresis
Products of Pb2+-triggered amplification reaction were analyzed by 20%
non-denaturating polyacrylamide gel electrophoresis (PAGE) in 1 x TBE buffer (90
mM Tris −HCl, 90 mM boric acid, 2 mM EDTA, pH 7.9) at a 45 V constant voltage
for 3 h at room temperature. The gel was silver-stained and imaged by a digital
camera.
Influence of Some Drugs on the sensing system
These drugs, gentamycin and benzyl penicillin, were demonstrated to have no
remarkable influence on the sensing system. The methylation process was carried out
in 10 μL 1×NEBuffer 2 containing 50 nM Dam-probe, 500 nM MB, 320 μM SAM,
20nM template, 100 nM Dam-probe, 400μM dNTP and 0.8unit of Dam MTase at 37
o
C for 30 min, and then at 65 oC for 20 min to inactivate Dam MTase. This 10 μL
resultant mixture was added to another 10 μL 1×NEBuffer 2 containing 2 unit of DpnI,
0.08 units of Nt.BbvCI, 2.5 units of Klenow Fragment and 10 μM different inhibitors.
The 20 μL reaction mixture was performed at 37 oC for 30 min before fluorescence
was measured.
Inhibition of Some Drugs on Dam MTase Activity
All the inhibition experiments were carried out in conditions similar to those of Dam
MTase activity assay except for the addition of 10 μM of different inhibitors. Briefly,
before the addition of 4 unit of Dam MTase, different inhibitors were introduced into
each sample. The solution was incubated at 37 oC for 40 min prior to the fluorescence
measurement. To investigate the dose-dependent inhibition, different gentamicin
concentrations were added into these samples. The following procedures were similar
as above.
Table S2 Comparison of linear and quadratic amplification mode
Recycle No.
Signal obtained in linear Signal obtained in quadratic
amplification mode
amplification mode
st
1
1
0
nd
2
2
0+1
rd
3
3
0+1+2
4th
4
0+1+2+3
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
th
N
N
(N-N) +…... + (N-3) + (N-2) + (N-1)
Finally, when the recycle No. is N, the signal obtained in linear amplification mode
and quadratic amplification mode are:
Y1= N
Y2 = (N-N) +…... + (N-3) + (N-2) + (N-1) = 0.5 N (N-1)
Y1 and Y2 represent the signal of linear amplification mode and quadratic
amplification mode, respectively. And N is the positive integer.
Figure S1 Principle of basic SDA and its application for Pb2+ detection.
Figure S2 The potential secondary structures and corresponding Gibbs free energy of
hairpin
probes
H1,
H2
and
H3
obtained
by
UNAFold
(http://www.idtdna.com/UNAFold?).
Figure S3 The effect of the stem length of hairpin probe on the on the fluorescence
response. The system contained Nt.BbvCI and 250 nM MB with varied hairpin probes
of 50 nM. F0 and F represent the fluorescence intensity in the absence and presence of
50 nM SDA product, respectively.
Figure S4 (A) Fluorescence spectra response to different concentrations of Pb2+ in the
basic SDA detection system. (B) Changes of fluorescence intensity at 518 nm upon
the addition of different concentrations of Pb2+.
Figure S5 The time-dependent effects of 5% (w/v) nitric acid (A) and 10 mM Mg2+ (B)
on the Pb2+- specific signal response.
Figure S6 Selectivity for Dam MTase detection over other enzymes. The
concentration of Dam MTase is 40 U/mL, whereas the concentration of M.SssI MTase
and T4 DNA ligase are each 175 U/mL and 100 U/mL.
Figure S7 Influence of benzylpenicillin and gentamycin on the sensing system for
Dam MTase detection.