of 21
Supplementary Materials For
A Compass at Weak Magnetic Fields using Thymine Dimer
Repair
Theodore J. Zwang,
1
Edmund C.M. Tse,
1
Dongping Zhong,
2
Jacqueline K.
Barton
1,
*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, CA 91125
2
Division of Chemistry and Physics, The Ohio State University, Columbus, OH 43210
Correspondence to jkbarton@caltech.edu
This PDF file includes:
Materials and M
ethods
Figures S1 to S1
4
2
3
MATERIALS and METHODS
DNA Synthesis
All materials for DNA synthesis were purchased from Glen Research. Oligonucleotides
were synthesized on an Applied Biosystems 3400 DNA synthesizer using phosphoramidite
chemistry on a controlled
-pore glass support. The two strands of a duplex are
synthesized
separately, purified, stored frozen, then annealed prior to electrochemical experiments. The 5’
-
end of one strand is modified with a C6 S
-S phosphoramidite that is later reduced before use.
High pressure liquid chromatography (HPLC) was performed using a reverse-
phase PLRP
-S
column (Agilent) using a gradient of acetonitrile and 50 mM ammonium acetate (5
-15%
ammonium acetate over 35 minutes).
Unmodified DNA.
DNA was synthesized using standard phosphoramidites and reagents.
After synthesis, the DNA was lyophilized overnight. It was then cleaved from the solid support
by incubation at 60
o
C with concentrated (28-
30%) NH
4
OH for 12 hours, filtered using CoStar
0.45 μm columns, then dried. The dried DNA film was resuspended in phosphate buffer (5 mM
phosphate, pH 7, 50 mM NaCl) and HPLC
-purified. The DMT (4,4’
-dimethoxytrityl) group
protecting the 5’
- end was then removed by incubation with 80% acetic acid for 45 minutes. The
reaction mixture was dried and resuspended in phosphate buffer. The DNA was isolated using
HPLC. The purified oligonucleotide was desalted using ethanol precipitation, dried, and the mass
was confirmed with m
atrix
-assisted laser desorption/ionization
- time of flight mass spectrometry
(MALDI
-TOF). Unmodified oligonucleotides were t
hen stored at -
20
o
C in phosphate buffer until
annealing with their complementary strand.
Cyclobutane pyrimidine dimer
generation
. Single stranded DNA (1 ml of 100-
200 μM )
with a single 5’
-TT-
3’, 5’
-UU-
3’, 5’
-TU
-3’, or 5’
-UT
-3’ was suspended in aqueous buf
fer
containing 1 mM acetophenone, 5 mM NaH
2
PO
4
, 50 mM NaCl, pH 7.5 and degassed with argon
in a glass container. The container was sealed and irradiated with a solar simulator (Oriel
instruments) or 302 nm UV light (spectroline transilluminator model TR
-302) for 10 minutes.
Following irradiation the DNA was purified using high performance liquid chromatography
(HPLC) using a reverse
-phase PLRP
-S column (Agilent) using a gradient of acetonitrile and 20
mM ammonium acetate (2
-3% acetonitrile over 10 minutes, then 3-
4% over the next 30 minutes)
at 80
o
C and a flow rate of 0.8 ml/min to separate the CPD from undimerized ssDNA. Both 16
and 29 bp ssDNA show a separation with CPD compared to without a CPD of approximately 4
minutes with the CPD containing strand el
uting first.
Thiolated DNA.
DNA was synthesized using standard phosphoramidites and reagents,
with the exception of a C6 S
-S phosphoramidite that was attached to the 5’
- end. After synthesis,
the DNA was lyophilized overnight. It was then cleaved from the solid support by incubation at
60
o
C with concentrated (28-
30%) NH
4
OH for 12 hours, filtered using CoStar columns, then
dried. The dried DNA film was resuspended in phosphate buffer (5 mM phosphate, pH 7, 50 mM
NaCl) and HPLC
-purified. The DMT (4,4’
-dimeth
oxytrityl) group protecting the 5’
- end was
then removed by incubation with 80% acetic acid for 45 minutes. The reaction mixture was dried
and resuspended in phosphate buffer. The DNA was isolated using HPLC as described above.
The purified oligonuceotide was desalted using ethanol precipitation, dried, and the mass was
confirmed with MALDI
-TOF. Within one week of annealing, the disulfide
-modified DNA was
reduced by resuspending in 50 mM Tris
-HCl, pH 8.4, 50 mM NaCl, 100 mM dithiothreitol
(Sigma) for 2 hour
s. The reduced thiol
-modified DNA was then purified by size exclusion
4
chromatography (Nap5 Sephadex, G
-25, GE Healthcare) with phosphate buffer as the eluent and
subsequently purified using HPLC.
Annealing Duplex DNA.
Duplex DNA for electrochemistry was prepared by first
quantifying the complementary strands with UV
-Visible spectroscopy, then mixing equimolar
(50
μ
M) complementary strands in 200
μ
l phosphate buffer. The DNA solution was then
deoxygenated by bubbling argon for at least 5 minutes per ml. Duplex DNA was then annealed
on a thermocycler (Beckman Instruments) by initial heating to 90
o
C followed by slow cooling
over 90 minutes.
DNA Sequences
:
16 bp DNA (with or without UV generated dimer)
5’-ACG TGA GTT GAG ACG T
-3’
3’-TGC ACT CAA CTC TGC A
-5’ – SH
Thymine Dimer with CA mismatch near surface
5’-ACG TGA GTT GA
A
ACG T
-3’
3’-TGC ACT CAA CT
C
TGC A-
5’ -
SH
29 bp DNA (with or without UV generated dimer)
3’-ATC ACG TCA TAT GAA CTG ACT GGA CGG TG-
5’
-SH
5’-TAG TGC AGT ATA C
TT GAC TGA CCT GCC AC
-3’
3’- ATC ACG TCA TAT CAA CTG TCT GCA CGC TG-
5’ -
SH
5’- TAG TGC AGT ATA GTT GAC AGA CGT GCG AC
-3’
The above sequences use the following abbreviations for modifications:
-SH = hexanethiol linker; TT = Pyrimidine Dimer
Protein preparation
Escherichia coli
photolyase wild type and mutants N378C, M345A, E274A, as well as
truncated
Arabidopsis Thaliana
cryptochrome 1 (
at
CRY1ΔC) without its C
-terminal domain
were provided by Prof. Dongping Zhong (
The Ohio State University). Proteins wer
e received at
180-
300 μM in a buffer containing 100 mM KCl, 50 mM Tris
-HCl at pH 7.5, 1 mM EDTA and
50% (v/v) glycerol. It is essential that these buffers do not contain concentrations of
dithiothreitol over 2 mM or other sulfur compounds that are typicall
y used to keep the flavin
cofactor reduced because they may disrupt gold-
thiol bonds crucial for the stability of DNA
monolayers used in electrochemistry. Proteins were thus generally received with a partially or
fully oxidized flavin that needed to be photoreduced for enzymatic activity.
E. coli
photolyase without the antenna cofactor was prepared as described previously.
12,
23
The mutant plasmids were constructed using QuikChange II XL kit (Stratagene) based on the
plasmid of wild
-type enzyme. All mutated
plasmids were sequenced to confirm the mutations.
The preparation of MBP
-tag fused
At
Cry1 with depletion of the C
-terminal tail
(
At
CRY1ΔC) was as described elsewhere with some modifications.
26
The
At
CRY1ΔC gene was
cloned into the pMal
-c2 vector (New Engl
and Biolabs) to obtain a construct that expresses
At
CRY1ΔC fused to the C
-terminus of maltose binding protein (MBP). The MBP
-tagged
At
CRY1ΔC was expressed in
E. coli
UNC523 and purified by affinity chromatography on
amylose resin.
5
All proteins were obtaine
d with stoichiometric flavin cofactor after purification and were
exchanged to a buffer containing 50 mM Tris at pH 7.5, 100 mM NaCl,1 mM EDTA, and 50%
(v/v) glycerol for further use.
Before experiments 50 uM
ec
PL or
at
CRY1ΔC were placed in tris buffer (50 mM Tris
-
HCl, 50 mM KCl, 1 mM EDTA, 10% glycerol, pH 7.5) and irradiated with blue light (405 ± 10
nm, <30 mW) from a diode laser pointer (Tmart) in an anaerobic chamber to photoreduce the
flavin to its active form (Fig
. S1
1). All solutions containing photolyase were degassed to remove
oxygen and kept in an anaerobic chamber (95% N
2
, 5% H
2
, <1 ppm O
2
) to prevent oxidation of
the flavin. During experiments with photolyase the protein was kept under constant blue light
irradiation. If the flavin was not fully photoreduced, or if oxygen was able to access the flavin
and oxidize it, the oxidized flavin peak would appear in cyclic voltammetry experiments
centered around
-420 mV vs AgCl/Ag. Further irradiation with blue light i
n anaerobic conditions
remove this peak. The presence of this peak did not appear to have a significant effect on
measurements of total charge transferred at later time points when it was removed by reduction
of the flavin with blue light.
Electrode fabri
cation
Multiplexed electrode surfaces were fabricated following a previously published
protocol.
8
In brief, one millimeter thick Si wafers with a 10 000 Å thick oxide layer were
purchased from Silicon Quest. First, wafers were cleaned thoroughly in 1165 R
emover
(Microchem) and vapor primed with hexamethyldisilizane (HMDS). S1813 photoresist
(Microchem) was spin
-cast at 4000 rpm and baked. The photoresist was patterned with a Karl
Suss MA6 contact aligner and a chrome photomask. Following post
-exposure baki
ng, wafers
were developed in MF
-CD26 developer for 40-
60
s and rinsed thoroughly with deionized water
then dried thoroughly. A 30 Å Ti adhesion layer and a 100 Å Au layer were deposited on the
chips with a CHA Mark 50 electron beam evaporator. Wafers were then immersed in P6
Remover overnight to complete metal lift-
off. Subsequently, the wafers were thoroughly baked
and cleaned by UV ozone treatment. SU
-8 2002 (Microchem) was spin-
cast at 3000 rpm, baked,
and photopatterned as above. Wafers were developed i
n SU
-8 Developer (Microchem) for 1 min
and baked for a permanent set of the photoresist. The wafers were subsequently diced into 1-
in.
by 1
-in. chips and used for electrochemistry experiments.
DNA
-modified electrode preparation
Multiplexed chips are gentl
y cleaned by sonicating with acetone then isopropanol before
drying with argon. They are then cleaned with UV/Ozone using a UVO cleaner for 20 minutes.
Immediately after cleaning the surface, a plastic clamp and rubber (BunaN) gasket are affixed to
the sur
face to create a well for liquid and 50 μM duplex DNA in phosphate buffer (5 mM
phosphate, pH 7, 50 mM NaCl) to make dsDNA films. The dsDNA was incubated on the
surface for 18
-24 hours. Once the dsDNA is on the surface, it cannot be dried without
compromi
sing the structure and therefore the measured properties of the film. The solution was
then exchanged 5x with 1 mM mercaptohexanol in phosphate buffer (pH 7, 5 mM phosphate, 50
mM NaCl, 5% glycerol) and incubated for 45 minutes. Lastly the surface was rins
ed at least 5x
with tris buffer (50 mM Tris
-HCl, 50 mM KCl, 1 mM EDTA, 10% glycerol, pH 7.5) that was
degassed by leaving open in an anaerobic chamber over many days.
6
Electrochemical measurements
The central well around the electrode surface created by th
e clamp was filled with
aqueous buffer containing degassed 50 mM Tris
-HCl, 50 mM KCl, 1 mM EDTA, 10% glycerol,
pH 7.5. An AgCl/Ag reference electrode (Cypress) was coated with a solidified mixture of 1%
agarose and 3M NaCl in water inside a long, thin pipe
tte tip. The tip was cut so that the salt
bridge could connect the electrode to the buffer from the top of the well. A platinum wire used as
an auxiliary electrode was also submerged in the buffer from the top of the well. The working
electrode contacted a dry part of unmodified gold surface. A CHI620D Electrochemical
Analyzer (CH Instruments) was used to control the electrochemical experiments. A picture of the
electrochemical set up i
s shown in Figure S2.
Magnetic field measurements
Magnetic field experiments w
ere conducted using 462 Gauss, 918 Gauss, 1803 Gauss,
and 6619 Guass surface strength magnetic field neodymium magnet (K&
J Magnetics). Alligator
clips were replaced with nonmagnetic stainless steel to minimize magnetic interference. All other
parts of the assembly were created using plastic to prevent extraneous objects the magnetic field
could influence. Additionally, the s
trong magnet was waved near the potentiostat during
operation, with no obvious change in signal that was apparent, to ensure that the magnet was not
interfering with the operation of the potentiostat. Each experiment that was conducted using a
magnetic fie
ld was compared to a similar quadrant on the same chip that was not tested under a
magnetic field and when compared to other experiments was normalized using this data. Each of
the four electrode quadrants were tested separately and consecutively (with dif
ferent orders of
the experimental condition). This separation is
achieved by separating each of the four electrode
sets from one another with a gasket and plastic clamp that creates separate wells around each set
of electrodes. Only the quadrant being test
ed is exposed to protein and has light focused on it
and, therefore, the magnetic field is only influencing the activity in that quadrant. During this
experiment the remaining quadrants are submerged in phosphate buffer without protein and are
not dried to ensure DNA structural integrity. Generally there was little variation seen when using
the same dsDNA on different surfaces, however using dsDNA made at different times could
result in different maximum values for repair, possibly due to slight differences
in surface
packing, purity, or efficiency of thymine dimer generation. Background magnetic field strength
and applied magnetic field strength were tested by measuring the x,y,z coordinates of the
magnetic field at the surface of the electrode using a gaus
s meter (F.W. Bell, 5100 series).
Thymine dimer repair activity
The repair of thymine dimer DNA by photolyase and cryptochrome was verified by
HPLC, mass spectrometry, gel shift, and digestion experiments. High pressure liquid
chromatography was performe
d on duplex DNA containing a thymine dimer before and after
incubation with cryptochrome and photolyase. C
ryptochrome (50 μM) or 50 μM photolyase was
irradiated for 1 hour with 35 μM duplex DNA. The DNA was then separated from the protein via
a spin column. The DNA was then resuspended in phosphate buffer. HPLC was performed with
a flow rate of 0.8 ml/min with an increasing gradient of acetonitrile in ammonium acetate. The
percent acetonitrile increased from 2% acetonitrile to 3% acetonitrile over 10 minute
s followed
by an increase from 3% acetonitrile to 4% acetonitrile over 30 minutes. The column was kept at
80
o
C to dehybridize the two strands.
7
Mass spectr
ometry and digestion experiments were conducted by first incubating duplex
DNA (33
μ
mol) in PDE activity buffer (20
μ
L, 100 mM Tris, pH 8.9, 100 mM NaCl, 14 mM
MgCl
2
) was added
At
CRY1ΔC (40 μM). The reaction mixture was irradiated with blue light in
the presence of a 6600 gauss magnetic field for 1 h under an inert atmosphere at RT.
Phosphodiesterase I (
Crotalus adamanteus
venom, USB, 0.1 unit) in PDE storage buffer (110
mM Tris, pH 8.9, 110 mM NaCl, 15 mM MgCl
2
, 50% glycerol) and 10
μ
L PDE activity buffer
were
added to the reaction mixture and heated to 37 °C for 1 h. CutSmart® Buffer (NEB, 1
μ
L)
and Cal
f Intestinal Alkaline Phosphatase (CIP, NEB, 10 unit) were added to the reaction mixture
in sequence. The reaction mixture was then incubated at 37 °C for 1 h. The total volume of the
reaction mixture was brought up to 100
μ
L and was subsequently centrifuged at 5000 ×
g. The
top 80
μ
L of solution was carefully transferred to a HPLC injection vial. HPLC injection volume
was 20
μ
L and a Chemcobond 5-
ODS
-H column (ChemcoPak, 5
μ
m, 4.6 × 150 mm) was used
for reversed
-phase HPLC. HPLC was performed using a flow
rate of 1 mL/min with an
increasing gradient of acetonitrile in ammonium acetate (50 mM) at RT. The percent acetonitrile
increased linearly from 3% acetonitrile to 10% acetonitrile over 30 min followed by a linear
increase from 10% acetonitrile to 20% acet
onitrile over 5 min, and then a linear ramp from 20%
acetonitrile to 50% acetonitrile over 10 min. The reaction products were purified by HPLC with
UV detection at 260 nm. Collected fractions were analyzed using LC
-TOF
-MS (Waters, Acquity
Ultra Performance
LC, Micromass Technologies, LCT Premier XE) on a BEH C18 column
(Waters, Acquity UPLC, 1.7
μ
m, 2.1 × 50 mm) at a flow rate of 0.39 mL/min with an increasing
gradient of acetonitrile with 0.1% formic acid in ammonium acetate (50 mM) at RT. The percent
acet
onitrile increased linearly from 5% acetonitrile to 65% acetonitrile over 3.3 minutes followed
by an isocratic flow at 65% acetonitrile for 0.5 min.
Data Availability
The data that support the findings of this study are available from the corresponding
author upon
reasonable request.
8
Figure S1
.
Plot of the reductive peak area in photolyase cyclic voltammetry scans before and
after the addition of competitor DNA. The addition of competitor dsDNA containing a thymine
dimer to a surface that has saturated the photolyase repair signal results in a s
ignificant decrease
in signal indicative of the photolyase dissociating from the surface and binding the competitor
dsDNA in solution. Data from multiple electrodes were normalized so that the maximum
reductive peak area was plotted as 100%. There is no observable difference with or without a
magnetic field for the signal decrease. Standard error was plotted with n≥4.
9
Figure S
2.
Measurement of multiplexed chip surface using SQUID magnetometer. (Top left)
Surface was incubated with dsDNA and photolyas
e as would be used in an experiment, then
dried to make compatible with SQUID measurement. The surface was then magnetized by
placing near a 6619 Gauss surface neodymium magnet to align any dipoles. The magnet did not
come into contact with the surface to prevent contamination. Note that this is the only
experiment with dried DNA because the duplex structure is not important for these
measurements. (Top right) The SQUID measurements show
that there is no magnetite present on
the multiplexed chip either due to the substrate or biological samples. The y
-axis is in volts and
the color scale represents 3 nT/V.
A small contamination was observed on the edge of the chip,
but even if these were assumed to be present in experiments the dipoles present are still too small
to influence the experiments at the field strengths we used
. (Bottom left) A photograph of a
typical experiment showing the multiplexed chip in its plastic assembly to create wells, a
neodymium magnet placed underneath the device, and working, refere
nce, and auxiliary
electrodes placed in the buffer in the top well. The light is irradiating the surface directly and can
be observed in the image near the center of the plastic device.
10
Figure S3
.
Plot of the reductive peak area from cyclic voltammetry
of photolyase on a 29 bp
T□T dsDNA
-
modified electrode under different magnetic field conditions. The dotted line
indicates the start of irradiation of the surface with blue light (405 ±10 nm). (Red) Photolyase
was photoreduced in solution without a magnet
present then added to the surface and monitored
with a 30 G magnetic field applied perpendicularly down towards the surface. (Blue) Photolyase
was photoreduced in solution with a 30 G magnetic field and then incubated with the surface
with the same field pointed perpendicularly down towards the surface. (Black) Photolyase was
photoreduced in solution with a 30 G magnetic field and then incubated with the surface without
an applied magnetic field. The presence or absence of an applied field during the redu
ction of
photolyase did not have any measurable effect on the amount of charge transferred at later time
points. Standard error was plotted with n=3.
11
Figure S
4.
Randles
-Sevcik plots of the peak currents of photolyase. Randles
-Sevcik plots of the
peak currents of photolyase without (top) and with (bottom) a 6000 G applied magnetic field
perpendicular to the surface of the electrode.
12
Figure S5
.
Plot of the reductive peak area from cyclic voltammetry of photolyase on a 29 bp
T□T dsDNA modified electrode
using opposite polarity magnetic fields. Applying a magnetic
field of 30 Gauss perpendicularly up (red) or down (blue) intersecting the plane of the electrode
did not show a measurable difference. Photolyase without a magnetic field (black) shows a
signif
icant increase in signal compared to both magnetic field directions. Standard error was
plotted with n=3.