The DNA substrate
used for the electrochemical characterization of DinG was either a
well-matched 20-mer DNA oligomer with a 15-mer 5′ to 3′
single-stranded overhang or the same substrate with the exception
of an abasic site being placed on the complementary strand four base
pairs from the bottom of the duplex (
Table S1). A 20-mer strand of DNA with a terminal thiol and 6-carbon linker
at the 5′ end of the strand was annealed to a 35-mer unmodified
strand of DNA to yield the electrochemical substrate. The electrochemical
substrate was designed to be competent to be unwound by DinG in a
helicase reaction. Single-stranded DNA stimulates the ATPase activity
of DinG, which requires at least a 15-mer single-stranded 5′
to 3′ overhang in order to unwind DNA substrates
in
vitro.(32 (
link)) In the electrochemical
cell, the DNA substrate is covalently tethered to the gold surface
via a gold–thiol bond.
The thiol-modified strand was
synthesized on a 3400 Applied Biosystems DNA synthesizer using standard
phosphoramidite chemistry. The complementary strands were purchased
from IDT. All phosphoramidites, including the terminal phosphoramidite
containing a 6-carbon disulfide linker, were purchased from Glen Research.
The thiol-modified and complementary strands were purified by HPLC
using an analytical C-18 column (Agilent). DNA strands were characterized
by MALDI mass spectroscopy. The DNA was quantified by UV–vis
absorbance, and equimolar amounts were annealed yielding the duplex
substrate.
To prepare DNA-modified single electrodes, a 50 μM
solution
of the DNA substrate was incubated overnight at ambient temperature
on a bare gold on mica surface (Agilent) in an electrochemical cell
with a capacity of 50 μL. Following incubation with the DNA
solution, the surface was rinsed and backfilled by incubating the
electrode with 1 mM 6-mercapto-1-hexanol for 45 min at room temperature.
Multiplex chip electrodes were prepared as described previously.
29 (link),34 (link) The well-matched electrochemistry substrate was used for all single
electrode experiments. For experiments with multiplex chip electrodes,
the well-matched and abasic-site substrates were laid down side-by-side
in separate quadrants on a single chip.
29 (link),34 (link)After
backfilling, the DNA-modified electrodes were rinsed with
the electrochemistry buffer (4 mM spermidine, 4 mM MgCl
2, 0.25 mM EDTA, 20% glycerol, 250 mM NaCl, 20 mM Tris-HCl, pH ∼8.5).
Protein concentration was measured by UV–vis absorbance using
an extinction coefficient at 410 nm of 17 000 M
–1 cm
–1.
22 (link) An aliquot
of 20 μM DinG was flash thawed by incubating it in a room temperature
water bath. The protein’s buffer was exchanged for the electrochemistry
buffer by diluting the protein two-fold into 2× spermidine buffer
(8 mM spermidine, 8 mM MgCl
2, 1 mM EDTA, 20 mM Tris-HCl,
pH ∼9.0).
Electrochemical measurements were made using
a CHI620D Electrochemical
Analyzer. For cyclic voltammetry, sweeps within a window from −0.4
V vs Ag/AgCl to 0.1 or 0.2 V were carried out at a scan rate of 50
mV/s for several hours. For electrochemistry measurements on single
electrodes with ATP, 1 mM ATP or 1 mM ATPγS (Sigma) was added
after the electrochemical signal grew in to an appreciable size (>20
nA). Cyclic voltammetry was then used to scan the electrode over several
hours.
Grodick M.A., Segal H.M., Zwang T.J, & Barton J.K. (2014). DNA-Mediated Signaling by Proteins with 4Fe–4S Clusters Is Necessary for Genomic Integrity. Journal of the American Chemical Society, 136(17), 6470-6478.