Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing

Article abstract of DOI:10.1021/jacs.1c02222

We report a series of 2′-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3′-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3′-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Full text of DOI:10.1021/jacs.1c02222

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Article
Carborane- or Metallacarborane-Linked Nucleotides for Redox
Labeling. Orthogonal Multipotential Coding of all Four DNA Bases
for Electrochemical Analysis and Sequencing
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+
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David Kodr, Cansu Pinar Yenice, Anna Simonova, Dijana Pavlovic Saftic, Radek Pohl,
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Veronika Sykorová, Mayreli Ortiz, Ludek Havran, Miroslav Fojta, Zbigniew J. Lesnikowski,
Ciara K. O’Sullivan,* and Michal Hocek*
Cite This: J. Am. Chem. Soc. 2021, 143, 7124−7134
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Supporting Information
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ABSTRACT: We report a series of 2′-deoxyribonucleoside triphos-
phates bearing dicarba-nido-undecaborate ([C2B9H11]1−), [3,3′-iron-
bis(1,2-dicarbollide)]− (FESAN, [Fe(C2B9H11)2]2−) or [3,3′-cobalt-
bis(1,2-dicarbollide)]− (COSAN, [Co(C2B9H11)2]2−) groups prepared
either through the Sonogashira cross-coupling or the CuAAC click
reaction. The modified dNXTPs were substrates for KOD XL DNA
polymerase in enzymatic synthesis of modified DNA through primer
extension (PEX). The nido-carborane- and FESAN-modified nucleotides
gave analytically useful oxidation signals in square-wave voltammetry and
were used for redox labeling of DNA. The redox-modified DNA probes
were prepared by PEX using tailed primers and were hybridized to
electrode (gold or glassy carbon) containing capture oligonucleotides.
The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP
allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and
ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox
coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in
one simple PEX experiment with electrochemical readout.
principle be applicable in redox coding of DNA bases7 for
INTRODUCTION
■
applications in sequencing or in the PEX-based electro-
Electrochemical analysis of nucleic acids1 is an attractive and
chemical determination of the nucleobase composition of an
cost-effective alternative to fluorescence-based techniques,
unknown sequence. A number of redox-active groups have
which are routinely used for sequencing of DNA2 or detection
already been reported for the labeling of DNA. Anthraqui-
of particular DNA or RNA sequences.3 For most electro-
none,7,8 nitrophenyl,9 benzofurazane,10 or azidophenyl11 were
chemical bioanalytical or diagnostic applications, redox label-
studied as reducible groups, whereas aminophenyl,9 methylene
ing of DNA bases4 is achieved through an attachment of some
blue,7,12 Nile blue,12,13 and phenothiazines,7 as well as
oxidizable or reducible molecules or functional groups with
ferrocene14−16 or substituted ferrocene labels17 can be oxidized
characteristic redox potentials,5 which need to be within a
on various types of electrodes. Previously, we attempted to
potential window compatible with the type of electrode used.
combine some of them, but only a combination of two
The redox label can be attached at position 5 of pyrimidine or
reducible groups, i.e. nitrophenyl with either benzofurazane or
at position 7 of 7-deazapurine 2′-deoxyribonucleoside
triphosphate (dNTP) and such modified dNTPs are typically
very good substrates for DNA polymerases in primer extension
azidophenyl,10 or a combination of two oxidizable groups
(ferrocene and ferrocenecarboxamide)17 were truly orthogonal
and ratiometric and thus useful for redox coding of two
(PEX) or polymerase chair reaction (PCR) synthesis of redox-
labeled DNA for subsequent electrochemical detection.
Recently, we have developed the enzymatic synthesis of
redox-labeled DNA probes, which are flanked by tails
Received: February 26, 2021
Published: April 30, 2021
complementary to capture oligonucleotides covalently attached
to gold electrodes and have exploited these in the construction
of electrochemical genosensors.6 A combination of several
orthogonal redox labels with differing redox potentials might in
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a
nucleobases. For a full set four redox labels for encoding of all
four nucleobases, it would be practical to have either all four
oxidizable or all four reducible so that all of them can be
simultaneously detected on one electrode using monodirec-
tional and highly sensitive voltammetric detection techniques
including square-wave and differential pulse voltammetry. It is,
however, very challenging to achieve an adequate resolution of
differentiable peaks, due to the limited potential windows
available on specific types of electrodes. Mercury electrodes,
for example, are excellent for the detection of signals due to
electrochemical reductions but poorly useful for oxidations due
to the anodic dissolution of mercury at potentials ≥+0.2 V
(Ag|AgCl|3 M KCl),18 while the use of gold electrodes in the
presence of chlorine ions becomes unreliable at potentials
higher than +0.9 V vs Ag/AgCl, but this potential window can
be extended to +1.2 to +1.3 V vs Ag/AgCl if a chloride-free
electrolyte such as strontium nitrate (Sr(NO3)2) is used.19 On
the other hand, carbon electrodes usually offer the widest
positive potential window (≥+1.2 V), making them the
electrodes of choice for the detect oxidation reactions.20 An
additional problem is the limitation of polymerase-mediated
enzymatic incorporation of high-density modified nucleotides.
However, recently we have shown that DNA polymerase can
be used even for synthesis of long sequences containing all four
modified bases using either PEX or asymmetric PCR
(aPCR).21
Carborane and metallacarborane clusters are highly hydro-
phobic sphere-shaped molecules that have been used in
medicinal chemistry,22 either as potential boron neutron-
capture-therapy (BNCT) agents23 or as components of
enzyme inhibitors,24 forming strong interactions with hydro-
phobic pockets of target proteins. They have also been
attached to nucleosides and some of them incorporated into
oligonucleotides25 by chemical synthesis on solid supports. We
have recently reported26 the polymerase incorporation of p-
carborane-linked dNTPs into DNA. Many carborane and
metallacarborane derivatives are also redox active.27−31 In
particular, 7,8-dicarba-nido-undekaborate anion,29 as well as
[3,3′-cobalt bis(1,2-dicarbollide)]− (COSAN)30 and [3,3′-
iron-bis(1,2-dicarbollide)]− (FESAN)31 clusters have been
used as oxidizable redox labels in the chemical synthesis of
labeled nucleic acids and their redox potentials are distinctly
different, presenting them as potential candidates for
orthogonal labeling. Therefore, we decided to explore these
carborane and metallacarborane clusters as potential oxidizable
redox labels in our quest for the development of a fully
orthogonal set for the redox coding of all four DNA
nucleobases.
Scheme 1. Synthesis of Alkyne-Linked Carboranes
a
Reagents and conditions: (i) NaH, toluene (dry), reflux, 4 h or 22
°C, 48 h.
The modified cytosine or 7-deazaadenine nucleoside
triphosphates (dCXTP and dAXTP) were prepared through a
direct one-step aqueous Sonogashira cross-coupling reac-
tion17,33,34 of halogenated nucleoside triphosphates (dCITP
or dAITP) with alkyne-linked carboranes 2a−c (Scheme 2).
The cross-coupling reactions were performed in the presence
of Pd(OAc)2, TPPTS (triphenylphosphine-3,3′,3″-trisulfo-
nate) ligand, CuI and Hu
̈
nig’s base in water/acetonitrile
(2:1). The desired modified triphosphates were isolated as
trisodium salts following HPLC purification and ion exchange,
achieving moderate to good yields (26−62%). The yields were
lowered due to the partial hydrolysis of triphosphates and
difficulty in isolation.
In the case of modified thymidine triphosphates (dTXTP),
we selected an alternative synthetic route using CuAAC click
reaction of known dUMeN3TP39 with the terminal alkynes 2a−c
(Scheme 2). This approach prevented possible undesired
cyclization of 5-alkynyluracils to furopyrimidines which often
occurs during the Sonogashira coupling of 5-iodouracil
derivatives with alkynes.15 The CuAAC reactions catalyzed
by CuSO4 in the presence of sodium ascorbate proceeded
smoothly giving the desired modified dTXTPs in good yields
(45−66%). In all cases modified dNXTPs were prepared in
sufficient amounts for the subsequent biochemical and
electrochemical experiments.
Enzymatic Incorporation Study of Modified dNXTPs.
Modified nucleotides were tested in primer extension experi-
ments (PEX) using 19-mer (tempA, tempC, tempT) or 31-mer
(temprnd16) templates (encoding for one or four modified
nucleotides) and 15-mer (primrnd) primer (for sequences of all
oligonucleotides see Table S1 in the Supporting Information).
KOD XL DNA polymerase was selected as this enzyme has
previously been reported to efficiently incorporate multiple
modifications.17,21 Primarily, we tested incorporation of just
one type of modified nucleotide (in the presence of the other 3
natural dNTPs). Figure 1 shows the PAGE analysis of the PEX
products. In all cases, it confirmed formation of full-length
products, where bands of modified products are typically
shifted slightly upward in comparison to the natural ones, due
to their bulkiness and higher molecular weight. In some cases
of multiply modified 31DNA_4Nx, we observed some
smearing, suggesting possible aggregation of modified single
RESULTS AND DISCUSSION
■
Synthesis. The attachment of the bulky (metalla)carborane
labels was envisaged through a flexible linker that would allow
the polymerase to incorporate the corresponding nucleotide
into DNA.32 We thus designed the diethylene glycol linked
(metalla)carborane terminal alkynes 2a−c suitable to be used
in either the Sonogashira reaction33,34 or Cu-catalyzed alkyne
azide cycloaddition (CuAAC).35 They were prepared (Scheme
1) through dioxane ring-opening from their dioxane-carborane
precursors 1a−c36−38 by treatment with in situ generated
sodium propargylalcoholate similar to a procedure previously
published36 for 8-(5-propargyl-3-oxa-pentoxy)-3,3′-cobalt bis-
(1,2-dicarbollide) (1c).
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a
Scheme 2. Synthesis of Modified dNXTPs
a
Reagents and conditions: (i) Pd(OAc)2, CuI, TPPTS, DIPEA H2O/CH3CN (2:1), 75 °C, 30 min; (ii) Pd(PPh3)2(Cl)2, CuI, PPh3, Et3N, H2O/
CH3CN (2:1), 75 °C, 1 h; (iii) alkyne, sodium ascorbate, CuSO4·5·H2O, H2O/t-BuOH (1:1), 22 °C, 16 h.
Figure 1. (1) PEX with KOD XL DNA polymerase using a single modified dNXTPs. (2) Denaturating PAGE analysis of PEX using 5′-(6-FAM)
labeled primrnd and (A) tempC, dCXTP; (B) tempA, dAXTP; (C) tempT, dTXTP [lanes (1) primer; (2) positive control, natural dNTPs; (3)
negative control, without studied dNTP; (4) reactions containing dNCB‑TP; (5) dNCBCoTP; (6) dNCBFeTP]. (3) Native PAGE analysis of PEX
using 5′-(6-FAM) labeled primerrnd, temprnd16, and (D) dCXTP; (E) dAXTP; (F) dTXTP [lanes (1) dsDNA ladder with denoted length; (2)
primer; (3) positive control, natural dNTPs; (4) negative control, without studied dNTP; (5) reactions containing dNCB‑TP; (6) dNCBCoTP; (7)
dNCBFeTP].
stranded ONs (ssONs) (data not shown). This could be
resolved by analysis on native PAGE (Figures S2−S4). The
identity of the products was also confirmed using MALDI-
TOF analysis (Table S2 in the Supporting Information) of
ONs after a larger scale PEX with a biotinylated template
followed by magnetoseparation purification.16 The successful
PEX incorporations of the modified dNXTPs shows that they
are reasonably good substrates for the KOD XL polymerase,
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and hence, they have potential for enzymatic incorporation of
carborane-labeled dNTPs.
We also attempted the polymerase chain reaction (PCR)
with the carborane modified dTXTPs under different
conditions, but no amplified product was observed. (Figure
S5) We assume that the bulky lipophilic nature of the modified
nucleotides in the template hinders the ability of the
polymerase to incorporate these bases, thus preventing
effective elongation.
Electrochemistry of Modified Nucleotides on Pyro-
lytic Graphite Electrode. Electrochemical experiments
studying carborane-linked dNXTPs were performed using
square-wave voltammetry (SWV) on a pyrolytic graphite
electrode (PGE) in acetate buffer (pH = 5.0). In the nido-
carborane-linked nucleotides dNCB‑TPs, we observed a signal
at +0.6 V (vs Ag/AgCl) corresponding to irreversible oxidation
of the boron cluster. In FESAN-derivatives dNCBFeTPs,
electrochemical peaks were detected at −0.4 V due to the
reversible oxidation of iron and at +1.2 V due to irreversible
boron cluster oxidation. For the COSAN nucleotides
dNCBCoTPs, the oxidation peak due to reversible oxidation of
Co was observed at very low potential −1.4 V. In the 7-
deazaadenine nucleotides dAXTPs, an additional peak at ca.
+1.2 V was also observed due to the oxidation of the
pyrrolopyrimidine moiety. The electrochemical signal of the
COSAN nucleotides (at −1.4 V) is already out of the potential
range for practical analytical applications, whereas the
oxidation signals of the nido-carborane and FESAN are within
the range of gold or carbon electrodes and they significantly
differ from one another, suggesting their applicability for
combinations in orthogonal redox coding. We thus have
further studied nido-carborane- and FESAN-linked nucleotides
as building blocks for the redox labeling of DNA (Figure 2).
Synthesis of Redox-Labeled DNA Probes and Anal-
ysis on Different Electrodes. In order to investigate the
electrochemical behavior of carborane-labeled DNA, the
modified DNA containing one (19DNA_1NX) or four
(31DNA_4NX) modifications were synthesized using a PEX
reaction followed by purification using the QIAquick
Nucleotide Removal Kit. These modified ONs were then
studied using SWV on the PGE (analogously to our previous
works9,16). Unfortunately, the observed signals were very weak
(Figure S9), which can probably be attributed to the low
amounts of the modified DNA adsorbed on the electrode
surface. Moreover, appearance of parasitic peaks at ca. +0.2 V
due to oxygenous surface groups at the PGE prevents the
combination with ferrocene-linked nucleotides.17
We subsequently turned our attention to our recently
reported approach6,17 based on the synthesis of tailed-PEX
products and their hybridization with electrode-tethered
oligonucleotides (Figure 3). We designed three 37-mer ON
templates flanked on its 5′-end with a 20-mer ssON which
were separated by a C3 spacer (−(CH2)3−). The templates
were designed to contain a range of mixtures (4:4; 2:6; 6:2) of
dT and dG, thus encoding for different ratios of modified dAX
and dCX nucleotides. In the first instance, we tested
combinations of either the nido-carborane- or FESAN-linked
dCCB‑TP or dCCBFeTP with our previously reported 7-
ferrocenylethynyl-7-deaza-dATP (dAEFcTP).16 The PEX re-
action was then used to generate 37-mer DNA bearing a
corresponding number of modifications and a 20-mer single
stranded tail. The C3 spacer prevented further polymerase
mediated extension, thus maintaining the single-stranded tail
Figure 2. Square-wave voltammograms of modified dNXTPs (40 μM)
measured at pyrolytic graphite electrode in 0.2 M acetate buffer (pH
= 5.0).
ON, which was complementary to a capture ON immobilized
on the electrode surface. In parallel, we have conducted a series
of analytical solution-based PEX experiments using 5′-(6-
FAM) labeled primer to demonstrate that full length PEX
products were formed, as expected. The presence of these
expected PEX products was indeed confirmed by PAGE and
MALDI-TOF analysis. (Figures S3−S4 and Table S2 in the
Supporting Information).
The capture ON was linked to the electrode either through
immobilization of 3′-thiol-linked ON on the surface of a gold
electrode6 or via the addition of thiolated probes to maleimide-
modified glassy carbon electrodes (GCEs).40 The tailed PEX
product was then hybridized with the modified electrode,
followed by thorough washing, thus removing any non-
hybridized amplicons and facilitating direct SWV measure-
ments without the need for any postextension purification step
of the PEX product. A 0.1 M Sr(NO3)2 solution was used as an
electrolyte solution containing divalent cations, due to the
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Figure 3. PEX synthesis of the modified DNA probes (bearing 2, 3, or 4 modified nucleotides) and their capture on the electrode.
ability of these cations to shrink and compact DNA,41 allowing
maximal hybridization between immobilized probes and their
complementary sequences.42
Figure 4 (part 1) shows the PAGE analyses of the different
PEX reactions using different templates and combinations of
dCCB-TP or dCCBFeTP with dAEFcTP. In all cases, we observed
formation of the full-length modified DNA products. The
voltammograms on glassy carbon electrode (Figure 4, part 2)
reveal clearly distinguishable peaks corresponding to the
oxidation FESAN at −0.1 V or nido-carborane at +0.6 V and
ferrocene at +0.2 V (all vs Ag/AgCl).
As each of the redox labels will transfer electrons at different
rates, with ferrocene transferring most rapidly, followed by the
ferrocarborane and then the metal-free nidocarborane, the
effect of frequency on the SWV response was evaluated in
order to obtain a reproducible and ratiometric signal. As can be
seen in Figure S10, the use of a 10 Hz frequency resulted in the
best ratiometric signal, which was also observed to provide
high intersynthesis batch (Figure S11) and interelectrode
reproducibility (Figure S12). As can be seen, in Figure S12, all
SWV were automatically background corrected. It should be
highlighted that while ratiometric responses are reproducibly
obtained for each DNA sequence under interrogation, the
signal intensities obtained are not absolute and cannot be used
as “calibrator values”. Thus, the signal intensities used to
measure the ratiometric ratios of redox labels in one ON
sequence cannot be extrapolated to another ON, but each
individual experiment reliably determines the ratios in the ON
being evaluated, as exemplified in Figure S13.
Remarkably, we were able to observe a proportional
correlation between the ratio of signal intensities with the
known ratio of incorporated labeled nucleotides. The SWV
observed with the same amplicons hybridized to probes
surface-tethered on gold electrodes also showed equivalent
differentiable and ratiometric signals (Figures S14).
Encouraged by the successful combinations of two redox-
labeled nucleotides, we advanced to testing a combination of
three orthogonally labeled nucleotides. We used a combination
of the above-mentioned dCCBFeTP and dAEFcTP with nido-
carborane modified thymidine dTCB‑TP. To this end, we
designed a 36-mer template which was used for the PEX
synthesis of a tailed DNA product containing 2 dCCBFe, 6
dAEFc, and 2 dTCB- nucleotides. Figure 5 shows the PAGE
analysis of the successful PEX reaction and SWV voltammo-
Since the first report43 of the use of the ratiometric
responses of electrochemical redox labels in electrochemical
sensors in 2014, there have been multiple approaches devised,
with the vast majority of these using square wave (SWV) or
differential pulse voltammetric (DPV) measurement.44 While
cyclic voltammetry can provide useful information regarding
reaction mechanisms, kinetics, and electron transfer rates,
SWV/DPV provides better developed signals and enhanced
sensitivity of detection of both reversible and nonreversible
redox labels, including those used in this study. Metal-
lacarboranes give (quasi)reversible redox electrochemistry
due to their metal components, while the carborane cages
are known to undergo irreversible electrochemical oxidation28
and indeed cyclic voltametric (CV) evaluation of the carborane
modified nucleotides demonstrated this expected behavior
(Figure S7). In addition, 7-deaza-dGTP, which was used for
redox coding of G was evaluated using CV and observed to
undergo irreversible oxidation (Figure S8), in agreement with
previous reports.45 SWV was thus selected as the preferred
method for the voltammetric measurement of the redox labels
used throughout this work.
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Figure 4. (1) Native PAGE analysis of PEX with KOD XL DNA polymerase using combination of modified dCXTP, dAEFcTP, 5′-(6-FAM) labeled
primC3, and (A) tempC34C/4A; (B) tempC36A/2C; (C) tempC36C/2A [lanes (1) dsDNA ladder with denoted length; (2) positive control, natural dNTPs;
(3) negative control, without studied dNTPs; (4) reactions containing dAEFcTP, dCCB‑TP; (5) dAEFcTP, dCCBFeTP]. (2) SWV of modified DNA
hybridized with complementary capture probe immobilized on GCE measured in 0.1 M Sr(NO3)2. Cyan, green, and violet designation corresponds
to dCCBFe, dAEFc, and dCCB‑, respectively. The black traces correspond to the experimental SWV signals of dAEFc/dCCB‑ or dAEFc/dCCBFe ratios
equal to (a1−2) 4/4; (b1−2) 6/2; (c1−2) 2/6, respectively. (3) Illustrative example of analyzed PEX products.
gram (GCE) confirming the response of the electrochemical
analysis correctly corresponding to the ratio of modified
nucleotides (observed ratio C/A/T 1/3.1/1.1). This ratio-
metric sensitivity was also observed using gold electrodes
(Figure S15).
redox labels. Integration of the signal intensities corresponded
very well with the known ratio of redox labels and hence with
the relative nucleobase composition of the DNA.
CONCLUSIONS
■
The final most challenging step was the extension to a
combination of four different redox-labeled nucleotides
covering the whole genetic alphabet. An empty “window” in
an applicable range of electrochemical spectra in the potential
window between 0.8 and 1.4 V vs Ag/AgCl led us to the
selection of previously published45 and commercially available
2-deoxy-7-deazaguanosine triphosphate (dG*TP). 7-Deaza-
guanine in DNA is known45 to show an intrinsic oxidation
peak at ca. 1.1 V which is significantly different from the signal
of guanine. As gold electrodes are not highly reliable at
potentials >+0.9 V vs Ag/AgCl when using chloride-containing
electrolytes, the experiments with the full set of all four
modified nucleotides could only be performed with the glassy
carbon electrodes. We thus performed the PEX reactions with
a combination of dCCBFeTP, dTCB‑TP, dAEFcTP, and dG*TP
using three different templates encoding for different ratios of
the four nucleotides in the extended DNA products. Figure 6
shows the PAGE analyses of the successful PEX reactions
showing the full-length products in all cases. The voltammo-
grams on glassy carbon electrodes in this figure also reveal four
clearly differentiated oxidation signals for each of the four
We have designed and synthesized a series of new dNTPs
bearing dicarba-nido-undecaborate, [3,3′-cobalt-bis(1,2-dicar-
bollide)]− or [3,3′-iron-bis(1,2-dicarbollide)]− groups tethered
to nucleobases via a flexible diethyleneglycol linker either
through the Sonogashira cross-coupling or the CuAAC click
reaction. Despite the very bulky and lipophilic substituents, the
modified dNXTPs were efficient substrates for KOD XL DNA
polymerase and were used in enzymatic synthesis of modified
DNA via the PEX reaction. Electrochemical study using SWV
revealed that each of these (metalla)carborane groups are
oxidized at distinctly different potentials. The cobaltacarborane
cluster showed oxidation at a very low potential, while the
other two clusters gave analytically useful signals at −0.4 V
(FESAN) and +0.6 V (nido-carborane) vs Ag/AgCl,
respectively.
In order to develop a multipotential redox coding of DNA
nucleobases, we primarily combined either the FESAN or nido-
carborane with our previously reported ethynyl ferrocene
modification. A combination of two modified dNXTPs
incorporated via PEX using a tailed template, followed by
hybridization with a capture ON immobilized on either gold or
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combination of a maximum of two different reducible10,11 or
two oxidizable17 labeled nucleotides at the same time (to
observe the ratio of two nucleobases), was observed to be
feasible. Here, we present a fully orthogonal combination of
four redox labels encoding all four canonical nucleobases,
which are fully ratiometric and facilitate the readout of the
proportion of each of the four nucleobases in a newly
synthesized DNA sequence using a single modified electrode.
While in this initial work the use of four different modifications
is limited by the maximum length of the newly synthesized
fully modified sequence to ca. 16 nucleotides, we anticipate
that further optimization and other combinations will allow
synthesis of longer stretches of hypermodified DNA.
Incorporation of a known labeled sequence for signal
normalization (e.g., via the primer tail) may be used to
facilitate determination of the absolute number/mol of each
label present in unknown sequences if needed for a specific
application.
While optical detection technologies such as fluorescence
are prone to background interference and require relatively
complex instrumentation, not typically compatible with
portable, or point-of-need application, there are increasing
numbers of examples of optical detection enabled smartphone
technologies.46 However, multiplexing still remains a grand
challenge due to the complex optics and multiple excitation
and emission wavelengths as well as the broad emission spectra
of fluorescent dyes, requiring either spatial separation of
sensors and detection using CCD as exploited in sequencing-
by-synthesis approaches,2,47 or use of individual detectors, such
as the zero-mode waveguide nanostructure arrays employed by
Pacific Biosciences,48 and indeed both of these approaches
have finally resulted in elegant, high-throughput, massively
parallelized, sequencing platforms. Surface-enhanced resonance
scattering (SERS) can overcome the problems due to the
overlapping, broad fluorescent spectra as the peaks obtained in
Raman spectra have narrow spectral widths, allowing for ease
of spectral separation and multiplexed detection,49 but despite
instrumental advances it still requires a complex optical setup
with an inherent cost impact.
Electrochemical detection, such as that detailed in this work,
on the other hand, is highly sensitive, simple, inexpensive,
compatible with miniaturization and integration with micro-
fluidics, and can exploit advanced semiconductor technologies,
facilitating rapid uptake and mass production, as evidenced by
the Ion-Torrent50 and Oxford Nanopore sequencing51
technologies. Ion-torrent sequencing remains a laboratory-
based instrument, requiring preamplification, and is highly
prone to homopolymer read errors. Nanopore’s sophisticated
MinION addresses portability and does not require preampli-
fication, but the platform still faces issues of high error rates
and stability of the protein nanopore-lipid bilayer membrane.52
While the work reported here is in its infancy, being just at
the proof-of-concept phase, the potential achievable via
upscaling and parallelization on electrode arrays is immense.
The ratiometry achievable could address problems encoun-
tered with homopolymers, the cost of the redox labeled dNTPs
is relatively low, massively multiplexed electrode arrays and
accompanying microfluidics can be facilely produced using
established microfabrication technologies. In addition, solid-
phase amplification could facilitate sequencing through syn-
thesis akin to the Pacific Biosciences approach.
Figure 5. (1) Native PAGE analysis of PEX with KOD XL DNA
polymerase using (A) combination of dCCBFeTP, dAEFcTP, dTCB‑TP,
2C/6A/2T
5′-(6-FAM) labeled primC3 and tempC3
[lanes (1) dsDNA
ladder with denoted length; (2) positive control, natural dNTPs; (3)
negative control, without studied dNTPs; (4) reaction containing
dAEFcTP, dCCBFeTP, dTCB‑TP; (2) SWV of modified DNA
hybridized with complementary capture probe immobilized on GCE
measured in 0.1 M Sr(NO3)2. Cyan, green, and violet designation
corresponds to dCCBFe, dAEFc, dTCB‑, respectively. The black trace
corresponds to the experimental SWV signals of dCCBFe/dAEFc
/
dCCB‑, ratio equals to (a) 2/6/2, respectively. (3) Illustrative example
of analyzed PEX product.
glassy carbon electrodes enabled facile electrochemical
measurements. The SWV showed that the signals of (metalla)-
carborane and ferrocene oxidations appeared at distinctly
different potentials and were ratiometric, corresponding to the
ratio of labeled nucleobases in the newly synthesized sequence.
Subsequently, a combination of three different redox labels
(FESAN, nido-carborane, and ferrocene) at each of three
different dNTPs (C, T, and A) were successfully and efficiently
incorporated into elongating DNA, and the subsequent SWV
readout clearly showed three distinctly different oxidation
signals, which were ratiometric with respect to the modified
nucleobases of each type. The final and most challenging goal
was the development of a full set of four orthogonal redox
labels for encoding the four canonical nucleobases. Here, we
chose 7-deazaguanine as the fourth redox label and we
succeeded in the PEX synthesis of DNA using all four modified
dNXTPs and the SWV analysis of the fully modified DNA on
glassy carbon electrode revealed four fully distinguished, and
ratiometric, oxidation peaks. The use of carbon electrodes
facilitated a wide enough potential window to detect all four
redox labels, while gold electrodes, although slightly easier to
functionalize with the thiolated surface-tethered probes, can
only be used for the simultaneous detection of three
orthogonal labels.
In previous works, either a combination of several labeled
dNTPs was used but only one modification at a time (to verify
which single nucleotide has been incorporated)7,9 or a
Furthermore, the approach reported can find niche
applications where more focused sequencing is required, and
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Figure 6. (1) Native PAGE analysis of PEX with KOD XL DNA polymerase using combination of dCCBFeTP, dAEFcTP, dTCB‑TP, dG*TP, 5′-(6-
FAM)-primC3, and (A) tempC32C/6A/2T/6G; (B) tempC32C/2A/2T/7G; (C) tempC31C/4A/3T/7G [lanes (1) dsDNA ladder with denoted length; (2) positive
control, natural dNTPs; (3) negative control, without studied dNTPs; (4) reactions containing dAEFcTP, dCCBFeTP, dTCB‑TP, dG*TP]. (2) SWV
of modified DNA hybridized with complementary capture probe immobilized on GCE measured in 0.1 M Sr(NO3)2. Cyan, green, violet and
orange designation corresponds to dCCBFedAEFc, dTCB‑, dG* respectively. The black traces correspond to the experimental SWV signals of dCCBFe
dAEFc/dCCB‑/dG* ratios equal to (a) 2/6/2/6; (b) 2/2/2/7; (c) 1/4/3/7, respectively. (3) Illustrative example of analyzed PEX products.
/
one can envisage a number of diverse applications including
minisequencing of short DNA stretches,53 detection of specific
sets of single-nucleotide polymorphisms,54 the detection of
multiple mutations in consecutive codons, or even the use of
the redox coding with electrochemical readout DNA-encoded
libraries.55 Furthermore, (metalla)carborane-linked nucleotides
may be used in synthesis of hypermodified DNA-based
polymers,21 synthesis of boron-rich oligonucleotides for
BNCT,23 and in SELEX to generate novel modified aptamers56
or other functional DNA constructs. All these applications are
currently being explored in our laboratories.
Barcelona, Spain; orcid.org/0000-0003-2603-2230;
Email: ciara.osullivan@urv.cat
Authors
David Kodr − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, CZ-16610 Prague
6, Czech Republic; orcid.org/0000-0002-5948-6426
Cansu Pinar Yenice − Departament d’Enginyeria Química,
Universitat Rovira i Virgili, 43007 Tarragona, Spain
Anna Simonova − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, CZ-16610 Prague
6, Czech Republic; Department of Organic Chemistry,
Faculty of Science, Charles University in Prague, Prague-2
12843, Czech Republic
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c02222.
́
́
Dijana Pavlovic Saftic − Laboratory of Medicinal Chemistry,
́
Institute of Medical Biology PAS, 92-232 Łódz, Poland
Radek Pohl − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, CZ-16610 Prague
6, Czech Republic
Full experimental details, methods, and procedures and
full characterization of all compounds, tables of
sequences of oligonucleotides, additional and full uncut
gels, additional electrochemical measurements, and
copies of MALDI and NMR spectra (PDF)
́
Veronika Sykorová − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, CZ-16610 Prague
6, Czech Republic
Mayreli Ortiz − Departament d’Enginyeria Química,
Universitat Rovira i Virgili, 43007 Tarragona, Spain;
orcid.org/0000-0002-9423-0055
AUTHOR INFORMATION
■
Corresponding Authors
̆
Ludek Havran − Institute of Biophysics, Czech Academy of
Michal Hocek − Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, CZ-16610 Prague
6, Czech Republic; Department of Organic Chemistry,
Faculty of Science, Charles University in Prague, Prague-2
12843, Czech Republic; orcid.org/0000-0002-1113-
2047; Email: hocek@uochb.cas.cz
Ciara K. O’Sullivan − Departament d’Enginyeria Química,
Universitat Rovira i Virgili, 43007 Tarragona, Spain;
Institució Catalana de Recerca i Estudis Avanca̧ ts, 08010
Sciences, 612 65 Brno, Czech Republic
Miroslav Fojta − Institute of Biophysics, Czech Academy of
Sciences, 612 65 Brno, Czech Republic
Zbigniew J. Lesnikowski − Laboratory of Medicinal
Chemistry, Institute of Medical Biology PAS, 92-232 Łódz,
Poland; orcid.org/0000-0003-3158-5796
́
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c02222
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of DNA suitable for electrochemical detection of DNA−protein
interactions. Chem. Sci. 2015, 6, 575−587.
Author Contributions
+D.K. and C.P.Y. contributed equally.
(12) (a) Dauphin-Ducharme, P.; Arroyo-Curras, N.; Plaxco, K. W.
High-precision electrochemical measurements of the guanine-,
mismatch- and length-dependence of electron transfer from
electrode-bound DNA are consistent with a contact-mediated
mechanism. J. Am. Chem. Soc. 2019, 141, 1304−1311. (b) Mahshid,
S. S.; Camiré, S.; Ricci, F.; Vallée-Bélisle, A. A Highly Selective
Electrochemical DNA-Based Sensor That Employs Steric Hindrance
Effects to Detect Proteins Directly in Whole Blood. J. Am. Chem. Soc.
2015, 137, 15596−15599. (c) Pheeney, C. G.; Barton, J. K.
Intraduplex DNA-mediated electrochemistry of covalently tethered
redox-active reporters. J. Am. Chem. Soc. 2013, 135, 14944−14947.
(d) Pheeney, C. G.; Guerra, L. F.; Barton, J. K. DNA sensing by
electrocatalysis with hemoglobin. Proc. Natl. Acad. Sci. U. S. A. 2012,
109, 11528−11533. (e) Cash, K. J.; Ricci, F.; Plaxco, K. W. An
electrochemical sensor for the detection of protein-small molecule
interactions directly in serum and other complex matrices. J. Am.
Chem. Soc. 2009, 131, 6955−6957.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Funding by the Czech Science Foundation (20-00885X to
D.K. and M.H.) is gratefully acknowledged. Other authors
were supported by the European Union’s Horizon 2020
research and innovation programme under grant agreement
no. 767325 POCOSTEO (to C.P.Y., M.O., and C.K.O’S.),
European Regional Development Fund; OP RDE (no.
CZ.02.1.01/0.0/0.0/16_019/0000729 to V.S.), National Sci-
ence Centre in Poland, grant no. 015/16/W/ST5/00413 (for
D.P.S. and Z.J.L.), and institutional support from the IBP CAS
(no. 68081707 to L.H. and M.F.). A Ph.D. scholarship for D.K.
from the Department of Chemistry of Natural Compounds of
the University of Chemistry and Technology Prague is also
acknowledged.
(13) (a) Zwang, T. J.; Hurlimann, S.; Hill, M. G.; Barton, J. K. Helix-
̈
Dependent Spin Filtering through the DNA Duplex. J. Am. Chem. Soc.
2016, 138, 15551−15554. (b) Gorodetsky, A. A.; Hammond, W. J.;
Hill, M. G.; Slowinski, K.; Barton, J. K. Scanning electrochemical
microscopy of DNA monolayers modified with Nile Blue. Langmuir
2008, 24, 14282−14288.
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