Article pubs.acs.org/Langmuir
Photoresponsive DNA Monolayer Prepared by Primer Extension Reaction on the Electrode Tadao Takada,* Mai Takemura, Yuta Kawano, Mitsunobu Nakamura, and Kazushige Yamana* Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan S Supporting Information *
ABSTRACT: We describe a simple and convenient method for the preparation of photoresponsive DNA-modified electrodes using primer extension (PEX) reactions. A naphthalimide derivative was used as the photosensitizer that was attached to the C5-position of 2′-deoxyuridine-5′-triphosphate (dUTPNI). It has been found that dUTPNI is a good substrate for the PEX reactions using KOD Dash and Vent (exo-) enzymes in solutions to incorporate naphthalimide (NI) moieties into the DNA sequences. On the electrode surface immobilized with the primer/ template DNA, the PEX reactions to incorporate dUTPNI molecules into the DNA sequence were found to efficiently proceed. With this solid-phase method, the DNA monolayers capable of generating photocurrent due to the photoresponsive NI molecule can be constructed. It was shown that the photocurrent generation was significantly suppressed by a single-nucleotide mismatch included in the primer/template DNA, which is applicable for the design of photoelectrochemical sensors to discriminate singlenucleotide sequences.
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attactive for the development of new types of biosensors.20 The photoelectrochemical biosensors have several advantages; i. e., the signal generation for the detection of target molecules can be controlled by photoirradiation, which eventually weakens the background signal.21−24 We and other research groups have already shown that the photocurrent generation in the modified DNA possessing a photosensitizer molecule can be applicable for use in a DNA mismatch sensor.25−29 In this work, we describe a simple and convenient method for the preparation of photoresponsive DNA-modified electrodes by PEX reactions. It has been shown that 2′-deoxyuridine5′-triphosphate (dUTPNI) possessing a photoresponsive naphthalimide molecule (NI) is a good substrate for the PEX reactions to incorporate NI into the DNA sequences. The important finding of our present research is that the NImodified DNA monolayer on a gold electrode shows a good photoresponsive property and can be applicable as a photoelectrochemical sensor for the detection of a single-nucleotide mismatch in DNA.
INTRODUCTION Modified nucleic acid derivatives with some useful functions have been used in biochemical and biomedical studies as diagnostic probes and sensors as well as in material applications.1 Most of them can be chemically synthesized by using a modified phosphoramidite reagent on a solid support or by a postsynthetic modification.1−4 Although the chemical synthesis of modified DNA sequences is usually facile and reliable, it faces problems, such as the incompatibility of modified phosphoramidites to standard DNA synthesis protocol, laborious synthesis procedures for modified phosphoramidite reagents, difficulties in the synthesis of long DNA strands (over 100-mer), and multiple labeling or modifications.1−3 An enzymatic method based on the incorporation of modified deoxyribonucleoside triphosphates by a polymerase reaction into the DNA sequence has proven to be an effective approach as an alternative to the chemical synthesis method.5−10 The primer extension (PEX) reaction enables us to control the number and arrangement of the functional molecules in the synthesized DNA, and it is especially useful for multiple modifications in long oligonucleotide sequences.11,12 The modified DNA with a variety of functional groups, such as fluorescent dyes and electrochemical reporters, have been prepared by PEX reactions using the appropriately modified nucleoside triphosphates as the substrates.13−15 Hocek and Fojta have developed a useful method using a PEX reaction to prepare redox-labeled DNAs that can be used as an electrochemical biosensor in detecting target nucleic acid sequences.10,16−19 Aside from the many electrochemical sensors based on redox-modified DNA probes, the modified DNA exhibiting photoelectrochemical signals has been shown to be © 2015 American Chemical Society
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RESULTS AND DISCUSSION Incorporation of Naphthalimide Molecules into DNA by Primer Extension Reaction. The enzymatic incorporation of NI molecules into a DNA sequence using a PEX reaction is illustrated in Figure 1. It is known that the modification at the C5-position of pyrimidine showed a tolerance to the enzymatic incorporation of a wide range of functional groups.14,15 Therefore, a NI molecule was attached at Received: December 28, 2014 Revised: March 8, 2015 Published: March 25, 2015 3993
DOI: 10.1021/la505013u Langmuir 2015, 31, 3993−3998
Article
Langmuir
Figure 2. Denaturing polyacrylamide gel electrophoresis (PAGE) experiments for the analysis of the incorporation efficiency of dUTPNI into DNA by the PEX reaction. A 14-mer primer (P1) is labeled with fluorescein (indicated by an asterisk (*)). Template DNAs with sequence of AT (T1), (AT)4 (T2), and (ATT)2 (T3) as the extension region. Lane 1, P1 without the PEX reaction; lanes 2−4, dATP and dUTP; lanes 5−10, dATP and dUTPNI. KOD Dash and Vent (exo-) as DNA polymerases were used for the PEX reaction.
that dUTPNI can work as a good substrate to incorporate NI molecules into the DNA sequences by PEX reactions. No fulllength product was obtained without dUTP or dUTPNI, and the PEX using dUTPNI proceeded even when the template included G and C in the primer extension region (Supporting Information Figure S1). Photoresponsive DNA Monolayer Prepared by PEX Reaction on the Electrode. A photoresponsive DNA monolayer on a gold electrode was then prepared by a PEX reaction as depicted in Figure 1c and Figure 3. A 13-mer primer DNA (P2) possessing a hexylthiol group at the 5′-end was immobilized on the gold electrode through the Au−S bond linkage by the usual method.28 A 10-mer oligo-dA (A10) sequence containing the hexylthiol group at the 5′-end was used as a diluter to modulate the surface density of the primer (P2) on the electrode. A template DNA (T4) designed for the single-nucleotide extension was hybridized to P2 on the electrode. After hybridization, an enzymatic reaction using Vent (exo-) and dUTPNI was carried out as shown in EPEX (Figure 3a). For comparison, the electrodes modified with the chemically synthesized NI-conjugated dsDNA were prepared for ENI (Figure 3b).29 The photocurrent responses of these electrodes were measured by excitation of the NI molecule upon irradiation at 340 nm (Figure 3 and Supporting Information Figure S2). The photocurrent generation on an electrode modified with NI-conjugated dsDNA has been reported.28,29 The excitation of NI upon irradiation generates a hole into DNA through charge separation process between excited NI and a nucleobase, and the generated hole migrates to the electrode through DNA and a reduced NI is recycled by a molecular oxygen dissolved in aqueous solution, resulting in the cathodic photocurrent response.28,29 After the PEX reactions using dUTPNI, the prompt photocurrents were observed for the electrodes with P2/T4. Little or no photocurrent was observed for the electrode without immobilization of P2. The photocurrent intensity increased with an increase in the amount of P2/T4
Figure 1. (a) Schematic illustration of the incorporation of naphthalimide (NI) molecules into DNA by PEX reaction; (b) chemical structure of 2′-deoxyuridine triphosphates labeled with NI at the C5-position of uridine (dUTPNI); (c) preparation of photoresponsive DNA-modified electrodes by the PEX reaction on the surface using dUTPNI as the substrate. Irradiation for the excitation of the NI molecules can generate a photocurrent through a DNAmediated charge transfer.
the C5-position of a uridine base through an ethynyl carbamoyl linker as shown in Figure 1b. The synthesis of dUTPNI was carried out by a condensation reaction between the aminomodified dUTP and N-hydroxysuccinimide (NHS)-derivatized NI, both of which can be prepared according to reported procedures.30,31 The progress of the PEX reactions using dUTPNI in solutions was monitored by denaturing polyacrylamide gel electrophoresis (PAGE) (Figure 2). We used KOD Dash and Vent (exo-) polymerases belonging to the B-type polymerase family because these enzymes possess a tolerance for pyrimidine nucleotide analogues carrying functional groups at the C5position.11 The sequences of the template DNAs (T1−T3) were designed to investigate the multiple incorporations of the NI molecules. As can be seen in lanes 5−7, the KOD Dash reactions of dUTPNI on T1−T3 gave single bands that are expected for their chain lengths. Because of the NI modifications in the DNA, these bands were slower migrating when compared to the corresponding DNA bands (lanes 2−4) obtained using the unmodified UTP. Essentially similar results were obtained when using the Vent (exo-) enzyme. Therefore, the PEX reactions using dUTPNI in the presence of T1−T3 as templates were efficient and the multiple incorporations of dUTPNI into the DNAs were successful. The results indicated 3994
DOI: 10.1021/la505013u Langmuir 2015, 31, 3993−3998
Article
Langmuir
We next examined the photocurrent responses during the multiple incorporations of the NI molecules into the DNA. The template DNA sequences used in this study have alternating TTA sequences (T5−T7) in the extension region (Figure 4
Figure 4. Photocurrent response of DNA monolayer prepared on the electrode using DNA templates (T4−T7) with the different number of incorporation sites of dUTPNI. The electrodes modified with 1:1 of P2 and A10 were used for the PEX reaction.
Table 1. Photocurrent Intensity (Ipc) of DNA-Modified Electrodes after PEX Reaction
Figure 3. (a) Photocurrent response of DNA monolayers prepared by PEX reaction using dUTPNI and template T4 on an electrode (EPEX). The photocurrent response was measured upon irradiation at 340 nm after the PEX reaction. Surface density of primer/template (P2/T4) was controlled by changing the ratio of P2 and A10 (2:1, 1:2, and 0:1). (b) The photocurrent response of the electrode modified with NIconjugated DNA prepared by phosphoroamidite synthesis (ENI).29 (c) Dependence of the photocurrent intensity on the surface density of P2/T4. Comparison of the photocurrent between the electrodes modified with the enzymatically (EPEX) and chemically synthesized NIDNA (ENI) is shown. The surface coverage of P2 controlled by changing the ratio of P2 and diluter A10 was determined by a chronocoulometric assay based on the assumption that the deposition efficiency of A10 was same as that of P2.32,33
T4 T5 T6 T7 M-A M-G M-G
sequencea,b
Ipcc/(nA·cm−2)
3′-TCTCTCTCTTTTT-A-5′ 3′-TCTCTCTCTTTTT-ATTA-5′ 3′-TCTCTCTCTTTTT-ATTATTA-5′ 3′-TCTCTCTCTTTTT-ATTATTATTA-5′ 3′-TCTCTCTCTTTTA-A-5′ 3′-TCTCTCTCTTTTG-A-5′ 3′-TCTCTCTCTTTTC-A-5′
31 ± 3.8 6.8 ± 2.2 3.6 ± 1.2
