Stranded 4 C5 0.14 ± 0.01 9 C4‐S 5 C3‐S 0.10 ± 0.00 10 C2‐S 6 C2‐S 0.07 ± 0.00 11 C3‐S 7 C3‐S 0.06 ± 0.01 12 C5‐S John Wiley & Sons, Ltd 3.3. Analysis of the interaction between the immobilized single‐stranded oligonucleotide and thrombin using SPR {#elsc1307-sec-0060} --------------------------------------------------------------------------------------------------------------- An interesting feature of the immobilized nucleic acid sensor chip is that after hybridizing with the target molecule, the immobilised strands are designed to have single‐stranded regions to create a free space for other hybridizing molecules. Here we show how this feature enables rapid analysis of two‐component systems. The immobilised Oligo‐A strand was complementary to the immobilized Oligo‐B strand, and thus could hybridize with a single‐stranded template strand (5'‐TTT TGT ATT TGC AGT TTT GCA AGT TAC CAT GTT TTA TTG TTA TCG TTT‐3') when diluted in the hybridization buffer (Supporting Information Figure [S2](#elsc1307-supl-0001){ref-type="supplementary-material"}). We used the template strand (Oligo‐B) for the analysis of interactions between two proteins. First, we investigated thrombin and its DNA aptamer (ODN5‐17) by immobilizing the template strand (Oligo‐B) on the Au surface, followed by addition of thrombin and ODN5‐17 (Figure [3A](#elsc1307-fig-0003){ref-type="fig"}). We analyzed these interactions using thrombin in two forms, thrombin (active form) and thrombin‐(Lys259‐Ala267)‐peptide, as previously reported [19](#elsc1307-bib-0019){ref-type="ref"}. When the template strand (Oligo‐B) was replaced with thrombin‐(Lys259‐Ala267)‐peptide, the SPR signal was low at all concentrations of thrombin. In contrast, when the template strand (Oligo‐B) was replaced with active thrombin, there was a correlation between the SPR signal and the concentration of thrombin. From this result, we concluded that the Oligo‐B strand binds to thrombin via its DNA aptamer. Furthermore, the binding of thrombin (active form) and thrombin‐(Lys259‐Ala267)‐peptide on the Oligo‐B strand to the Au surface was almost equivalent, which suggests that the recognition sequence in the aptamer is not important for binding to thrombin. {#elsc1307-fig-0003} Next, we investigated a mutant of thrombin in which we removed residues Lys259 and Ala267 from the α‐helix III region of the DNA binding site and replaced these with alanine residues, as described in Figure [2A](#elsc1307-fig-0002){ref-type="fig"}. However, in the case of this thrombin mutant, the immobilized Oligo‐B strand did not bind to the active form (residues 263--282) or the inactive form of thrombin (Lys259‐Ala267). 3.4. Analysis of the binding between the immobilized single‐stranded oligonucleotide and thrombin using fluorescent resonance energy transfer {#elsc1307-sec-0070} --------------------------------------------------------------------------------------------------------------------------------------------- In this experiment, we designed the template strand to include single‐stranded regions in which thrombin could bind; therefore, analysis using fluorescent resonance energy transfer (FRET) was a suitable method to investigate the interaction. In this approach, we used FRET between a fluorophore‐labeled oligonucleotide and a quencher‐labeled oligonucleotide, which enables measurements of the change in FRET signals when thrombin binds to the immobilized oligonucleotide. When thrombin interacts with the immobilized single‐stranded oligonucleotide, the FRET signal would change. Using this approach, we investigated thrombin binding to the single‐stranded oligonucleotide Oligo‐D (Supporting Information Figure [S4](#elsc1307-supl-0001){ref-type="supplementary-material"}). Here, the signal change was detected only when the fluorophore‐labeled DNA strand Oligo‐C (5'‐FAM‐TTC TCG CGT AGT TGA TTG CGC TGC CGC CGC‐3') was introduced, and this suggested that the single‐stranded region in Oligo‐D had been recognized. The results of our FRET analysis using thrombin and its mutants (Figure [3B](#elsc1307-fig-0003){ref-type="fig"}--[C](#elsc1307-fig-0003){ref-type="fig"}) are summarized in Table [2](#elsc1307-tbl-0002){ref-type="table"}. First, we investigated the binding of thrombin to the single‐stranded oligonucleotide Oligo‐D. We used a blank control, with no thrombin, to detect background noise. Then, thrombin was introduced, and when this was mixed with the single‐stranded template strand (Oligo‐D) and fluorescence‐labeled Oligo‐C, the signal change was detected. Next, we investigated the interaction between thrombin mutants and the single‐stranded oligonucleotide. For this purpose, each thrombin mutant was mixed with the single‐stranded template strand (Oligo‐D), the fluorescent‐labeled Oligo‐C, and thrombin‐(Lys259‐Ala267)‐peptide, and the signal change was compared with that with active thrombin. From these results, we concluded that thrombin interacts with the single‐stranded template strand (Oligo‐D) when it has active form, and this interaction is not affected by the presence of Lys259 and Ala267. ###### Results of the FRET analysis of the interactions between thrombin and its mutants Template strand Thrombin mutants Binding signal change ----------------- -------------------- ----------------------- Oligo‐D Active thrombin \+ Thrombin‐(Lys)‐ \+ (Ala267)‐peptide Thrombin‐(Lys)‐(Ala)‐+ Active thrombin ++ Oligo‐D Inactive thrombin \+ Thrombin‐(Lys)‐ \+ (Ala267)‐peptide Thrombin‐(Lys)‐(Ala)‐+ Inactive thrombin ++ John Wiley & Sons, Ltd 4. DISCUSSION {#elsc1307-sec-0080} ============= Here we have demonstrated a sensor chip with high affinity for single‐stranded oligonucleotides that can be used to investigate various interactions. We introduced single‐stranded regions on the surface of an Au sensor chip through the functionalization of a SAM on the Au surface by a 1,12‐diaminododecane spacer containing a reactive amine group. The functionalization of the Au chip was necessary to immobilize the oligonucleotide on the Au surface for analysis. In the case of a sensor chip containing a random DNA‐containing surface, which we created by mixing a DNA chip with the Au chip [20](#elsc1307-bib-0020){ref-type="ref"}, we could not immobilize the DNA on the Au surface due to the steric hindrance generated by the oligonucleotide [20](#elsc1307-bib-0020){ref-type="ref"}. The interaction between DNA strands was confirmed using the immobilized Oligo‐A strand on the Au sensor chip, and this interaction increased the number of DNA strands on the surface of the Au chip. Using the template strand (Oligo‐B) in a 1:1 molar ratio with a complementary single‐stranded strand (ODN5‐17) enabled the immobilization of the DNA strands on the Au sensor chip. The presence of single‐stranded DNA on the Au sensor chip was confirmed using Oligo‐A (Supporting Information Figure [S3](#elsc1307-supl-0001){ref-type="supplementary-material"}). In order to make possible experiments for multi‐component systems, the surface of the Au sensor chip was covered with a nitrocellulose membrane. Then, several small