{
  "abstract": "DNA aptamers are attractive alternatives to antibodies and advances in selection technologies have yielded several candidate sequences, creating a strong need for efficient methods to evaluate their binding specificity. We previously established a photoaffinity electrophoretic mobility shift assay (EMSA) for analyzing DNA-protein interactions. In this study, we investigated whether this method can be applied to assess the binding specificities of α-thrombin-binding DNA aptamers. The binding specificities of a 15-mer and a 29-mer aptamer were evaluated via photoaffinity EMSA using reaction mixtures enriched in zymogen prethrombin-2, whose structure closely resembles α-thrombin but lacks fibrinogen-clotting activity. Photoaffinity EMSA enabled quantification of the relative binding strengths of α-thrombin and prethrombin-2 resolved in the same sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) lane, revealing that the specificity for α-thrombin was approximately 2.5-3.5-fold higher than that for prethrombin-2 for both aptamers. In the case of the 15-mer aptamer, introducing the photoreactive group at the 9th nucleotide reduced its binding specificity for α-thrombin. This finding indicates that the position of the modification affects aptamer-protein recognition and offers clues about the structural basis of binding. Photoaffinity EMSA also enabled us to distinguish the binding sites of the 15-mer and 29-mer aptamers on α-thrombin using competition experiments with dabigatran and heparin, while allowing selective detection of α-thrombin in human plasma. Together, these results demonstrate that photoaffinity EMSA is a simple and informative approach that can evaluate the binding specificities of multiple DNA aptamers for a target protein in a single SDS-PAGE-based assay.",
  "abstract_shown": true,
  "affiliations": [],
  "article_type": null,
  "arxiv": null,
  "author_total": 5,
  "authors": [
    {
      "name": "Yutaka Sadakane"
    },
    {
      "name": "Akari Tsubokura"
    },
    {
      "name": "Mitsuki Kasai"
    },
    {
      "name": "Yuka Ito"
    },
    {
      "name": "Chihiro Hirai"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/23hkhvyag1h3t.bib",
  "cited_by": 0,
  "doi": "10.1248/cpb.c25-00751",
  "fulltext_url": "https://www.jstage.jst.go.jp/article/cpb/74/8/74_c25-00751/_article",
  "id": "23hkhvyag1h3t",
  "issns": [
    "1347-5223",
    "0009-2363"
  ],
  "landing_url": "https://www.jstage.jst.go.jp/article/cpb/74/8/74_c25-00751/_article",
  "language": null,
  "licence": "CC-BY-NC",
  "licence_label": "CC BY-NC",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by-nc/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "jstage-journal",
      "chemistry/medicinal"
    ],
    [
      "openalex",
      "1312|Advanced biosensing and bioanalysis techniques"
    ]
  ],
  "openalex": "W7171946679",
  "pdf_url": "https://www.jstage.jst.go.jp/article/cpb/74/8/74_c25-00751/_pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-08-01",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/23hkhvyag1h3t.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:34Z",
      "native_id": "10.1248/cpb.c25-00751",
      "source": "jstage",
      "url": "https://www.jstage.jst.go.jp/article/cpb/74/8/74_c25-00751/_article"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Advanced biosensing and bioanalysis techniques",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Photoaffinity Electrophoretic Mobility Shift Assay for Analyzing the Binding Specificity of DNA Aptamers to α-Thrombin",
  "title_text": "Photoaffinity Electrophoretic Mobility Shift Assay for Analyzing the Binding Specificity of DNA Aptamers to α-Thrombin",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:17:34Z",
  "url": "https://research.draconitic.com/paper/23hkhvyag1h3t",
  "venue": "Chemical and Pharmaceutical Bulletin",
  "versions": []
}