Photoaffinity Electrophoretic Mobility Shift Assay for Analyzing the Binding Specificity of DNA Aptamers to α-Thrombin
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.
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BibTeX
@article{sadakane2026photoaffinity,
title = {{Photoaffinity Electrophoretic Mobility Shift Assay for Analyzing the Binding Specificity of DNA Aptamers to α-Thrombin}},
author = {Yutaka Sadakane and Akari Tsubokura and Mitsuki Kasai and Yuka Ito and Chihiro Hirai},
journal = {Chemical and Pharmaceutical Bulletin},
year = {2026},
month = aug,
doi = {10.1248/cpb.c25-00751},
url = {https://www.jstage.jst.go.jp/article/cpb/74/8/74_c25-00751/_article},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - Photoaffinity Electrophoretic Mobility Shift Assay for Analyzing the Binding Specificity of DNA Aptamers to α-Thrombin
AU - Yutaka Sadakane
AU - Akari Tsubokura
AU - Mitsuki Kasai
AU - Yuka Ito
AU - Chihiro Hirai
PY - 2026
DA - 2026/08/01
JO - Chemical and Pharmaceutical Bulletin
DO - 10.1248/cpb.c25-00751
UR - https://www.jstage.jst.go.jp/article/cpb/74/8/74_c25-00751/_article
AB - 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.
SN - 1347-5223
SN - 0009-2363
ER -