title: Aptamer-mediated universal enzyme assay based on target-triggered DNA polymerase activity.
abstract: We herein describe an innovative method for a universal fluorescence turn-on enzyme assay, which relies on the target enzyme-triggered DNA polymerase activity. In the first target recognition step, the target enzyme is designed to destabilize detection probe derived from an aptamer specific to DNA polymerase containing the overhang sequence and the complementary blocker DNA, which consequently leads to the recovery of DNA polymerase activity inhibited by the detection probe. This target-triggered polymerase activity is monitored in the second signal transduction step based on primer extension reaction coupled with TaqMan probe. Utilizing this design principle, we have successfully detected the activities of two model enzymes, exonuclease I and uracil DNA glycosylase with high sensitivity and selectivity. Since this strategy is composed of separated target recognition and signal transduction modules, it could be universally employed for the sensitive determination of numerous different target enzymes by simply redesigning the overhang sequence of detection probe, while keeping TaqMan probe-based signal transduction module as a universal signaling tool.
authors: Department of Chemical and Biomolecular Engineering (BK 21+ program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-338, Republic of Korea; Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
authors: Ki Soo
authors: KS
authors: Park
authors: Department of Chemical and Biomolecular Engineering (BK 21+ program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-338, Republic of Korea.
authors: Chang Yeol
authors: CY
authors: Lee
authors: Department of Chemical and Biomolecular Engineering (BK 21+ program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-338, Republic of Korea.
authors: Kyoung Suk
authors: KS
authors: Kang
authors: Department of Chemical and Biomolecular Engineering (BK 21+ program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-338, Republic of Korea. Electronic address: hgpark@kaist.ac.kr.
authors: Hyun Gyu
authors: HG
authors: Park
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods; D019914:Taq Polymerase / Q000737:chemistry*; D013824:Thermus / Q000201:enzymology; D051981:Uracil-DNA Glycosidase / Q000032:analysis* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D051981:Uracil-DNA Glycosidase
keywords: DNA aptamer; DNA polymerase; Enzyme assay; Fluorescent biosensors