Fluorescence Detection of DNA by the Catalytic Activation of an Aptamer/Thrombin Complex

Abstract

A conjugate consisting of a thrombin aptamer tethered to the thrombin, Th, with a sensing nucleic acid (1) is used for the optical detection of DNA. The thrombin/aptamer complex blocks the biocatalytic functions of Th. Hybridization of the analyte DNA (2) to the sensing nucleic acid 1 yields a rigid duplex that detaches the aptamer from Th, a process that activates the protein toward the hydrolysis of bis(p-tosyl-Gly-Pro-Arg)-R110 (3) to the rhodamine 110 fluorophore (4). The system allows the DNA sensing with a sensitivity limit of 1 x 10-8 M. The aptamer/Th conjugate is also immobilized on glass slides for the optical detection of DNA. The dissociation of the aptamer/Th complex upon hybridization and the subsequent dehybridization of the duplex and the regeneration of the catalytically inactive Th/aptamer complex duplicate machinery functions.

Protein detection via direct enzymatic amplification of short DNA aptamers

“…(9)(10)(11)(12) A wealth of sensitive aptamer-based biodetection approaches have been reported in which aptamers were labeled with molecules such as redox probes, fluorescent dyes, or nanocrystals to be an integral part of signal transduction. (13)(14)(15)(16)(17)(18)(19)(20)(21) In addition, researchers have recently taken advantage of PCR to amplify DNA aptamers for sensitive detection of protein targets. Le and coworkers describe the isolation of a protein-aptamer complex via capillary electrophoresis, followed by PCR amplification of the bound ssDNA and subsequent visualization and quantification by gel electrophoresis.…”

Nukleinsäure‐basierte molekulare Werkzeuge

“…Durch die Zugabe des vollständigen Komplements zu R, des AntiFreigabestrangs, konnte das System zurückgesetzt werden. [143] Bei [144] Sowohl [5] in geöffnetem Zustand wird von drei DNA-Strängen A-C gebildet. Hybridisierung mit einem Treibstoffstrang F bringt die Pinzette in eine geschlossene Konformation.…”

Aptamer Sensors Combined with Enzymes for Highly Sensitive Detection

“…Since TBA is well characterized, some researchers, including ourselves, have used thrombin as a detection enzyme, utilizing ability of TBA inhibiting thrombin activity (Pavlov et al, 2005;Yoshida et al, 2006a). Protease activity was measured using a synthetic peptide labeled with a fluorescent molecule as the substrate.…”