Fluorescence detection of DNA by the catalytic activation of an aptamer/thrombin complex.
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.
Pavlov
Valeri
V
Institute of Chemistry, The Farkas Center for Light-Induced Processes, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Shlyahovsky
Bella
B
Willner
Itamar
I
D002384:Catalysis; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000032:analysis / Q000737:chemistry; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D009841:Oligonucleotides / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D012235:Rhodamines; D004247:DNA; D013917:Thrombin