Allosteric aptamers controlling a signal amplification cascade allow visual detection of molecules at picomolar concentrations.
A broadly applicable homogeneous detection system has been developed. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition (precipitation) of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF(165)) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF(165), reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus, triggering the cascade and enabling the phase transition. As few as 5 fmol of VEGF(165) could be detected by the naked eye within an hour. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout and can be modified to detect molecules to which aptamers can be obtained.
Chelyapov
Nickolas
N
Laboratory for Molecular Science, University of Southern California, Los Angeles, California 90089, USA. chelyapo@usc.edu
D000494:Allosteric Regulation; D052157:Aptamers, Nucleotide / Q000494:pharmacology*; D001483:Base Sequence; D001777:Blood Coagulation / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008863:Microspheres; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D052157:Aptamers, Nucleotide; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D008666:Metalloendopeptidases; C033858:russellysin