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.
Laboratory for Molecular Science, University of Southern California, Los Angeles, California 90089, USA. chelyapo@usc.edu
Nickolas
N
Chelyapov
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