Allosteric Aptamers Controlling a Signal Amplification Cascade Allow Visual Detection of Molecules at Picomolar Concentrations

Abstract

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.

Functional Nucleic Acid Sensors

“…A DNA aptamer was selected by Chelyapov to bind and inhibit RVV-X, and the aptamer was fused to an aptamer for human vascular endothelial growth factor (VEGF 165 ) to construct an allosteric aptamer 584. Upon addition of VEGF 165 , the allosteric aptamer was incapable of binding to RVV-X.…”

Aptamer Sensors Combined with Enzymes for Highly Sensitive Detection

“…We succeeded in designing a type of split AES for sensing adenosine (Yoshida et al, 2006a), IgE and insulin ). Chelyapov and Fletcher et al reported similar sensing systems for AESs (Chelyapov, 2006;Fletcher et al, 2010). Chelyapov used an aptamer that inhibited Russell's viper venom factor X activator (RVV-X), and Fletcher et al used an aptamer that inhibited EcoRI.…”

“…In the case of a protease such as thrombin and RVV-X factor X activator, we can measure protease activity via observation of the coagulation that results from enzymatic activity. Chelyapov constructed a biosensor that can evaluate RVV-X activity with the naked eye, using microspheres for signal amplification (Chelyapov, 2006). Chelyapov succeeded in the detection of VEGF with a low detection limit of 5 fmol.…”

Aptamer‐Based Bioanalytical Assays: Amplification Strategies

“…The random sequence region undergoes selection (allosteric selection) to rise with the desired binding specificity (Soukup and Breaker, 1999;Breaker, 2002). Aptazyme-based assays have been developed demonstrating the advantages of these tools in array systems (Seetharaman et al, 2001) or as amplification systems to reach lower limits of detection (Hesselberth et al, 2003;Chelyapov et al, 2006). Seetharaman et al (2001) optimized an aptazyme array that discriminated between a variety of metabolites and drug analytes.…”