Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction.

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 µM theophylline in as little as 15 min.

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry.

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

A Simple Colorimetric System for Detecting Target Antigens by a Three-Stage Signal Transformation-Amplification Strategy.

Inexpensive, straightforward, and rapid medical diagnostics are becoming increasingly important for disease identification in time- and resource-limited settings. Previous attempts to link oligonucleotide-based aptamers and hammerhead ribozymes to form ligand-induced ribozymes have been successful in identifying a variety of small molecule and protein targets. Isothermal exponential amplification reactions (EXPAR) amplify minute amounts of nucleic acid templates without requiring special instrumentation. We introduce a colorimetric assay that we engineered using an aptamer, hammerhead ribozyme, EXPAR, and peroxidase activity in conjunction with a 3,3',5,5'-tetramethylbenzidine (TMB) substrate. This is a modular signal enhancer system that can be easily modified to detect virtually any chosen analyte target within 5-10 min with minimal technical requirements. Ligand-aptamer binding causes the ribozyme to change conformation and self-cleave. The cleaved ribozyme triggers exponential amplification of a reporter sequence during EXPAR. The amplification products fold into single-stranded DNA guanine quadruplexes that exhibit peroxidase-like activity and can oxidize a colorless TMB substrate into a colored reaction product for visual detection. As a proof of concept, we examined the bronchodilator theophylline versus its chemical analogue, caffeine. We demonstrate linear changes in absorption readout across a wide range of target concentrations (0.5-1000 µM) and the ability to visually detect theophylline at 0.5 µM with an approximately 35-fold increased specificity versus that of caffeine. This three-stage detection system is a versatile platform that has the potential to improve the rapid identification of target analytes.