Toehold-Exchange-Based Activation of Aptamer Switches Enables High Thermal Robustness and Programmability

Abstract

Aptamer switches are attractive nature-inspired tools for developing smart materials and nanodevices. However, the thermal robustness and programmability of current aptamer switches are often limited by their activation processes that are coupled with high reaction enthalpy. Here, we present an enthalpy-independent activation approach that harnesses toehold-exchange as a general framework to design aptamer switches. We demonstrate mathematically and experimentally that this approach is highly effective in improving thermal robustness and thus leads to better analytical performances of aptamer switches. Enhanced programmability is also demonstrated through fine-grained and dynamic tuning of effective affinities and dynamic ranges, as well as the construction of a synthetic DNA network that resembled biological signaling cascades. Our study not only enriches the current toolbox for engineering and controlling synthetic molecular switches but also offers new insights into their thermodynamic basis, which is critical for diverse synthetic biological designs and applications.

Comparison of Aptamer Signaling Mechanisms Reveals Disparities in Sensor Response and Strategies to Eliminate False Signals

“…Despite the great promise of aptamer-based sensors, they collectively still face challenges in terms of achieving sufficient sensitivity and specificity to detect analytes at relevant concentrations in real-world conditions. Considerable effort has been dedicated to improving the sensitivity of these sensors by lowering the background signal, − amplifying the target signal, − and suppressing matrix effects, , as well as increasing their thermal robustness and tuning their dynamic range . Unfortunately, little attention has been paid to studying and augmenting the specificity of the sensors themselves, which is problematic because sensors that lack selectivity are vulnerable to false-positive and false-negative results.…”

Sensitive microscale thermophoresis assay for rapid ochratoxin A detection with fluorescently labeled engineered aptamer

“…30 A recent NMR study demonstrated that OTA binding induces the folding of the aptamer into a duplex and G-quadruplex structure, and OTA binds at the junction of the duplex and G-quadruplex. 31 Taking advantage of the aptamer affinity, aptamer-based assays and sensors for OTA detection have been developed, including fluorescence, colorimetry, and electrochemical methods, [20][21][22][32][33][34][35][36][37] but some of them encounter limitations such as a complex fabrication process, long detection time, and cumbersome experimental steps. Aptamer-based MST methods have recently attracted intense attention for rapid sensitive target detection; they rely on the fact that the aptamer and the aptamer-target complex generate different MST signals.…”

Construction of Targeting-Peptide-Based Imaging Reagents and Their Application in Bioimaging

“…However, the major limitation to the clinical use of Nbs is the risk of nephrotoxicity associated with its high kidney uptake . Functional nucleic acids (FNAs), such as aptamers, are another type of recognition element that have the advantage of programmability to visualize a biological process. , Besides, there are some small molecules which have been designed and synthesized for binding to biological macromolecules such as proteins and DNA and are now emerging as a recognition element. , Several reviews have discussed recent developments in the application of the above targeting ligand molecules in the field of molecular imaging. − …”