Kinetic optimization of a protein‐responsive aptamer beacon

Abstract

Aptamers have been utilized as biosensors because they can be readily adapted to sensor platforms and signal transduction schemes through both rational design and selection. One highly generalizable scheme for the generation of the so-called aptamer beacons involves denaturing the aptamer with antisense oligonucleotides. For example, rational design methods have been utilized to adapt anti-thrombin aptamers to function as biosensors by hybridizing an antisense oligonucleotide containing a quencher to the aptamer containing a fluorescent label. In the presence of thrombin, the binding equilibrium is shifted, the antisense oligonucleotide dissociates, and the beacon lights up. By changing the affinity of the antisense oligonucleotide for the aptamer beacon, it has proven possible to change the extent of activation of the beacon. More importantly, modulating interactions between the antisense oligonucleotide and the aptamer strongly influences the kinetics of activation. Comparisons across multiple, designed aptamer beacons indicate that there is a strong inverse correlation between the thermodynamics of hybridization and the speed of activation, a finding that should prove to be generally useful in the design of future biosensors. By pre-organizing the thrombin-binding quadruplex within the aptamer the speed of response can be greatly increased. By integrating these various interactions, we were ultimately able to design aptamer beacons that were activated by threefold within 1 min of the addition of thrombin.

Aptamers Embedded in Polyacrylamide Nanoparticles: A Tool for in Vivo Metabolite Sensing

“…23 Kinetic properties of a thrombin-binding aptamer displacement beacon has been evaluated, and the response time was found to be in seconds. 24 The data in Figure 4B confirm the instant response of AptaNPs in a cytosol buffer as reported previously. 17 We determined the response time to be less than 2 s under the experimental setting used in this study.…”

Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches

“…The key feature of this design is that it offers two distinct control parameters: displacement strand length (LDS) and loop length (Lloop). This is in contrast to alternative constructs such as aptamer beacons, which have just a single control parameter-displacement strand length-that confers only coarse-grained control over affinity and couples the construct's kinetics to its thermodynamics 16 . We show mathematically and experimentally that the two control parameters of the ISD design enable us to precisely and independently tune the thermodynamics and kinetics of the resulting aptamer switches.…”

Development of an aptamer-functionalized molecular recognition gating membrane targeting a specific protein on the basis of the aggregation phenomena of DNA–PNIPAM

“…The use of aptamer beacons is an easy-to-use method based on fluorescence that can monitor the dissociation of dsDNA composed of an aptamer induced by the aptameretarget molecule binding [48]. The dsDNA used for an aptamer beacon is composed of 5 0 -terminal fluorophore-modified TBA (F-TBA) and 3 0 -terminal quencher-modified complementary DNA (Q-cDNA).…”