pHAST (pH-Driven Aptamer Switch for Thrombin) Catch-and-Release of Target Protein

Abstract

A pH-driven DNA nanomachine based on the human α-thrombin binding aptamer was designed for the specific catch-and-release of human α-thrombin at neutral and acidic pH, respectively. In neutral conditions, the thrombin aptamer component of the nanomachine is exposed and exists in the G-quadruplex conformation required to bind to the target protein. At slightly acidic pH, the polyadenine tail of the nanomachine becomes partially protonated and A+(anti)•G(syn) mispairing results in a conformational change, causing the target protein to be released. Förster resonance energy transfer (FRET) was used to monitor conformational switching over multiple pH cycles. Electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy were used to show pH dependent protein binding and release by the nanomachine. This approach could be applied generally to existing G-rich aptamers to develop novel biosensors, theranostics, and nanoswitches.

A direct selection strategy for isolating aptamers with pH-sensitive binding activity

“…These results demonstrate that our screening method can be used to generate high-affinity aptamers with pH-responsive functionality without relying exclusively on previously-identified pH-sensitive motifs. 17,18 As such, we believe this approach will prove highly valuable for generating environmentally-responsive aptamers for drug delivery, biosensors, and a variety of other applications.…”

“…15,16 In the context of molecular detection or controlled drug release, it would be especially advantageous to have aptamers for which the affinity is modulated by environmental pH, but only a small number of pH-sensitive aptamers have been reported to date. 17,18 These were produced by engineering known pH-responsive motifs into existing aptamers. For example, the Ricci group designed a cocaine-binding aptamer that incorporates a pH-dependent triplex, and were able to modulate the affinity of the aptamer for cocaine through pH changes.…”

pH-Control in Aptamer-Based Diagnostics, Therapeutics, and Analytical Applications

“…There are several existing methods for engineering pH-responsive aptamer binding. The first strategy involves conformational change of the aptamer in response to pH: by incorporating pH-responsive motifs or non-canonical binding into the structure, the aptamer can be induced to fold into or out of binding conformation in response to specific pH levels [ 12 , 40 ]. A variation of this can be achieved using a pH-responsive strand that is partially complementary to the aptamer, controlling conformation through base pairing without altering the aptamer itself [ 37 ].…”

“…McConnell et al investigated in 2016 a method of introducing pH dependence into an aptamer using the A + (anti)G(syn) motif, which would require less alteration of aptamers that already include a high proportion of G [ 40 ]. A thrombin aptamer was selected as a well-characterized example of the common G-quadruplex aptamer conformation [ 40 ].…”

Investigations of Printed Flexible pH Sensing Materials Based on Graphene Platelets and Submicron RuO2Powders

“…Measuring the proton activity (pH) of environment is one of the basic chemical measurements required in various fields of human interest, like biotechnology [20], medicine [21], environmental science [22], water-support system monitoring [23], food safety [24], and many others. Apart from direct pH measurement, sensors detecting proton activity can be employed as a basis for modification, for example, with enzymes [25], nucleic acids [26], or microorganisms [27], yielding biosensors for even more applications. Given this need for sensing of pH and pH-affecting markers or reactions, improvement and development of appropriate production technologies is a relevant task for research.…”