Optimizing the Specificity Window of Biomolecular Receptors Using Structure-Switching and Allostery

To ensure maximum specificity (i.e., minimize cross-reactivity with structurally similar analogues of the desired target), most bioassays invoke "stringency", the careful tuning of the conditions employed (e.g., pH, ionic strength, or temperature). Willingness to control assay conditions will fall, however, as quantitative, single-step biosensors begin to replace multistep analytical processes. This is especially true for sensors deployed in vivo, where the tuning of such parameters is not just inconvenient but impossible. In response, we describe here the rational adaptation of two strategies employed by nature to tune the affinity of biomolecular receptors so as to optimize the placement of their specificity "windows" without the need to alter measurement conditions: structure-switching and allosteric control. We quantitatively validate these approaches using two distinct, DNA-based receptors: a simple, linear-chain DNA suitable for detecting a complementary DNA strand and a structurally complex DNA aptamer used for the detection of a small-molecule drug. Using these models, we show that, without altering assay conditions, structure-switching and allostery can tune the concentration range over which a receptor achieves optimal specificity over orders of magnitude, thus optimally matching the specificity window with the range of target concentrations expected to be seen in a given application.