Learning from\nClassic: DNA-Based Conditional Equilibrium\nConstant To Regulate Affinity “On-the-Fly” for Bioassays

Artificial programming of affinity is beneficial to optimize\nresponsiveness\nin biomolecules for various applications. In one classical theory,\none comprehensive parameter, conditional equilibrium constant (<i>K</i>’<sub>EDTA</sub>), can accurately and quantitatively\ndefine the affinity of ethylene diamine tetraacetic acid (EDTA) for\nmetal ions. Learning from the classic, we have proposed a novel DNA-based\nconditional equilibrium constant (<i>K</i>’<sub>DNA</sub>) to regulate DNA probes' affinity and response “on-the-fly”,\nlong after the probe design and synthesis. Artificial regulation of\naffinity over several magnitudes has been simply realized via short\noligonucleotides with different lengths, concentrations, and combinations.\nThe thermodynamic response can be quantitatively simulated by one\nDNA-based conditional equilibrium constant (<i>K</i>’<sub>DNA</sub>), acting as an analogue to the classical EDTA system. The\nproof of concept of affinity programming also allows improved discrimination\nof single-nucleotide variants as well as assaying ribonuclease and\ndoxycycline in a homogeneous solution. Therefore, the theory of DNA-based\nconditional equilibrium constant (<i>K</i>’<sub>DNA</sub>) will enable to engineer versatile DNA switches with programmable\naffinity in assays and bionanotechnology.