Learning from Classic: DNA-Based Conditional Equilibrium Constant To Regulate Affinity “On-the-Fly” for Bioassays

Artificial programming of affinity is beneficial to optimize responsiveness in biomolecules for various applications. In one classical theory, one comprehensive parameter, conditional equilibrium constant (<i>K</i>'_EDTA), can accurately and quantitatively define the affinity of ethylene diamine tetraacetic acid (EDTA) for metal ions. Learning from the classic, we have proposed a novel DNA-based conditional equilibrium constant (<i>K</i>'_DNA) to regulate DNA probes' affinity and response "on-the-fly", long after the probe design and synthesis. Artificial regulation of affinity over several magnitudes has been simply realized via short oligonucleotides with different lengths, concentrations, and combinations. The thermodynamic response can be quantitatively simulated by one DNA-based conditional equilibrium constant (<i>K</i>'_DNA), acting as an analogue to the classical EDTA system. The proof of concept of affinity programming also allows improved discrimination of single-nucleotide variants as well as assaying ribonuclease and doxycycline in a homogeneous solution. Therefore, the theory of DNA-based conditional equilibrium constant (<i>K</i>'_DNA) will enable to engineer versatile DNA switches with programmable affinity in assays and bionanotechnology.