Systematic Scanning Mutagenesis Unveils the Structure-Function Landscape Enabling Aptamer Evolution

Aptamers are valuable recognition elements for biosensing and therapeutic applications; however, an accessible strategy to link structure with function during postselection engineering remains challenging. Here, we introduce the concept of a structure-function landscape for aptamers, constructed by systematic single-point mutagenesis that maps the contribution of each nucleotide to target binding. Using OTC2, an aptamer that recognizes multiple tetracycline antibiotics, as a model, we identified critical binding sites and variable regions within the landscape. Guided by this landscape, we engineered OTC2 through combinatorial mutagenesis of only five nucleotides, yielding an evolved aptamer (OTC2M1) with enhanced affinity and substantially improved binding kinetics. The improved performance translated directly into sensing applications: an aptamer switch based on OTC2M1 achieved a 40-fold improvement in detection sensitivity for tetracycline antibiotics compared to the original aptamer. This work establishes the structure-function landscape as an informative and accessible platform for rational aptamer engineering and highlights its potential to guide the development of high-performance biosensors.