고처리 스크리닝을 위한 RNA 기반의 합성 바이오 센서의 진화적 공학기법 연구

Abstract

Artificial selection, mimicking natural selection, is employed in vitro to enhance the survival of the fittest and eliminate undesirable variants, facilitating a more efficient design-build-test-learn cycle. Biosensors play a crucial role in this artificial selection system, introducing logic by translating intracellular product levels into cellular responses. The use of biosensor-guided high-throughput screening has significantly advanced metabolic and protein engineering, overcoming challenges of system complexity and low throughput. However, the limited repertoire of natural biosensors has led to the development of synthetic biosensors. Despite the efficiency, biosensor development through evolutionary engineering is time-consuming, requiring multiple rounds of selection. The effectiveness of biosensors in guiding directed evolution depends on meeting specific criteria, including operational range and signal-to-noise ratio. Challenges in biosensor diversity and development have prompted the exploration of novel aptamer selection methods, focusing on optimized strategies for riboswitch development to save time and effort while ensuring quality. In this study, capture-SELEX was developed to screen conformation-changing aptamers, mimicking ligand binding-dependent strand displacement in riboswitches. The random aptamer library was also subjected to two different strategies. Point mutations and random sequences were inserted into the ligand-binding sequence, reflecting the secondary backbone structure of previously identified aptamers. Additionally, a translation-initiation module was added after the random sequence to link ligand binding-induced conformation changes to ribosome binding site (RBS) sequestration. The enrichment profile and characterization of the developed riboswitches support the successful application of these novel strategies in aptamer selection. These approaches have effectively enriched ligand-specific aptamers and reduced development time by bypassing the need for transducer selection processes.