High-throughput screening of biomolecules using cell-free systems.

High-throughput screening (HTS) has revolutionized the identification and evaluation of biomolecules by enabling the parallel testing of large libraries of compounds, nucleic acids, and proteins against biological targets. Traditionally conducted in live cells, HTS faces limitations such as cellular toxicity, metabolic interference, and regulatory constraints. Cell-free systems (CFS), which operate in vitro using reconstituted transcription-translation machinery, have emerged as powerful alternatives. These systems circumvent the constraints of cellular physiology, allowing for rapid and tunable expression of biomolecules directly from DNA or RNA templates. This chapter explores the principles, platforms, and applications of CFS-based HTS, highlighting its transformative impact on synthetic biology, drug discovery, diagnostics, and protein engineering. Several cell-free systems are detailed, including those derived from E. coli, wheat germ, rabbit reticulocytes, and the defined PURE system. The integration of CFS with high-throughput platforms such as microplates, droplet microfluidics, and paper-based devices enables cost-effective, scalable, and multiplexed assays. Analytical readouts, including fluorescence, luminescence, mass spectrometry, and digital PCR, provide real-time, sensitive detection of biochemical outputs. Furthermore, automation and machine learning are increasingly incorporated through robotic liquid handling and data-driven DBTL cycles, accelerating discovery and design processes. Despite challenges such as high reagent costs and limited post-translational modifications, innovations such as lyophilized CFS kits, artificial cells, and AI-integrated closed-loop platforms are expanding the frontiers of HTS. Altogether, CFS-based HTS offers a flexible, rapid, and accessible approach for next-generation biomolecular screening and therapeutic development.