Parallel and Automated PNA Synthesis in μSPOT Format.

Peptide nucleic acids (PNA) are versatile RNA- and DNA-binding molecules with unique advantages over traditional antisense technologies. Their synthetic peptide backbone imparts resistance to enzymatic degradation and enhances hybridization specificity by eliminating ionic interactions, making them highly stable and effective in binding target sequences. PNA also benefit from low toxicity and immunogenicity, broadening their utility in therapeutic and diagnostic applications. Importantly, PNA synthesis leverages existing peptide synthesis infrastructure and protocols, making them an accessible and scalable option for laboratories equipped for peptide research. Here we present an optimized protocol for the parallel synthesis of PNA libraries via μSPOT using functionalized cellulose supports in automated solid-phase peptide synthesis (SPPS). The protocol encompasses cellulose functionalization, automated synthesis, cleavage, and quality control using liquid chromatography-mass spectometry (LC-MS), as well as array printing and evaluation. This accessible methodology offering a streamlined, cost-effective approach for systematic screening and development of PNA-based molecules accelerating innovation in antisense therapeutics and molecular tools.