Abstract
Non-poly(A) messenger ribonucleic acid (mRNA) purification remains a critical bottleneck for cell-free translation platforms like transcription-translation coupled with the association of puromycin linker (TRAP) display for peptide screening, where sub-optimal thermal release can compromise results. Herein, we demonstrate a “complex system response” approach for optimizing thermal elution from probe-coated magnetic beads. Using an orthogonal array composite design with two optimization rounds (17 three-factor + 9 two-factor experiments), we systematically explored temperature (60–95°C), time (5–30 min), pH (7–9), and their interactions, revealing an unanticipated optimum of 60°C for 23.5 min at pH 9 (vs. conventional protocols of 90°C for 10 min). These conditions were associated with a maximum release efficiency of 71 ± 7% with 10 pM RNA, sufficient for downstream TRAP library construction.