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<p>Non-poly(A) messenger ribonucleic acid (mRNA) purification remains a critical bottleneck for cell-free translation platforms like <i>tr</i>anscription-translation coupled with the <i>a</i>ssociation of <i>p</i>uromycin 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&#xa0;°C), time (5–30&#xa0;min), pH (7–9), and their interactions, revealing an unanticipated optimum of 60&#xa0;°C for 23.5&#xa0;min at pH 9 (vs. conventional protocols of 90&#xa0;°C for 10&#xa0;min). These conditions were associated with a maximum release efficiency of 71 ± 7% with 10 pM mRNA, sufficient for downstream TRAP library construction.</p>

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Optimization of mRNA thermal release on an integrated microfluidic platform by a complex system response method

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月22日

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  • Swati T. Gurme,
  • Yi-Cheng Tsai,
  • Bhushan Koparde,
  • Da-Jeng Yao,
  • Lily Hui-Ching Wang,
  • Gwo-Bin Lee

摘要

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 mRNA, sufficient for downstream TRAP library construction.