Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
Nat Commun. 2023 May 11;14(1):2657. doi: 10.1038/s41467-023-38244-8.
Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide maximum capping efficiency of around 80-90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.
从 2020 年两种疫苗的临床应用开始，mRNA 疗法目前正在针对各种应用进行研究。去除转录的 mRNA 中的免疫原性未加帽的 mRNA 对于 mRNA 研究和临床应用至关重要。常用的加帽方法为广泛使用的 Cap-0 和 Cap-1 型 mRNA 提供了约 80-90%的最大加帽效率。然而，未加帽和加帽的 mRNA 具有几乎相同的物理化学性质，这给它们的物理分离带来了挑战。在这项工作中，我们开发了疏水光封闭标签修饰的帽类似物，通过反相高效液相色谱将加帽的 mRNA 与未加帽的 mRNA 分离。随后的光照射恢复了无足迹的天然加帽的 mRNA。即使在 Cap-2 型 mRNA 中，这种方法也能提供 100%的加帽效率，具有适用于 650nt 和 4,247nt mRNA 的多功能性。我们发现，在培养的细胞和动物中，Cap-2 型 mRNA 的翻译活性比用标准加帽方法制备的 Cap-1 型 mRNA 高 3 到 4 倍。
