A safe and effective delivery system is considered a key to the success of nucleic acid therapeutics. It has been reported that pulmonary surfactants or their components could facilitate the uptake of small interfering RNA (siRNA) into the lung epithelial cells. Previously, our group investigated the use of KL4 peptide, a synthetic cationic peptide that simulates the structural properties of surfactant protein B (SP-B), as siRNA delivery vector. Although KL4 peptide exhibits good in vitro siRNA transfection efficiency on lung epithelial cells, its therapeutic potential is limited by its poor aqueous solubility due to the presence of a high proportion of hydrophobic leucine residues. In this study, we aim to address the solubility issue, designing five different modified peptides by replacing the hydrophobic leucine with alanine or valine, and assess their potential as siRNA delivery vectors. While the modified peptides retain the overall cationic property, their siRNA binding is also affected and their transfection efficiency is inferior to the parent KL4 peptide. A closer examination of the conformation of these peptides by circular dichroism shows that substitution of leucine residues leads to the change of the secondary structure from α-helical content to either β-sheet or more disordered, β-turn conformations. Relatively conservative amino acid substitutions, in terms of hydrophobicity bulk, lead to substantial conformational alteration, heavily impacting siRNA binding and release, cellular uptake, and transfection efficiency. Although the peptide modification strategy employed in this study was unsuccessful in developing an improved version of KL4 peptide for siRNA delivery, it highlights the importance of the α-helical conformation for efficient siRNA transfection, providing useful insights for future development of peptide-based RNA delivery system.

中文翻译：

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KL4 肽的修饰揭示了 α 螺旋结构对高效 siRNA 递送的重要性

安全有效的递送系统被认为是核酸治疗成功的关键。据报道，肺表面活性剂或其成分可以促进小干扰 RNA (siRNA) 摄入肺上皮细胞。此前，我们小组研究了KL4肽（一种模拟表面活性蛋白B（SP-B）结构特性的合成阳离子肽）作为siRNA递送载体的用途。虽然 KL4 肽在体外表现良好siRNA 对肺上皮细胞的转染效率，由于存在高比例的疏水性亮氨酸残基，其治疗潜力受限于水溶性差。在这项研究中，我们旨在解决溶解度问题，通过用丙氨酸或缬氨酸替换疏水性亮氨酸来设计五种不同的修饰肽，并评估它们作为 siRNA 递送载体的潜力。虽然修饰的肽保留了整体的阳离子特性，但它们的 siRNA 结合也会受到影响，并且它们的转染效率不如亲本 KL4 肽。通过圆二色性对这些肽的构象进行更仔细的检查表明，亮氨酸残基的取代导致二级结构从α-螺旋内容变为β-折叠或更无序的β-转角构象。相对保守的氨基酸取代，就疏水性而言，会导致大量的构象改变，严重影响 siRNA 的结合和释放、细胞摄取和转染效率。尽管本研究中采用的肽修饰策略未能成功开发用于 siRNA 递送的 KL4 肽的改进版本，但它强调了 α-螺旋构象对于有效 siRNA 转染的重要性，为未来基于肽的 RNA 递送的发展提供了有用的见解系统。