Kliuchnikov Evgenii, Maksudov Farkhad, Pierson Daniel, Marx Kenneth A, Chanda Arani, Barsegov Valeri
Department of Chemistry, University of Massachusetts, Lowell, MA 01854, USA.
Technical Operations, Sarepta Therapeutics, Cambridge, MA 02142, USA.
Mol Ther Nucleic Acids. 2025 Aug 14;36(3):102685. doi: 10.1016/j.omtn.2025.102685. eCollection 2025 Sep 9.
We combined circular dichroism (CD) and viscosity measurements with molecular dynamics (MD) simulations and classification and regression approaches to machine learning to characterize solution structures of 22-mer, 25-mer, and 30-mer peptide- (-GlyArg6) conjugated phosphorodiamidate morpholino oligonucleotides (PPMOs). PPMO molecules form non-canonical folded structures with 1.4- to 1.5-nm radius of gyration, 4-6 base pairs and 5-11 base stacks, characterized by -49 to -71 kcal/mol free energy of folding. The 4.5-6.1 cm/g intrinsic viscosity and Huggins constant of 4.5-9.7 indicate PPMO-PPMO interactions at higher concentrations. The random-coil 3'-end conjugated -GlyArg portion does not alter molecular properties of phosphorodiamidate morpholino oligonucleotide (PMO) components, which explains why CD spectra, viscosity-concentration profiles, and inhibitor activities of 22-mer, 25-mer, and 30-mer PPMOs and PMOs are similar but the peptide enhances the PPMO cellular uptake. PPMOs' viscosity is lower than PMOs' viscosity, due to PMO-peptide position-dependent interactions, especially in 25-mer PPMO, explaining differences in CD and high-concentration viscosity. These results reiterate the importance of the conformational ensemble view of non-canonical PPMO structures in solution, in agreement with our previous PMO study. The addition of -GlyArg does not alter the structure and molecular properties of the PMO components of the PPMO structures but impacts the viscosity of the PPMO-based aqueous solution formulations.
我们将圆二色性（CD）和粘度测量与分子动力学（MD）模拟以及机器学习中的分类和回归方法相结合，以表征22聚体、25聚体和30聚体肽-（-GlyArg6）共轭磷酰二胺吗啉代寡核苷酸（PPMO）的溶液结构。PPMO分子形成具有1.4至1.5纳米回转半径、4至6个碱基对和5至11个碱基堆叠的非经典折叠结构，其特征在于折叠自由能为-49至-71千卡/摩尔。4.5至6.1厘米/克的特性粘度和4.5至9.7的哈金斯常数表明在较高浓度下PPMO之间存在相互作用。随机卷曲的3'-末端共轭-GlyArg部分不会改变磷酰二胺吗啉代寡核苷酸（PMO）组分的分子性质，这解释了为什么22聚体、25聚体和30聚体PPMO以及PMO的CD光谱、粘度-浓度曲线和抑制剂活性相似，但肽增强了PPMO的细胞摄取。由于PMO-肽位置依赖性相互作用，PPMO的粘度低于PMO的粘度，尤其是在25聚体PPMO中，这解释了CD和高浓度粘度的差异。这些结果重申了溶液中非经典PPMO结构的构象集合观点的重要性，这与我们之前对PMO的研究一致。添加-GlyArg不会改变PPMO结构中PMO组分的结构和分子性质，但会影响基于PPMO的水溶液配方的粘度。
