Structural Optimization of Guanidinium-Linked Morpholino Phosphorodiamidate Morpholino Oligonucleotide Chimeras to Improve In Vitro Antisense Efficacy

IF 4.5 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Bioconjugate Chemistry Pub Date : 2026-08-19 Epub Date: 2026-07-22 DOI:10.1021/acs.bioconjchem.6c00176

Mohammed Qasim, Sharmeen Naaz, Subhamoy Pratihar, Swrajit Nath Sharma, Surajit Sinha

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引用次数: 0

Abstract

Although phosphorodiamidate morpholino oligomers (PMOs) are potent and stable antisense agents, their neutral backbone limits cellular uptake and biodistribution, requiring specialized delivery strategies to improve therapeutic efficacy. Chimeric guanidinium-linked morpholino oligomers (GMOs) with PMOs are designed to enhance cellular delivery yet compromise hybridization with the target sequence due to backbone rigidity. To address this, we designed flexible GMO-PMO chimeras by incorporating sarcosine, β-alanine, and ethanamine as internucleotide spacers in the oligonucleotide. Thermal melting studies on a 19-mer polythymidine (polyT) sequence demonstrate that these flexible GMO-PMOs partially restore duplex stability in a modification-dependent manner. This structural restoration is also observed in biologically relevant mixed GMO-PMO sequences targeting Nanog, a gene linked to stemness and cancer biology, while retaining their B-type global geometry as confirmed by circular dichroism analysis. The optimized ethanamine-modified Nanog chimera elicited strong, dose-dependent silencing, with up to 95% repression in MCF7 and ∼73% in HCT116 cells at 1 μM after 48 h. Importantly, both rigid and flexible GMO-PMOs exhibit similar lysosomal sequestration, varying in a cell-line-dependent manner. Collectively, flexible GMO-PMO chimeras mitigate the long-standing trade-off between carrier-free PMO delivery and duplex stability, establishing a robust platform for antisense gene silencing and potential therapeutic applications.

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提高体外反义效果的胍-键合氨基磷二酯寡核苷酸嵌合体结构优化。

虽然磷酸二酯morpholino低聚物（PMOs）是一种有效且稳定的反义药物，但其中性骨架限制了细胞的摄取和生物分布，需要专门的递送策略来提高治疗效果。与PMOs的嵌合胍-连接的morpholino oligomers （GMOs）被设计用于增强细胞递送，但由于骨干刚性而损害与目标序列的杂交。为了解决这个问题，我们设计了灵活的转基因生物- pmo嵌合体，将肌氨酸、β-丙氨酸和乙胺作为寡核苷酸的核苷酸间隔物。对19聚胸腺嘧啶（polyT）序列的热熔融研究表明，这些柔性转基因生物- pmos以修饰依赖的方式部分恢复了双相稳定性。这种结构恢复也在靶向Nanog（一种与干性和癌症生物学相关的基因）的生物学相关的混合转基因生物- pmo序列中观察到，同时保留了它们的b型全局几何形状，这一点得到了圆二色分析的证实。优化的乙胺修饰的Nanog嵌合体诱导了强的剂量依赖性沉默，在1 μM后48小时，MCF7细胞抑制高达95%，HCT116细胞抑制约73%。重要的是，刚性和柔性GMO-PMOs都表现出相似的溶酶体隔离，以细胞系依赖的方式变化。总的来说，灵活的转基因生物-PMO嵌合体缓解了长期以来无载体PMO传递和双相稳定性之间的权衡，为反义基因沉默和潜在的治疗应用建立了强大的平台。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioconjugate Chemistry 生物-化学综合

CiteScore

9.00

自引率

2.10%

发文量

236

审稿时长

1.4 months

期刊介绍： Bioconjugate Chemistry invites original contributions on all research at the interface between man-made and biological materials. The mission of the journal is to communicate to advances in fields including therapeutic delivery, imaging, bionanotechnology, and synthetic biology. Bioconjugate Chemistry is intended to provide a forum for presentation of research relevant to all aspects of bioconjugates, including the preparation, properties and applications of biomolecular conjugates.