A side-by-side comparison of peptide-delivered antisense antibiotics employing different nucleotide mimics. A side-by-side comparison of peptide-delivered antisense antibiotics employing different nucleotide mimics

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes bear great potential in the fight against antimicrobial resistance and for precision microbiome editing. The development of such antisense antibiotics has primarily focused on using phosphoramidite morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based therapy in general. Here, we compare seven chemically distinct 10mer-ASOs, all designed to target the same essential acpP mRNA upon delivery with a KFF-peptide carrier into Salmonella, comparing PNA, PMO, phosphorothioate modified-DNA (PTO), 2’-methoxylated RNA (RNA-OMe), 2’-methoxyethylated RNA (RNA-MOE), 2’-fluorinated RNA (RNA-F) and 2’-locked RNA (LNA). Our systematic analysis using a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and bacterial killing suggests that only PNA and PMO make it into Salmonella to cause bacterial growth inhibition. However, given their high target binding and translational repression activity in vitro, ASOs based on LNA and RNA-MOE appear to be promising candidates for antisense antibiotics that will require the identification of an efficient carrier. Overall design: Comparative gene expression profiling analysis of RNA-seq data for Salmonella cells after 15 min exposure to several nucleotide mimics fused to the KFF pepide targeting the essential gene acpP. 2 replicates and 10 conditions

靶向细菌必需基因mRNA的反义寡核苷酸（Antisense oligomer, ASO）类抗生素，在对抗抗菌耐药性及精准微生物组编辑领域具有巨大应用潜力。此类反义抗生素的开发主要聚焦于采用磷酰胺酰吗啉代（phosphoramidite morpholino, PMO）与肽核酸（peptide nucleic acid, PNA）骨架，却很大程度上忽略了近年来推动反义寡核苷酸疗法广泛发展的诸多新型化学修饰策略。
本研究对比了7种化学结构各异的10聚体反义寡核苷酸，所有序列均设计为靶向同一段必需基因acpP的mRNA，并通过KFF肽载体递送至沙门氏菌（Salmonella）；所测试的化学骨架涵盖PNA、PMO、硫代磷酸修饰DNA（phosphorothioate modified-DNA, PTO）、2'-甲氧基化RNA（2’-methoxylated RNA, RNA-OMe）、2'-甲氧乙基化RNA（2’-methoxyethylated RNA, RNA-MOE）、2'-氟化RNA（2’-fluorinated RNA, RNA-F）与2'-锁核酸（2’-locked RNA, LNA）。
我们通过多种体外与体内实验方法，系统评估了这些反义寡核苷酸的摄取效率、靶标结合能力与杀菌活性，结果显示仅PNA与PMO能够进入沙门氏菌并实现细菌生长抑制。然而，鉴于LNA与RNA-MOE类反义寡核苷酸在体外具备优异的靶标结合与翻译抑制活性，它们有望成为反义抗生素的候选分子，但仍需开发高效的递送载体。
整体实验设计：对经KFF肽融合的多种核苷酸模拟物处理15分钟后的沙门氏菌细胞，开展RNA测序数据的比较基因表达谱分析，实验共设置10种条件与2个生物学重复。