Development of Long Asymmetric siRNA Structure for Target Gene Silencing and Immune Stimulation in Mammalian Cells.

IF 4 2区 医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Nucleic acid therapeutics Pub Date : 2023-10-01 DOI:10.1089/nat.2023.0003
Soonkap Kim, Young Gyu Kang, Jaejin Kim, Pooja Dua, Dong-Ki Lee
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Abstract

Post-transcriptional regulation of transcript abundances by RNA interference (RNAi) is a widely conserved regulatory mechanism to control cellular processes. We previously introduced an alternative siRNA structure called asymmetric siRNA (asiRNA), and showed that asiRNA exhibits comparable gene-silencing efficiency with reduced off-target effects compared with conventional siRNAs. However, to what extent the length of the guide strand affects the gene-silencing efficiency of asiRNAs is still elusive. In this study, we analyzed in detail the gene-silencing ability of asiRNAs along the guide strand length and immunostimulatory capacity of asiRNAs. We generated asiRNAs containing various guide strand lengths ranging from 25 to 29 nt, called long asiRNA (lasiRNA). We found that the gene-silencing activity of lasiRNAs decreased as the length of the guide strand increased. Nonetheless, the 3'-end overhangs that are complementary to the target gene have higher efficiency for gene silencing compared with mismatched overhangs. In addition, we found that the silencing efficiency of lasiRNAs correlates with their Ago2-binding affinity. Finally, replacing the mismatched overhang with a TLR7- or TLR9-associated immune response motif induced a toll-like receptor (TLR)-specific immune response and retained gene-silencing activity. Our findings demonstrate that lasiRNA structures can be tailored to function as bifunctional siRNA, which trigger a specific immune response combined with target gene silencing. Taken together, we anticipate that our findings provide a road map for the subsequent development of immune-stimulating lasiRNA, which bear the potential to be applied for therapeutic benefits.

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用于哺乳动物细胞靶基因沉默和免疫刺激的长不对称siRNA结构的开发。
RNA干扰对转录物丰度的转录后调节是一种广泛保守的控制细胞过程的调节机制。我们之前介绍了一种称为不对称siRNA(asiRNA)的替代siRNA结构,并表明与传统siRNA相比,asiRNA表现出相当的基因沉默效率,并降低了脱靶效应。然而,引导链的长度在多大程度上影响asiRNA的基因沉默效率仍然难以捉摸。在本研究中,我们详细分析了asiRNAs沿引导链长度的基因沉默能力和asiRNAs的免疫刺激能力。我们产生了含有25至29个不同引导链长度的asiRNA nt,称为长asiRNA(lasiRNA)。我们发现lasiRNA的基因沉默活性随着引导链长度的增加而降低。尽管如此,与错配的悬突相比,与靶基因互补的3'端悬突具有更高的基因沉默效率。此外,我们发现lasiRNA的沉默效率与其Ago2结合亲和力相关。最后,用TLR7或TLR9相关免疫反应基序取代不匹配的悬突诱导了toll样受体(TLR)特异性免疫反应并保留了基因沉默活性。我们的研究结果表明,lasiRNA结构可以被定制为双功能siRNA,它可以触发与靶基因沉默相结合的特异性免疫反应。总之,我们预计我们的发现为免疫刺激性lasiRNA的后续开发提供了路线图,该技术具有应用于治疗的潜力。
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来源期刊
Nucleic acid therapeutics
Nucleic acid therapeutics BIOCHEMISTRY & MOLECULAR BIOLOGY-CHEMISTRY, MEDICINAL
CiteScore
7.60
自引率
7.50%
发文量
47
审稿时长
>12 weeks
期刊介绍: Nucleic Acid Therapeutics is the leading journal in its field focusing on cutting-edge basic research, therapeutic applications, and drug development using nucleic acids or related compounds to alter gene expression. The Journal examines many new approaches for using nucleic acids as therapeutic agents or in modifying nucleic acids for therapeutic purposes including: oligonucleotides, gene modification, aptamers, RNA nanoparticles, and ribozymes.
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