Amide Internucleoside Linkages Suppress the MicroRNA-like Off-Target Activity of Short Interfering RNA.

IF 3.5 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-15 DOI:10.1021/acschembio.4c00824

Chandan Pal, Michael Richter, Jayamini Harasgama, Eriks Rozners

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Abstract

RNA interference (RNAi) has rapidly matured as a novel therapeutic approach. In this field, chemical modifications have been critical to the clinical success of short interfering RNAs (siRNAs). Notwithstanding the significant advances, achieving robust durability and gene silencing in extrahepatic tissues, as well as reducing off-target effects of siRNA, are areas where chemical modifications can still improve siRNA performance. The present study developed the challenging synthesis of amide-linked guanosine dimers (G_AM1G and G_AM1A) and completed an "amide walk" one by one, systematically replacing every internucleoside phosphate with an amide linkage in a guide strand targeting the PIK3CB gene. Dual-luciferase and RT-qPCR assays in HeLa cells showed that, in a model system of unmodified siRNAs, the amide linkage at position 3 (between nucleosides 3 and 4) suppressed the cleavage of off-target YY1 and FADD mRNAs similarly to the industry gold standard modification glycol nucleic acid (GNA). These results suggest that amide linkages in the seed region have strong potential to improve the specificity of siRNAs by suppressing the microRNA-like off-target activity.

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酰胺核苷间键抑制短干扰RNA的microrna样脱靶活性。

RNA干扰（RNAi）作为一种新的治疗方法已经迅速成熟。在这一领域，化学修饰对短干扰rna （sirna）的临床成功至关重要。尽管取得了重大进展，但在肝外组织中实现强大的耐久性和基因沉默，以及减少siRNA的脱靶效应，仍然是化学修饰可以提高siRNA性能的领域。本研究开发了具有挑战性的酰胺连接鸟苷二聚体（GAM1G和GAM1A）的合成，并完成了一个接一个的“酰胺行走”，系统地将每个核苷间磷酸替换为针对PIK3CB基因的导链中的酰胺连接。HeLa细胞的双荧光素酶和RT-qPCR分析显示，在未修饰sirna的模型系统中，位置3（核苷3和4之间）的酰胺连锁抑制脱靶YY1和FADD mrna的切割，类似于行业金标准修饰乙二醇核酸（GNA）。这些结果表明，种子区的酰胺键通过抑制microrna样脱靶活性来提高sirna的特异性具有很强的潜力。

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来源期刊

ACS Chemical Biology 生物-生化与分子生物学

CiteScore

7.50

自引率

5.00%

发文量

353

审稿时长

3.3 months

期刊介绍： ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology. The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies. We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.

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