锁定核酸寡核苷酸在siRNA临床前生物分析中的应用。,Scientific Reports

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锁定核酸寡核苷酸在siRNA临床前生物分析中的应用。
Scientific Reports ( IF 3.8 ) Pub Date : 2019-03-05 , DOI: 10.1038/s41598-019-40187-4
Mai B Thayer ₁ , Julie M Lade ₁ , David Doherty ₁ , Fang Xie ₁ , Babak Basiri ₁ , Omar S Barnaby ₂ , Noor S Bala ₁ , Brooke M Rock ₁

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尽管siRNA具有极好的治疗潜力，但尚未充分阐明造成强烈的间接暴露-应答关系的机制。要了解与有效活性相关的siRNA特性，需要对siRNA的位置进行表征。表征中的技术挑战是从生物样品中检测和定量siRNA。本文所述，锁定核酸（LNA）杂交-连接ECL ELISA设计用于对siRNA有义链和反义链的超灵敏定量，而与结构修饰无关。该测定法用于测量临床前物种血清和组织匀浆中的siRNA。我们观察到从系统循环中快速清除siRNA，这与组织内长时间积累形成对比。该测定法还能够从与治疗性siRNA相关的RNA诱导的沉默复合物（RISC）和Argonaute 2（Ago2）中区分和定量游离siRNA。我们利用正交方法LC-MS来研究3'核酸外切酶对反义链代谢的活性。综上所述，我们证明了LNA杂交-连接ECL ELISA是一种可靠的分析方法，可直接用于在整个生物基质中无缝测量siRNA治疗剂的暴露。

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Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics.

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3' exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

更新日期：2019-03-05

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