PDF(Pubmed)
Abstract：
The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively. Key features • Adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other unmodified and modified oligonucleotides. • Does not necessitate any Biological Safety Level clearance and offers a rapid, cost-effective, and entirely in vitro procedure. • Allows researchers to evaluate multiple modification patterns that, when coupled with targeting activity, allow for selecting the best modification pattern prior to in vivo analysis.
摘要：
寡核苷酸治疗领域正在迅速发展，特别是对抗孤儿疾病和癌症。然而,寡核苷酸的内在不稳定性，尤其是RNA,面对细胞内和循环中遇到的恶劣条件，提出了重大挑战。因此,在开发寡核苷酸疗法时，评估血清中寡核苷酸的稳定性具有重要意义。该协议概述了一种可靠且可重复的制备寡核苷酸双链体的方法，再加上凝胶电泳确认。随后，该方案定义了一种机制来评估血清中寡核苷酸双链体的稳定性。该协议旨在为研究人员建立标准化的参考，使他们能够比较各种修饰对寡核苷酸稳定性的影响，并有效评估降解动力学。主要特点•适用于小干扰RNA(siRNA),microRNA(miRNA),反义寡核苷酸（ASO），和其他未修饰和修饰的寡核苷酸。•不需要任何生物安全级别的清除，并提供了一个快速，成本效益高,完全在体外程序。•允许研究人员评估多种修改模式，当与目标活动相结合时，允许在体内分析之前选择最佳的修饰模式。
