POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices
POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices
复制标题
DOI：
10.1038/s41598-020-66829-6
复制
发表时间：
2020-06-26
影响因子：
4.6
通讯作者：
Rock, Brooke M.
中科院分区：
文献类型：
--
作者：
Thayer, Mai B.;Humphreys, Sara C.;Rock, Brooke M.
寡核苷酸疗法使用短干扰 RNA (siRNA) 或反义寡核苷酸 (ASO) 分子来利用内源系统中和靶 RNA，以阻止后续的蛋白质翻译。虽然潜在的临床应用广泛，但递送效率和肝外靶向具有挑战性。生物分析对于理解这些复杂的关系非常重要。目前文献缺乏对测量复杂生物基质中 siRNA 和 ASO 的稳健且灵敏的方法的描述。本文描述的是基于非酶杂交的免疫测定，其能够对血清、尿液、胆汁以及肝脏和肾脏匀浆中的单个siRNA链（反义或有义）进行定量。分析实用性也在 ASO 中得到了证明。该检测方法在之前的工作基础上进行了改进，取消了酶促步骤，并进一步结合锁核酸 (LNA) 核苷酸修饰，以增加分析物杂交亲和力并提高灵敏度、特异性和稳健性。我们报告了一种血清中 siRNA 的超灵敏动态范围为 0.3 至 16,700 pM 的检测方法。该测定已提交给血清和组织基质的准确性和精密度的全面鉴定，并使用单一和混合分析物评估测定性能。可靠的基于 LNA 杂交的方法消除了基质样品提取、富集或扩增步骤的需要，而这些步骤可能会受到更先进的化学修饰的阻碍。
Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems-neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications.