寡核苷酸药物及寡核苷酸制备技术研究进展

Therapeutic oligonucleotides: a review

摘要:

寡核苷酸是生物医学和生命科学研究中调节基因表达的基本工具，并被开发为基因靶向治疗药物，用于治疗病毒、肿瘤和遗传病。寡核苷酸药物主要包括反义寡核苷酸、小干扰RNA、核酶、脱氧核酶、反基因、CpG寡核苷酸、转录因子诱饵和核酸适配体等。天然的寡核苷酸在体内很容易被降解，特异性低，且有毒副作用。因此，药物寡核苷酸通常带有特定的修饰基团，如硫代磷酸二酯键、氟代、甲基以及锁核酸等，以增强寡核苷酸在体内的稳定性，提高特异性，并降低其毒副作用。目前，寡核苷酸主要采用化学方法合成，但化学合成的寡核苷酸初产物纯度低，而纯化十分困难。大规模核酸合成仪和纯化设备十分昂贵，因而大量合成和纯化寡核苷酸的成本高昂，大大限制了寡核苷酸药物的研究和应用。尽管已经涌现了多种多样的核酸扩增和检测方法，但用于扩增寡核苷酸的方法极少，且均不适合大量制备寡核苷酸。一种新的基于热循环的寡核苷酸扩增方法，称为“聚合酶-内切酶扩增反应” (Polymerase-endonuclease amplification reaction, PEAR)，能够使寡核苷酸等小分子核酸在双酶催化下，利用独特的“滑动-切割机制”进行自我复制，并实现指数扩增。PEAR反应简单、高效、稳定。该方法已成功制备硫代和氟代修饰的寡核苷酸，与化学合成法相比，该技术不依赖于大规模DNA合成仪，降低了生产成本，适合大量生产高纯度的寡核苷酸，将有助于推动寡核苷酸药物的研究和应用。

Abstract:

Oligonucleotides are widely used as effective tools to regulate gene expression and drugs for targeted gene therapy. Therefore, they are potentially useful for the treatment of viral, tumor and hereditary diseases. Therapeutic oligonucleotides include antisense oligonucleotide, Small interference RNA (siRNA), Ribozyme, DNAzyme, anti-gene, CpG, decoy and aptamer. Therapeutic oligonucleotides usually carry certain modifications, such as phosphorothioates, fluoro or locked nucleic acids, to enhance the stability and specificity, and reduce the side-effects, because natural oligonucleotides have poor stability in vivo, low specificity and side effects. Now oligonucleotides are usually manufactured by chemical synthesis, with low purity and high cost. Here, we review a novel thermocyclic reaction for the amplification of oligonucleotides, referred to as Polymerase-endonuclease Amplification Reaction (PEAR) catalyzed by two thermostable enzymes. PEAR is simple, efficient, and stable. Comparing with traditional chemical synthesis, PEAR-based enzymatic production of oligonucleotides could be a robust alternative method for the large-scale production of therapeutic or non-therapeutic oligonucleotides.