Department of Medicinal and Applied Chemistry, Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Bioconjug Chem. 2010 Sep 15;21(9):1642-55. doi: 10.1021/bc1001505.
Chemical conjugations of nucleic acids with macromolecules or small molecules are common approaches to study nucleic acids in chemistry and biology and to exploit nucleic acids for medical applications. The conjugation of nucleic acids such as oligonucleotides with peptides is especially useful to circumvent cell delivery and specificity problems of oligonucleotides as therapeutic agents. However, current approaches are limited and inefficient in their ability to afford peptide-oligonucleotide conjugates (POCs). Here, we report an effective and reproducible approach to prepare POCs and other nucleic acid conjugates based on a newly developed nucleic acid phosphoramidation method. The development of a new nucleic acid phosphoramidation reaction was achieved by our successful synthesis of a novel amine-containing biotin derivative used to systematically optimize the reactions. The improved phosphoramidation reactions dramatically increased yields of nucleic acid-biotin conjugates up to 80% after 3 h reaction. Any nucleic acids with a terminal phosphate group are suitable reactants in phosphoramidation reactions to conjugate with amine-containing molecules such as biotin and fluorescein derivatives, proteins, and, most importantly, peptides to enable the synthesis of POCs for therapeutic applications. Polymerase chain reactions (PCRs) to study incorporation of biotin or fluorescein-tagged DNA primers into the reaction products demonstrated that appropriate controls of nucleic acid phosphoramidation reactions incur minimum adverse effects on inherited base-pairing characteristics of nucleotides in nucleic acids. The phosphoramidation approach preserves the integrity of hybridization specificity in nucleic acids when preparing POCs. By retaining integrity of the nucleic acids, their effectiveness as therapeutic reagents for gene silencing, gene therapy, and RNA interference is ensured. The potential for POC use was demonstrated by two-step phosphoramidation reactions to successfully synthesize nucleic acid-tetraglycine conjugates. In addition, phosphoramidation reactions provided a facile approach to prepare nucleic acid-BSA conjugates with good yields. In summary, the new approach to phosphoramidation reactions offers a universal method to prepare POCs and other nucleic acid conjugates with high yields in aqueous solutions. The methods can be easily adapted to typical chemistry or biology laboratory setups which will expedite the applications of POCs for basic research and medicine.
核酸与大分子或小分子的化学偶联是研究化学和生物学中核酸以及将核酸应用于医学的常用方法。将寡核苷酸等核酸与肽偶联尤其有助于解决寡核苷酸作为治疗剂的细胞递药和特异性问题。然而，目前的方法在提供肽-寡核苷酸偶联物（POC）的能力方面存在局限性和效率低下。在这里，我们报告了一种基于新开发的核酸磷酰胺化方法制备 POC 和其他核酸偶联物的有效且可重复的方法。通过成功合成了一种新型含胺生物素衍生物，我们实现了核酸磷酰胺化反应的新发展，该衍生物用于系统优化反应。改进的磷酰胺化反应可将核酸-生物素偶联物的产率在 3 小时反应后提高至 80%。任何带有末端磷酸基团的核酸都是磷酰胺化反应的合适反应物，可以与含胺分子（如生物素和荧光素衍生物、蛋白质）偶联，最重要的是可以与肽偶联，从而合成用于治疗应用的 POC。聚合酶链反应（PCR）研究了生物素或荧光素标记的 DNA 引物掺入反应产物的情况，证明了对核酸磷酰胺化反应的适当控制对核酸中核苷酸的遗传碱基配对特性几乎没有不利影响。在制备 POC 时，磷酰胺化方法保持了核酸杂交特异性的完整性。通过保持核酸的完整性，确保了它们作为基因沉默、基因治疗和 RNA 干扰治疗试剂的有效性。通过两步磷酰胺化反应成功合成核酸-四肽偶联物，证明了 POC 的应用潜力。此外，磷酰胺化反应还提供了一种制备具有良好产率的核酸-BSA 偶联物的简便方法。总之，新的磷酰胺化反应方法为在水溶液中以高产率制备 POC 和其他核酸偶联物提供了一种通用方法。该方法可以很容易地适应典型的化学或生物学实验室设置，这将加速 POC 在基础研究和医学中的应用。
