Swenson Colin S, Argueta-Gonzalez Hector S, Sterling Sierra A, Robichaux Ryan, Knutson Steve D, Heemstra Jennifer M
Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States.
Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States.
ACS Omega. 2022 Dec 22;8(1):238-248. doi: 10.1021/acsomega.2c03568. eCollection 2023 Jan 10.
The deamination of adenosine to inosine is an important modification in nucleic acids that functionally recodes the identity of the nucleobase to a guanosine. Current methods to analyze and detect this single nucleotide change, such as sequencing and PCR, typically require time-consuming or costly procedures. Alternatively, fluorescent "turn-on" probes that result in signal enhancement in the presence of target are useful tools for real-time detection and monitoring of nucleic acid modification. Here we describe forced-intercalation PNA (FIT-PNA) probes that are designed to bind to inosine-containing nucleic acids and use thiazole orange (TO), 4-dimethylamino-naphthalimide (4DMN), and malachite green (MG) fluorogenic dyes to detect A-to-I editing events. We show that incorporation of the dye as a surrogate base negatively affects the duplex stability but does not abolish binding to targets. We then determined that the identity of the adjacent nucleobase and temperature affect the overall signal and fluorescence enhancement in the presence of inosine, achieving an 11-fold increase, with a limit of detection (LOD) of 30 pM. We determine that TO and 4DMN probes are viable candidates to enable selective inosine detection for biological applications.
腺苷脱氨生成肌苷是核酸中的一种重要修饰，它在功能上可将核碱基的身份重新编码为鸟苷。目前用于分析和检测这种单核苷酸变化的方法，如测序和聚合酶链反应（PCR），通常需要耗时或昂贵的程序。另外，在靶标存在时会导致信号增强的荧光“开启”探针是用于实时检测和监测核酸修饰的有用工具。在此，我们描述了强制插入肽核酸（FIT-PNA）探针，其设计用于与含肌苷的核酸结合，并使用噻唑橙（TO）、4-二甲基氨基萘二甲酰亚胺（4DMN）和孔雀石绿（MG）荧光染料来检测A到I的编辑事件。我们表明，将染料作为替代碱基掺入对双链体稳定性有负面影响，但不会消除与靶标的结合。然后我们确定，相邻核碱基的身份和温度会影响在肌苷存在下的整体信号和荧光增强，实现了11倍的增加，检测限（LOD）为30 pM。我们确定TO和4DMN探针是用于生物应用中实现选择性肌苷检测的可行候选物。
