苝/氟硼荧-TFO诊疗剂的设计合成及构效关系研究

One of the few existing therapeutic agents that able to regulate the expression of certain gene at transcription level is triplex forming oligo-nucleotides (TFOs). In this project we plan to synthesize a series of fluorophore (perylene and Bodipy) intercalated X-TFO strands, which were designed to specifically recognize and hybridize with selected dsDNA (TAOS1 sequence) in order to optically detect and therapeutically intervene the target gene. The X-TFO agents were linear, structurally and optically suitable fluorophores were linked to the strand via a D-threoninol spacer, which forms wedge-like intercalation between native base pairs meanwhile ensures the fluorescent signal was only switched-on upon hybridization with target duplex. Alternation of the fluorophore structure, numbers of introduced fluorophores and numbers of base pair intervals will be systematically studied for optimization of the X-TFOs theranostic properties. ..To the best of our knowledge, this prototype of X-TFO theranostic agents targeting dsDNA has not been reported yet. Once synthesized and optimized, this new X-TFO agent could achieve quantitate, multi-modality, non-invasive and dynamic monitoring and regulation of the target gene in sequence-specific manner. This highly-sensitive synthetically facile theranostic agent could be employed for early diagnosis and anti-gene therapy, and can apply to various gene targets at low cost, assisting the in-depth study of molecular mechanisms of disease and immune responses, shedding a new light on early diagnostics and personalized medicine. The X-TFO theranostic agent is promising applying in precision medicine research.

本课题拟研制系列以苝与氟硼荧基团修饰的三螺旋形成寡核苷酸X-TFO诊疗剂， 检测其对靶dsDNA序列的特异识别诊断作用与转录抑制治疗效果。根据项目组成员之前在食道癌方面的研究，从TAOS1基因筛选确定了标靶序列， 并设计了与之相应的X-TFO配列。结构适宜的荧光基团以D-苏氨醇连接支架导入配列中， 确保了X-TFO只在检测到标靶形成楔型嵌合时发出荧光讯号。本课题的一项重要研究内容是系统地设计调整所合成荧光化合物结构与导入配列的模式(荧光基团数目，荧光基团之间间隔的天然碱基数目)，综合分析诊疗剂构型对其灵敏度及抑制靶dsDNA转录效果的影响，以优化其诊疗效能。尽我们所知, 以苝-/氟硼荧-TFO进行光学标测并抑制致癌基因转录复制的研究尚未见报道。此诊疗剂具有序列特异识别的灵敏度，合成相对简单而低本高效，可望拓展于多种靶基因相关病变的诊治，转化精准医学应用前景可期。

1. 在光电相应分子探针与诊疗机能分子前药研究方面，申请人与团队根据合理设计，优化光物理性质和治疗活性，制备了一系列新型氟硼荧与Ir(III)Pt(II)络合物，含有氟化苯丙胺和丙烯酰胺辅助配体，成果分别发表在Applied Organometallic Chemistry (2019)，Inorganic Chemistry Communications (2018)。亚细胞分布成像结果和分子对接计算一致显示诊疗剂可靶向dsDNA分布于细胞核内。诊疗剂分子体系也具有细胞的微粘度和微极性成像探针的功能。在RT-CT综合疗法中诊疗剂放疗敏化SER值1.63~2.10，即使在放疗耐受细胞系中也观察到效果。此外，复合物已显示出抗病毒活性对HSV I型与II型病毒均具有细胞周期G2阻滞的抑制作用。此类分子元件可拓展应用作为XNA核酸适配体的人工碱基导入所合成的靶基因配列中，发挥机能达到精准基因检测与调控。本课题设计合成了一系列可靶向核酸的分子探针诊疗剂， 通过优化荧光化合物的结构从而提高与dsDNA的结合程度，筛选出了光物理信号响应优化的细胞微环境分子探针，在放疗增敏和抑制病毒核酸复制两方面进行了功能验证。.2. 在基于核酸适配体探针元件的核酸检测方面，申请人与团队对核酸元件与纸基界面桥联的反应装配方法具有研究基础，成果分别发表在Biotechnology & Bioengineering (2019)，ACS Applied Materials & Interfaces (2018)，Trends in Analytical Chemistry (2017)。结合XNA核酸适配体元件的新兴发展，阐述了使用核酸适配体作为信号传感元件的纸基即时检测最新发展，除信号传感器机制和方法外，还揭示了实际操作与效能的详细评估。.这些研究成果可协助合成化学，生物医学工程的研究人员和临床实践的医生在设计、制造和选择使用合适的核酸制剂和技术方法方面获得新的见解，响应国家卫健委的分级医疗与社区化介护的政策，“基层首诊接得住”的方针可由创新核酸制剂与分子体外检测方法研究切实推进。