Synthesis and investigation of modified oligonucleotides as probes of DNA repair

作为 DNA 修复探针的修饰寡核苷酸的合成和研究

• 批准号: 299384-2011
• 负责人: Wilds, Christopher
• 金额: $ 2.55万
• 依托单位: Concordia University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=594934
• 关键词: Synthesis; investigation; modified; oligonucleotides; probes

Interstrand cross-links (ICL) can be formed in DNA as a result of endogenous processes such as lipid peroxidation and represent some of the most toxic lesions encountered by cells because they obstruct unwinding of the two strands of DNA which is critical to the processes of replication, transcription and recombination. Interference with these events in the cell by ICLs can be exploited in cancer chemotherapy regimens that employ bifunctional alkylating agents. However, the potency of these agents is diminished by the ability of cancer cells to repair the same lesions these agents induce and this causes the onset of resistance. A number of DNA repair pathways exist in cells including direct repair, base-excision repair, nucleotide-excision repair (NER), homologous recombination (HR), nonhomologous end joining and DNA-mismatch repair. Repair pathways such as NER, HR and translesion DNA synthesis (TLS) have all been implicated in ICL repair in mammalian cells, however, there is an increasing realization that ICL processing may depend on the nature of the lesion (atoms attached, linker length and orientation) making it challenging to generalize how specific ICLs are repaired. A detailed understanding of the processes by which DNA damage are recognized and repaired may lead to the design of more effective alkylating therapeutics that induce lesions that can evade repair. Our group has developed methods using a combination of solution and solid-phase synthesis to prepare sufficient quantities of ICL containing oligonucleotides for biophysical, structural and repair studies. These studies will assess whether such lesions induce a structural change in DNA that may play a role in recognition by repair enzymes. In addition, these ICL DNA will be used in experiments with DNA repair proteins to observe whether these ICL are either susceptible or evade repair processes. Correlations made between structure and repair susceptibility will aid our understanding of the processing of these therapeutically relevant lesions and will contribute to the field of DNA repair.

链间交联（ICL）可以在DNA中形成，作为内源性过程的结果，如脂质过氧化，并且代表了细胞遇到的一些最具毒性的病变，因为它们阻碍了对复制，转录和重组过程至关重要的两条DNA链的解绕。icl对细胞中这些事件的干扰可以在使用双功能烷基化剂的癌症化疗方案中利用。然而，这些药物的效力被癌细胞修复这些药物诱导的相同病变的能力所削弱，这导致了耐药性的发生。细胞中存在多种DNA修复途径，包括直接修复、碱基切除修复、核苷酸切除修复（NER）、同源重组（HR）、非同源末端连接和DNA错配修复。修复途径如NER、HR和翻译DNA合成（TLS）都与哺乳动物细胞的ICL修复有关，然而，越来越多的人认识到ICL的加工可能取决于病变的性质（附着的原子、连接体的长度和方向），因此很难概括出特异性ICL是如何修复的。对DNA损伤被识别和修复过程的详细了解可能会导致设计出更有效的烷基化疗法，从而诱导可以逃避修复的病变。我们的团队已经开发出使用溶液和固相合成相结合的方法来制备足够数量的含有寡核苷酸的ICL，用于生物物理、结构和修复研究。这些研究将评估这些损伤是否会引起DNA的结构变化，这种变化可能在修复酶的识别中起作用。此外，这些ICL DNA将用于DNA修复蛋白的实验中，以观察这些ICL是否易受影响或逃避修复过程。结构和修复易感性之间的相关性将有助于我们理解这些治疗相关病变的处理过程，并将为DNA修复领域做出贡献。