Biosensing Properties of Fluorescent DNA Base Analogs

化学修饰的DNA碱基可以促进导致严重健康影响(如癌症)的突变，或者产生有用的荧光衍生物，这些衍生物是表征生物分子相互作用的理想候选者。我的NSERC资助的研究项目是关于设计和合成含有荧光碱基类似物的结构修饰的DNA寡核苷酸。这些寡核苷酸被用来扩大我们对大体积DNA加合物(加成产物)介导的突变性的理解，并作为新的生物分子工具用于传感应用。 这笔赠款的第一部分集中于由人类致癌物质产生的大量DNA加合物的结构和荧光特性。具体地说，2‘-脱氧鸟苷(DG)的C8位上连接的大量芳基会产生C8-芳基-DG损伤，这是一种常见的DNA加合物类型。对于这些病变，加合物构象可以决定诱变能力。新型C8-芳基-DG加合物具有荧光性质，可作为DNA双链内加合物构象的灵敏指示剂，以及当加合物被DNA聚合酶处理时。这个项目的短期目标是将它们的荧光与加合物构象和体外诱变性联系起来。长期目标是能够利用细胞DNA中的荧光C8-芳基-DG加合物来了解加合物构象如何影响细胞过程，如DNA复制和DNA修复。 这项资助的第二部分研究了DNA适配子中可见荧光染料的生物传感特性。适配子是一种单链寡核苷酸，具有高度的亲和力和特异性，能与多种靶标结合。掺入DNA适配子的染料被设计成对目标结合具有发射敏感性，这是一个诊断信号。该项目的短期目标是建立将荧光染料结合到DNA适配子中的策略，用于检测食品毒素和蛋白质，作为建立食品安全和检测与疾病相关的生物标记物的概念验证。这涉及到：染料稳定性、特定部位的发射响应和检测极限的优化。该项目的长期目标是开发各种荧光适配传感器，以集成到负担得起、易于使用的手持式检测试剂盒中，用于医疗保健诊断。 概述的工作是由一群有才华的本科生和研究生完成的，他们学习核酸合成的基础知识，以及如何使用各种仪器技术表征得到的修饰DNA的分子识别特性。这些学生不仅接受这项申请的首席研究员的指导，还接受两名具有核酸化学和染料合成专业知识的博士后研究员的指导。

Chemically modified DNA bases can facilitate mutations that cause serious health effects such as cancer, or produce useful fluorescent derivatives that are ideal candidates for characterizing biomolecular interactions. My NSERC-funded research program is concerned with the design and synthesis of structurally modified DNA oligonucleotides that contain fluorescent base analogs. Such oligonucleotides are utilized to expand our understanding of mutagenicity mediated by bulky DNA adducts (addition products) and serve as new biomolecular tools for sensing applications. Part 1 of this grant focuses on the structural and fluorescent properties of bulky DNA adducts that are produced by human carcinogens. Specifically, bulky aryl groups attached to the C8-site of 2'-deoxyguanosine (dG) produce C8-aryl-dG lesions, which is a common DNA adduct type. For these lesions, adduct conformation can dictate mutagenic potency. Novel C8-aryl-dG adducts are designed to possess fluorescent properties that can serve as a sensitive indicator of adduct conformation within the DNA duplex and when the adduct is processed by DNA polymerases. The short-term goal of this project is relate their fluorescence to adduct conformation and in vitro mutagenicity. The long-term goal is to be able to utilize fluorescent C8-aryl-dG adducts within cellular DNA to gain an understanding of how adduct conformation impacts cellular processes, such as DNA replication and DNA repair. Part 2 of this grant examines the biosensing properties of visible fluorescent dyes within DNA aptamers. Aptamers are single-stranded oligonucleotides that bind a variety of targets with high affinity and specificity. The dyes incorporated into the DNA aptamers are designed to possess emission sensitivity to target binding, which serves as a diagnostic signal. The short-term goal of this project is to establish strategies for incorporation of fluorescent dyes site-specifically into DNA aptamers for detection of food toxins and proteins as a proof-of-concept for establishing food safety and detecting biomarkers related to disease. This involves optimization of: dye stability, site-specific emission response and detection limits. The long-term goal of this project is the development of various fluorescent aptasensors for incorporation into affordable, easy-to-use hand-held detection kits for point-of-care diagnostics. The work outlined is carried out by a talented group of undergraduate and graduate students who learn the basics of nucleic acid synthesis and how to characterize the molecular recognition properties of the resulting modified DNAs using a wide variety of instrumental techniques. The students receive guidance not only from the principal investigator of this application, but also from two postdoctoral fellows with expertise in nucleic acid chemistry and dye synthesis.