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物。 我的NSERC资助的研究计划是关于设计和合成的结构修饰的DNA寡核苷酸，含有荧光碱基类似物。 这样的寡核苷酸被用来扩大我们的理解介导的庞大的DNA加合物（加成产物）的致突变性，并作为新的生物分子的传感应用工具。 该资助的第1部分重点关注由人类致癌物产生的大体积DNA加合物的结构和荧光特性。 具体地，连接到2 '-脱氧鸟苷（dG）的C8-位点的大体积芳基产生C8-芳基-dG损伤，这是常见的DNA加合物类型。 对于这些病变，加合物构象可以决定致突变效力。 新型C8-芳基-dG加合物被设计成具有荧光性质，其可以用作DNA双链体内的加合物构象的灵敏指示剂，并且当加合物被DNA聚合酶加工时。 本项目的短期目标是将它们的荧光与加合物构象和体外致突变性联系起来。 长期目标是能够利用细胞DNA内的荧光C8-芳基-dG加合物，以了解加合物构象如何影响细胞过程，如DNA复制和DNA修复。 适体是以高亲和力和特异性结合多种靶标的单链寡核苷酸。 掺入DNA适体中的染料被设计成对目标结合具有发射灵敏度，这可用作诊断信号。 该项目的短期目标是制定将荧光染料定点掺入DNA适体的策略，用于检测食物毒素和蛋白质，作为建立食品安全和检测与疾病相关的生物标志物的概念验证。 这涉及优化：染料稳定性，位点特异性发射响应和检测限。 该项目的长期目标是开发各种荧光适体传感器，以纳入负担得起的、易于使用的手持式检测试剂盒，用于护理点诊断。 概述的工作是由一群才华横溢的本科生和研究生进行的，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所得修饰DNA的分子识别特性。 学生们不仅接受本申请的主要研究者的指导，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.