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Molecular engineering of nucleic acid probes for intracellular imaging and bioanalysis.

机译：用于细胞内成像和生物分析的核酸探针的分子工程。

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摘要

The ability to monitor biological processes in the context of living cells with good spatial and temporal resolution offers significant potential for understanding many biological problems. The key to the successful signaling of these processes is the use of molecular probes. Currently, there are limitations for intracellular probes, which include low sensitivity, reduced selectivity, and poor stability. The major goal of this research is to integrate molecular engineering techniques with new signaling materials and approaches to design more sensitive and effective nucleic acid probes.; In an attempt to develop sensitive molecular probes, novel materials were explored for signal amplification and background reduction. Conjugated polymers (CPs) are good candidates for signal amplification because of their excellent light harvesting and superquenching properties. Using a solid phase synthesis method, CP labeled molecular beacons (MBs) were prepared. In addition, superquenchers, a series of macromolecules with exceptional quenching capabilities, were generated through a molecular assembling approach and used for labeling MBs.; To overcome the problem of autofluorescence from biological fluids, a new signaling approach called the excimer light switching signaling technique was developed. An aptamer that selectively binds to platelet derived growth factor (PDGF) was labeled with pyrene molecules on both ends, resulting in a light-switching aptamer. The probe changes its emission from a blue monomer emission to a green excimer emission upon binding to PDGF. Taking advantage of the long fluorescence lifetime of pyrene, time-gated measurements were performed to eliminate biological background signals.; Finally, the stability and false signals of nucleic acid probes were addressed. When used inside cells, normal nucleic acid probes are prone to enzymatic digestion, protein binding, and elicitation of RNase H action, all of which lead to nonspecific signals. The possibility of alleviating these issues by using locked nucleic acid (LNA) bases in the molecular probe design was investigated. With different stem lengths and LNA base ratios, LNA-MBs were designed and their thermodynamic properties, hybridization kinetics, enzymatic resistance, as well as interactions with DNA binding proteins, were studied. In addition to using base modification, we designed a new type of molecular probes called hybrid molecular probe, which does not generate false signal upon digestion by nuclease, binding to SSB. HMP is capable of selectively detecting targets from cellular samples.; The new materials, synthesis methods, and signaling techniques developed in this research have the potential for developing sensitive and effective molecular probes for bioanalysis and intracellular imaging. Future endeavors will include the application of these probes to single living cell gene expression studies.
机译：以良好的时空分辨率在活细胞中监测生物过程的能力为理解许多生物学问题提供了巨大的潜力。这些过程成功发出信号的关键是使用分子探针。当前，细胞内探针存在局限性，包括灵敏度低，选择性降低和稳定性差。该研究的主要目的是将分子工程技术与新的信号材料和方法相结合，以设计更灵敏有效的核酸探针。为了开发敏感的分子探针，探索了用于信号放大和背景降低的新型材料。共轭聚合物（CPs）具有出色的光收集和超猝灭特性，因此是信号放大的良好候选者。使用固相合成方法，制备了CP标记的分子信标（MBs）。此外，通过分子组装方法产生了具有超强淬灭能力的一系列大分子超猝灭剂，并用于标记MB。为了克服生物液体中自发荧光的问题，开发了一种新的信号传导方法，称为准分子光开关信号传导技术。选择性结合血小板衍生生长因子（PDGF）的适体在两端均被pyr分子标记，从而产生了光开关适体。探针与PDGF结合后，其发射将从蓝色单体发射变为绿色准分子发射。利用of的长荧光寿命，进行时间门控测量以消除生物学背景信号。最后，解决了核酸探针的稳定性和错误信号。当在细胞内使用时，正常的核酸探针易于酶消化，蛋白质结合和引起RNase H的作用，所有这些都会导致非特异性信号。研究了在分子探针设计中使用锁定核酸（LNA）碱基缓解这些问题的可能性。设计了具有不同茎长和LNA碱基比的LNA-MB，并研究了它们的热力学性质，杂交动力学，酶抗性以及与DNA结合蛋白的相互作用。除了使用碱基修饰外，我们还设计了一种称为杂交分子探针的新型分子探针，该探针在被核酸酶消化后不会与SSB结合产生假信号。 HMP能够从细胞样本中选择性检测靶标。这项研究中开发的新材料，合成方法和信号技术具有开发灵敏有效的分子探针进行生物分析和细胞内成像的潜力。未来的工作将包括将这些探针应用于单个活细胞基因表达研究。

著录项

  • 作者

    Yang, Chaoyong James.;

  • 作者单位

    University of Florida.;

  • 授予单位 University of Florida.;
  • 学科 Biology Molecular.; Chemistry Analytical.; Health Sciences Oncology.
  • 学位 Ph.D.
  • 年度 2006
  • 页码 202 p.
  • 总页数 202
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;化学;肿瘤学;
  • 关键词

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