Real-time monitoring of interactions between naturally occurring proteins and DNA (or RNA) quadruplexes using whispering gallery mode resonators.

使用回音壁模式谐振器实时监测天然蛋白质和 DNA（或 RNA）四链体之间的相互作用。

基本信息

批准号：
1649726
负责人：
金额：
--
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1649726
关键词：
Real time monitoring interactions between

项目摘要

Protein-DNA interactions (PDIs) are critical for regulating many cellular processes including transcription, replication and DNA repair. PDIs are traditionally characterised based on methods including electrophoretic mobility-shift assays and nuclease footprinting. More recently, microarray-based approaches have appeared allowing for rapid, high-throughput characterisation of in vitro DNA-binding specificities. However, fewer methods are available to accurately measure interaction affinities. Among them, Surface Plasmon Resonance (SPR) technology remains the gold standard in direct biomolecular interaction sensing. It suffers, however, from a large number of drawbacks including limitations due to mass transport affecting kinetic analysis, non-specific interactions of the ligands with the matrix or elevated running cost. Herein, we propose to develop an easy-to-use, cheap and highly sensitive sensor for monitoring PDIs in real time. We will characterise the interactions between selected proteins and DNA (or RNA) targets immobilised on a whispering gallery mode (WGM) resonator. WGM sensing offers attractive prospects over more conventional technologies. Advantages of WGM sensors include real-time sub-second acquisitions, label-free, can be made using CMOS processing methods and accessibility to a non-expert (which implies that it can be used for point-of-care diagnostic applications). The operating mechanism of WGM sensing is simple: essentially resonance modes in dielectric optical micro-cavities can be used to detect minute changes in the surrounding local environment. Binding of an analyte to the cavity, or even a change in conformation of analyte already bound, will result in a shift in the resonance wavelength. Importantly real-time information such as association, dissociation, folding, and unfolding trajectories can easily be obtained making this an exceptionally valuable technique. Within the scope of this PhD, we will be focusing on biologically relevant PDIs between naturally occurring proteins and four-stranded G-quadruplex structures (or G4) found at the end of telomeres and also widespread in human gene promoters. Formation of intramolecular G4s was first proposed to occur at the 3'-end of telomeres, thus preventing telomere maintenance by the enzyme telomerase in tumour cells. Convergent bioinformatics studies subsequently revealed the high prevalence of putative G-quadruplex forming sequences across the human genome, with a strong enrichment in untranslated regions, within 1kb upstream of the transcription start site (TSS). Severe conditions like cancer, fragile X syndrome, Bloom syndrome and Werner syndrome are related to genomic defects that involve G-quadruplex forming sequences. For this reason, G-quadruplex recognition and processing (e.g. stabilisation or unwinding) by naturally occurring proteins represent a key target to modulate physiological or pathological pathways. A large number of small molecules have appeared in the literature that bind G4s in a structure-specific manner and show biological activity both in vitro and in vivo. However, very little is known about the way these proteins recognise G4s. Whilst most G4-targeting therapeutic small molecules are assessed based on their ability to bind and/or stabilise these structures, little is known about their ability to interfere with these structure-specific PDIs. This multidisciplinary project will therefore deliver valuable tools for monitoring, in real-time and with medium- to high-throughput, the interaction between proteins and naturally occurring DNA and RNA G4s at the telomeres or in gene promoters. It will also deliver small molecules drugs that can interfere with these PDIs and as a consequence have great therapeutic (e.g. anti-cancer) potential via either inhibition of telomerase activity in cancer cells or specific transcriptional regulation of proto-oncogenes.

蛋白质-DNA 相互作用 (PDI) 对于调节许多细胞过程（包括转录、复制和 DNA 修复）至关重要。 PDI 的传统表征方法包括电泳迁移率变化分析和核酸酶足迹分析。最近，出现了基于微阵列的方法，可以快速、高通量地表征体外 DNA 结合特异性。然而，可用于准确测量交互亲和力的方法较少。其中，表面等离子共振（SPR）技术仍然是直接生物分子相互作用传感的黄金标准。然而，它存在许多缺点，包括由于影响动力学分析的传质而产生的限制、配体与基质的非特异性相互作用或运行成本升高。在此，我们建议开发一种易于使用、廉价且高灵敏度的传感器来实时监测 PDI。我们将表征选定的蛋白质与固定在回音壁模式 (WGM) 谐振器上的 DNA（或 RNA）目标之间的相互作用。与更传统的技术相比，WGM 传感具有更有吸引力的前景。 WGM 传感器的优点包括实时亚秒级采集、无标签、可以使用 CMOS 处理方法进行，并且非专家也可以使用（这意味着它可以用于护理点诊断应用）。 WGM 传感的工作机制很简单：本质上可以利用介电光学微腔中的谐振模式来检测周围局部环境的微小变化。分析物与腔的结合，或者甚至已经结合的分析物构象的变化，将导致共振波长的变化。重要的是，可以轻松获得诸如关联、解离、折叠和展开轨迹等实时信息，这使得这是一项非常有价值的技术。在本博士研究范围内，我们将重点研究天然存在的蛋白质和端粒末端发现的、也广泛存在于人类基因启动子中的四链 G-四链体结构（或 G4）之间的生物学相关 PDI。首先提出分子内 G4 的形成发生在端粒的 3' 端，从而阻止肿瘤细胞中端粒酶对端粒的维持。随后的收敛生物信息学研究揭示了人类基因组中假定的 G-四链体形成序列的高流行性，并且在转录起始位点 (TSS) 上游 1kb 内的非翻译区域中高度富集。癌症、脆性 X 综合征、布卢姆综合征和沃纳综合征等严重疾病与涉及 G-四链体形成序列的基因组缺陷有关。因此，天然存在的蛋白质对 G-四链体的识别和处理（例如稳定或解旋）代表了调节生理或病理途径的关键目标。文献中已经出现了大量以结构特异性方式结合G4并在体外和体内表现出生物活性的小分子。然而，人们对这些蛋白质识别 G4 的方式知之甚少。虽然大多数 G4 靶向治疗小分子是根据其结合和/或稳定这些结构的能力进行评估的，但对其干扰这些结构特异性 PDI 的能力知之甚少。因此，这个多学科项目将提供有价值的工具，以中高通量实时监测端粒或基因启动子中蛋白质与天然存在的 DNA 和 RNA G4 之间的相互作用。它还将提供可以干扰这些 PDI 的小分子药物，因此通过抑制癌细胞中的端粒酶活性或对原癌基因进行特异性转录调节，具有巨大的治疗（例如抗癌）潜力。