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-DNA相互作用（PDI）对于调节许多细胞过程至关重要，包括转录，复制和DNA修复。传统上，PDI是根据包括电泳迁移率转移测定和核酸酶足迹的方法来表征的。最近，似乎基于微阵列的方法似乎可以快速，高通量表征体外DNA结合特异性。但是，更少的方法可以准确测量相互作用的亲和力。其中，表面等离子体共振（SPR）技术仍然是直接生物分子相互作用传感的金标准。但是，它遭受了许多缺点，包括由于影响动力学分析的大规模运输，配体与基质的非特异性相互作用或升高的运行成本的限制。在此，我们建议开发一个易于使用，便宜且高度敏感的传感器，以实时监视PDI。我们将表征所选蛋白与DNA（或RNA）靶标之间的相互作用，该靶标在耳语画廊模式（WGM）谐振器上固定。 WGM传感为更多传统技术提供了有吸引力的前景。 WGM传感器的优点包括可以使用CMOS处理方法和对非专家的可访问性制成的实时次秒采集，无标签（这意味着它可以用于护理点诊断应用程序）。 WGM传感的工作机制很简单：基本上是介电光学微型腔中的共振模式，可用于检测周围本地环境的微小变化。分析物与空腔的结合，甚至已经结合的分析物的构象变化，都会导致共振波长的变化。重要的是，可以轻松获得相关，解离，折叠和展开轨迹等实时信息，从而使其成为一种非常有价值的技术。在该博士学位的范围内，我们将重点关注自然存在的蛋白质与四链的G四链体结构（或G4）之间的生物学相关PDI（或G4），并且在人类基因促进剂中也广泛。首先提出分子内G4S的形成是在端粒的3末端发生，从而阻止肿瘤细胞中酶端粒酶维持端粒。随后，收敛的生物信息学研究揭示了整个人类基因组推定的G四链体形成序列的高流行率，在未翻译的区域中，在转录起始位点上游（TSS）的1KB中具有很强的富集。严重的疾病，例如癌症，脆弱的X综合征，Bloom综合征和Werner综合征与涉及G Qu-四链体形成序列的基因组缺陷有关。因此，天然存在的蛋白质是调节生理或病理途径的关键靶标，G-四链体识别和加工（例如稳定或放松）。在文献中出现了许多小分子，这些分子以结构特异性的方式结合G4，并在体外和体内显示生物学活性。但是，这些蛋白质识别G4的方式知之甚少。尽管大多数靶向G4靶向的治疗性小分子是根据它们结合和/或稳定这些结构的能力来评估的，但对它们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，用于实时和中等直通，在端粒或基因启动子中，蛋白质与天然发生的DNA和RNA G4之间的相互作用。它还将提供小分子药物，这些药物可以干扰这些PDI，因此，通过抑制癌细胞中端粒酶活性或原始基因的特定转录调控，具有巨大的治疗性（例如抗癌）潜力。