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

蛋白质-DNA相互作用（pdi）是调控许多细胞过程的关键，包括转录、复制和DNA修复。传统上，pdi的特征是基于电泳迁移位移测定和核酸酶足迹等方法。最近，基于微阵列的方法已经出现，允许快速，高通量表征体外dna结合特异性。然而，很少有方法可以准确地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。然而，它有很多缺点，包括由于质量传输影响动力学分析的局限性，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，廉价和高灵敏度的传感器，用于实时监测pdi。我们将描述选定的蛋白质和DNA（或RNA）目标之间的相互作用，这些目标固定在窃窃私语画廊模式（WGM）谐振器上。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采集，无标签，可以使用CMOS处理方法进行，非专业人员也可以使用（这意味着它可以用于即时诊断应用）。WGM传感的工作机制很简单：本质上，介质光学微腔中的共振模式可以用来检测周围局部环境的微小变化。分析物与腔体的结合，或者甚至已经结合的分析物构象的改变，将导致共振波长的移位。重要的实时信息，如结合、解离、折叠和展开轨迹可以很容易地获得，使其成为一种非常有价值的技术。在本博士学位的范围内，我们将专注于自然发生的蛋白质和端粒末端发现的四链g -四重结构（或G4）之间的生物学相关的PDIs，也广泛存在于人类基因启动子中。分子内G4s的形成首次被提出发生在端粒的3'端，从而阻止端粒酶在肿瘤细胞中维持端粒。随后，趋同的生物信息学研究表明，假定的g -四重体形成序列在整个人类基因组中非常普遍，在转录起始位点（TSS）上游1kb内的非翻译区域高度富集。癌症、脆性X染色体综合征、Bloom综合征和Werner综合征等严重疾病都与涉及g -四重体形成序列的基因组缺陷有关。因此，天然蛋白对g -四重体的识别和加工（如稳定或解绕）是调节生理或病理途径的关键靶点。文献中已经出现了大量以结构特异性方式结合G4s的小分子，并在体外和体内均显示出生物活性。然而，人们对这些蛋白质识别G4s的方式知之甚少。虽然大多数靶向g4的治疗性小分子是根据其结合和/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，用于实时和中高通量监测蛋白质与端粒或基因启动子中自然发生的DNA和RNA G4s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，从而通过抑制癌细胞中的端粒酶活性或对原癌基因的特定转录调节，具有巨大的治疗（例如抗癌）潜力。

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.