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（PDIs）对于调节包括转录、复制和DNA修复在内的许多细胞过程至关重要。PDI传统上基于包括电泳迁移率变动测定和核酸酶足迹法的方法来表征。最近，出现了基于微阵列的方法，允许快速，高通量表征体外DNA结合特异性。然而，更少的方法可用于精确测量相互作用亲和力。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，它遭受了大量的缺点，包括由于影响动力学分析的质量传递的限制，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，廉价和高灵敏度的传感器，用于监测真实的时间的PDI。我们将研究固定在回音壁模式（WGM）谐振器上的选定蛋白质和DNA（或RNA）靶之间的相互作用。WGM传感提供了比传统技术更有吸引力的前景。WGM传感器的优势包括实时亚秒级采集、无标记、可以使用CMOS处理方法进行，以及非专家的可访问性（这意味着它可以用于即时诊断应用）。WGM传感的操作机制很简单：基本上可以使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的构象的变化，将导致共振波长的偏移。重要的是，可以很容易地获得实时信息，如缔合、解离、折叠和展开轨迹，这是一种非常有价值的技术。在这个博士学位的范围内，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-四链体结构（或G4）之间的生物相关PDI，并且在人类基因启动子中也很普遍。分子内G4的形成首先被提出发生在端粒的3 '端，从而阻止肿瘤细胞中端粒酶对端粒的维持。聚合生物信息学研究随后揭示了在人类基因组中推定的G-四链体形成序列的高流行率，在转录起始位点（TSS）上游1kb内的非翻译区中具有强烈富集。严重的疾病如癌症、脆性X综合征、布卢姆综合征和沃纳综合征与涉及G-四链体形成序列的基因组缺陷有关。由于这个原因，G-四链体识别和处理（例如稳定或解旋）的天然存在的蛋白质代表了一个关键的目标，以调节生理或病理途径。大量的小分子已经出现在文献中，其以结构特异性方式结合G4，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G4的方式知之甚少。虽然大多数G4靶向治疗性小分子基于其结合和/或稳定这些结构的能力进行评估，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，用于实时和中高通量地监测蛋白质与端粒或基因启动子中天然存在的DNA和RNA G4之间的相互作用。它还将递送可以干扰这些PDI的小分子药物，并且因此通过抑制癌细胞中的端粒酶活性或原癌基因的特异性转录调节而具有巨大的治疗（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.