Super-resolution imaging of RNA structures and processes

RNA 结构和过程的超分辨率成像

• 批准号: 2589738
• 金额: --
• 依托单位: University of St Andrews
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2589738
• 关键词: Super; resolution; imaging; RNA; structures

In addition to carrying genetic information, the flexibility and dynamics of RNA molecules allows them to fold into specific structures that dictate their fate and role in many crucial biological processes. Traditional structure determination methods such as X-ray, NMR or cryo-EM generate exquisitely resolved static 3D-structures, but they cannot be applied to study the structure of long and dynamic RNA sequences. The first step to determine the structure of long RNA molecules is to be able to discriminate stretches of single-stranded RNA from secondary and tertiary folded segments. Super-resolution optical imaging of long DNA sequences using fluorescence intercalators or fluorescently labelled DNA-binding proteins has been recently demonstrated as a tool to reveal the structure of the duplex DNA. In contrast, the application of super-resolution imaging of RNA sequences has been limited to detecting their presence, but not their structure, using in-situ hybridization or the incorporation of fluorescent aptamers within the RNA sequence. Current intercalator-based methods used commonly for DNA staining cannot discriminate between single- or duplex nucleic acid sequences, therefore they have limited application to image RNA structure.Our aim is to demonstrate super-resolution structural imaging of i) fully transcribed RNA sequences ii) and co-transcriptionally emerging RNA molecules, using a novel approach combining fluorescently labelled single-strand binding proteins (SSBs) and STED imaging. As part of our current BBSRC funding to investigate DNA repair pathways, we have identified a monomeric single-strand binding protein from an archaeal organism that binds single-strand RNA with ~1000-fold higher affinity (KD~ 4 nM) than duplex RNA (KD ~ 5 M). The 14 kDa SSB protein binds only four RNA nucleotides and can be tagged with indocarbocyanine derivatives that have been shown to behave as excellent probes to STED imaging. Importantly, single-molecule FRET microscopy has shown a fast binding and unbinding dynamics of the SSB proteins to the nucleic acid single stranded region and this can be further modulated by the ionic strength of the medium [Morten et al, 2017]. The stochastic binding of fluorescently labelled SSBs to the nucleic acid single strand sequences can be considered as a point accumulation for imaging nanoscale topography (PAINT) method that uses a protein-based reporter instead of a short DNA sequence (DNA-PAINT). Because the interaction of the SSB protein with the RNA is not sequence specific, SSB stochastic binding will light up the entire single-strand RNA sequence which is desirable for many applications. Thus, the combination of SSB-PAINT and STED will allow, for the first time, to discriminate entire single-strand RNA regions from duplex RNA segments, which is currently impossible, and monitor transcriptional processes with an unprecedented spatial resolution. We expect the fast exchange of labelled SSB monomers on the nucleic acid strand to minimize the impact of sample photobleaching by the STED depletion laser and thus increase resolution. SSB-PAINT will be an invaluable tool to researchers working in a wide range of RNA-related processes within BBSRC remit including transcription, RNA-protein interactions, splicing and the structure of viral RNAs, both in vitro and in vivo.

除了携带遗传信息外，RNA分子的灵活性和动态性使它们能够折叠成特定的结构，这些结构决定了它们在许多关键生物过程中的命运和作用。传统的结构测定方法，如x射线、核磁共振或冷冻电镜，可以生成精细分辨的静态3d结构，但它们不能用于研究长链和动态RNA序列的结构。确定长链RNA分子结构的第一步是能够从二级和三级折叠片段中区分单链RNA的延伸。利用荧光插层器或荧光标记的DNA结合蛋白对长DNA序列进行超分辨率光学成像，最近被证明是揭示双工DNA结构的一种工具。相比之下，RNA序列的超分辨率成像的应用仅限于检测它们的存在，而不是它们的结构，使用原位杂交或在RNA序列中掺入荧光适体。目前用于DNA染色的基于插入器的方法不能区分单核酸序列或双核酸序列，因此它们在图像RNA结构中的应用有限。我们的目标是利用一种结合荧光标记单链结合蛋白（SSBs）和STED成像的新方法，展示i)完全转录RNA序列ii)和共转录新兴RNA分子的超分辨率结构成像。作为我们目前BBSRC资助研究DNA修复途径的一部分，我们已经从一种古细菌生物中鉴定出一种单体单链结合蛋白，它与单链RNA的亲和力（KD~ 4 nM）比双链RNA （KD~ 5 M）高1000倍。14 kDa的SSB蛋白仅结合4个RNA核苷酸，可以用吲哚碳菁衍生物标记，这些衍生物已被证明是STED成像的优秀探针。重要的是，单分子FRET显微镜显示了SSB蛋白与核酸单链区域的快速结合和解结合动力学，这可以通过介质的离子强度进一步调节[Morten等，2017]。荧光标记的ssb与核酸单链序列的随机结合可以被认为是成像纳米尺度地形（PAINT）方法的点积累，该方法使用基于蛋白质的报告基因而不是短DNA序列（DNA-PAINT）。由于SSB蛋白与RNA的相互作用不是序列特异性的，SSB随机结合将照亮整个单链RNA序列，这在许多应用中是理想的。因此，SSB-PAINT和STED的结合将首次允许区分整个单链RNA区域和双链RNA片段，这是目前不可能的，并以前所未有的空间分辨率监测转录过程。我们期望标记的SSB单体在核酸链上的快速交换可以最大限度地减少STED耗尽激光对样品光漂白的影响，从而提高分辨率。SSB-PAINT将成为研究人员在体外和体内广泛研究BBSRC范围内rna相关过程的宝贵工具，包括转录、rna -蛋白相互作用、剪接和病毒rna结构。