A new approach for imaging RNA at the single cell level

分子生物学的中心法则支配着地球上的生命；它最简单的表达是DNA - RNA -蛋白质。RNA，核糖核酸，在DNA中隐藏的基因组信息与蛋白质共同表达的表型之间架起了桥梁。在大多数情况下，细胞内RNA的动态直接反射蛋白质的表达，因此具有音型特性。到目前为止，已经开发了多种方法，包括基因芯片微阵列，实时PCR，基于珠状荧光激活分选和高通量测序。这些方法是基于对足够数量的RNA的分析，通常来自异质细胞或组织的集合。虽然这些信息在描述群体水平上的转录是有用的，但关于组织中每种细胞类型或/和群体中的单个细胞的重要信息往往缺乏。为了充分了解细胞对生理和病理信号的反应机制，很明显，细胞中RNA的动力学也必须在单细胞和单分子水平上进行分析。只有在单个细胞水平上，我们才能开始理解单个细胞对相同刺激的不同反应，并准确地建立种群网络。只有在单分子水平上，我们才能有意义地精确地感知细胞中RNA的动态。荧光显微镜是一种非侵入性、非破坏性的技术，能够从单个分子、细胞、组织到人的各个层面进行成像。在空间分辨率、灵敏度、选择性和动力学方面，没有任何其他方法能比它更接近活细胞中的分子。为了利用荧光成像技术在RNA检测中的潜力，我们提出开发一种新的能量转移纳米探针用于RNA成像，该探针结合了金纳米颗粒（Au NPs）和荧光蛋白（FPs），以实现活细胞中敏感的高分辨率原位RNA成像。由于在可见光波段的选择性发射，FPs广泛应用于荧光显微镜，而Au NPs的光学性质强烈依赖于它们的形状和可调谐的物理特征。表面等离子体增强局部场对附近荧光团的影响，使得从短距离金属诱导猝灭到远距离金属增强荧光等丰富的物理过程得以开发。最近，我们发现在近红外双光子激发下，表面等离子体增强了金纳米棒（NRs）与DAPI（一种常用的DNA染色剂）之间的共振能量转移。一旦FPs和Au NPs的光学性质匹配良好，增强的能量传递和双光子成像，可以显著提高信噪比，从而实现高分辨率、低光损伤和深穿透的敏感成像。所提出的纳米探针充分利用了Au NPs的独特特性，具有很高的猝灭效率、较长的猝灭距离（尤其有利于多rna检测）、光稳定性、生物相容性以及无需转染剂即可进入细胞的能力。此外，双光子发光特性使其成为生物成像中极好的荧光探针，是成像时间和特殊细胞内运输的理想选择。近十年来，对Au NPs的深入研究表明，它们在成像、传感、药物传递和热治疗等领域具有广泛的应用潜力。本文提出的能量转移纳米探针将为多重传感和治疗的进一步整合提供一个新的平台。

The central dogma of molecular biology governs life on earth; its simplest expression is DNA - RNA - protein. RNA, Ribonucleic acid, bridges genome information harboured within DNA to phenotypes collectively expressed by protein. In most cases, the dynamics of RNA in the cell directly reflexes the expression of protein, hence the phonotypical properties. So far, a variety of methods have been developed, including gene-chip microarrays, real-time PCR, bead-based fluorescence-activated sorting and high-throughput sequencing. These methods are based on analysis of sufficient quantity of RNA, often, from a collection of heterogeneous population of cells or tissues. Although such information is useful in describing transcriptions at the population level, important information on each cell type in a tissue or/and single cell in a population are often lacking. To fully understand the mechanisms that cells respond to physiological and pathological cues, it is evident that the dynamics of RNA in the cell must also be analysed at the single cell and single molecule levels. Only at the single cell level we can start to understand differential response of individual cells to the same stimulus, and to accurately build up the network of the population. Only at the single molecule level, can we sense the dynamics of RNA in the cell to a meaningful precision.Fluorescence microscopy is a non-invasive, non-destructive technique, capable of imaging at levels from a single molecule, cell, tissue, to a man. No other method can interrogate molecules in living cells with anything remotely approaching its combination of spatial resolution, sensitivity, selectivity and dynamics. To exploit the potential of fluorescence imaging technique in RNA detection, we propose to develop novel energy transfer nanoprobes for RNA imaging that combine gold nanoparticles (Au NPs) and fluorescent proteins (FPs) to enable sensitive high resolution in situ RNA imaging in living cells. FPs are widely used in fluorescence microscopy due to the selective emission over visible band, whereas optical property of Au NPs strongly depends on their shape and physical features that can be tuned. The influence of surface plasmon enhanced local field on fluorophores nearby make it possible to exploit rich physical processes from metal induced quenching at a short separation to metal enhanced fluorescence in distance separation. Recently, we found surface plasmon enhanced resonance energy transfer between Au nanorods (NRs) and DAPI, a commonly used DNA stain, under two-photon excitation in the near infrared range. Once both the optical properties of FPs and Au NPs are well matched, enhanced energy transfer and two-photon imaging, could significantly increase signal/noise ratio, leading to sensitive imaging of high resolution, less photo damage and deep penetration. The proposed nanoprobe takes full advantage of the unique properties of Au NPs, which possess great quenching efficiency, increased quenching distance (especially beneficial for multi-RNA detection), photostable, biocompatible, and the ability to enter cells without the use of transfection agents. Moreover, two-photon luminescence makes them excellent fluorescence probes in biological imaging on its own, which is ideal for imaging temporal and special intracellular trafficking. Intensive research on Au NPs in the last decade has demonstrated their great potential in broad applications including imaging, sensing, drug delivery and thermal therapy. The energy transfer nanoprobe proposed here will provide a new platform for further integration of multiplex sensing and therapeutics.