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;它最简单的表达就是DNA - RNA -蛋白质.核糖核酸，将DNA中的基因组信息与蛋白质共同表达的表型联系起来。在大多数情况下，细胞中RNA的动态直接反映了蛋白质的表达，因此具有语音特征。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，基于珠的荧光激活分选和高通量测序。这些方法是基于分析足够量的RNA，通常来自细胞或组织的异质群体的集合。虽然这些信息在群体水平上描述transmittance是有用的，但通常缺乏关于组织中每种细胞类型或/和群体中单个细胞的重要信息。为了充分理解细胞对生理和病理线索的反应机制，显然还必须在单细胞和单分子水平上分析细胞中RNA的动力学。只有在单细胞水平上，我们才能开始理解单个细胞对相同刺激的不同反应，并准确地建立群体网络。只有在单分子水平上，我们才能以有意义的精确度感知细胞中RNA的动态。荧光显微镜是一种非侵入性、非破坏性的技术，能够在从单个分子、细胞、组织到人体的水平上成像。没有其他方法可以在活细胞中询问分子，其空间分辨率、灵敏度、选择性和动态性的组合远远接近。为了利用荧光成像技术在RNA检测中的潜力，我们提出开发用于RNA成像的新型能量转移纳米探针，其结合联合收割机金纳米颗粒（Au NPs）和荧光蛋白（FP）以实现活细胞中的灵敏的高分辨率原位RNA成像。由于在可见光波段的选择性发射，FP被广泛用于荧光显微镜，而Au NPs的光学性质强烈地依赖于它们的形状和可调节的物理特征。表面等离子体增强的局域场对附近荧光团的影响使得利用从短距离金属诱导猝灭到远距离金属增强荧光的丰富物理过程成为可能。最近，我们发现了表面等离子体增强的共振能量转移之间的Au纳米棒（NR）和DAPI，一种常用的DNA染料，在双光子激发下，在近红外范围。一旦FP和Au NPs的光学性质很好地匹配，增强的能量转移和双光子成像可以显著增加信噪比，导致高分辨率、较少的光损伤和深穿透的灵敏成像。所提出的纳米探针充分利用了Au NPs的独特性质，其具有很高的淬灭效率、增加的淬灭距离（特别有利于多RNA检测）、光稳定性、生物相容性以及在不使用转染剂的情况下进入细胞的能力。此外，双光子发光使它们本身成为生物成像中的优秀荧光探针，这对于成像时间和特殊的细胞内运输是理想的。近十年来对Au纳米粒子的深入研究表明，它们在成像、传感、药物输送和热治疗等领域具有巨大的应用潜力。本文提出的能量转移纳米探针将为进一步整合多重传感和治疗提供一个新的平台。

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.