A new approach for imaging RNA at the single cell level

Project: Research

Description

"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."

Key findings

We have successfully developed a new type of mRNA nanoprobe that is based on functionalized gold nanorod with single strand DNA in a hairpin structure in conjunction with a fluorescence label for mRNA biomarker detection at single cell level.
StatusFinished
Effective start/end date29/07/1328/01/15

Funding

  • BBSRC (Biotech & Biological Sciences Research Council): £119,257.00

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RNA
Imaging techniques
Nanoprobes
Fluorescence
Energy transfer
Quenching
Proteins
Molecules
Photons
Fluorescence microscopy
DNA
Cells
Tissue
Nanorods
Gold
Optical properties
Genes
Metals
Messenger RNA
Molecular biology