A new approach for imaging RNA at the single cell level

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.

We propose to develop a new approach for RNA imaging at the single cell level through energy transfer imaging microscopy based on novel energy transfer nanoprobes. These nanoprobes utilize Au NPs and FPs linked with single stranded DNA (ssDNA). The ssDNA will be designed to form a hairpin structure of which the loops contain complementary sequences to the RNAs to be detected. In the absence of target RNAs, FP is proximal to the gold surface due to the hairpin DNA structure, and the fluorescence is quenched. In the presence of target RNA, the hairpin structures will be broken via RNA-DNA base pairing. As a result, FP and gold surface fall distant apart and fluorescence is recovered or even enhanced under a favourable conditions. The intensity and lifetime of the fluorescence from FPs will register the spatial and temporal dynamics of targeted RNA in the cell.
We will optimise an approach to syntheses FP-Au NP probes. We will modify the existing method to conjugate FPs with ssDNA through a cysteine residue, by genetic engineering, at the C-terminus of the FPs, covalently linked with amino-modified ssDNA. The FP-ssDNA will be immobilised to the Au NP surface by a ligand exchange process, which replace CTAB on Au NP with thiolated ligands that permit DNA conjugation.
We will examine the fluorescence coupling process between FP and Au NPs to optimise the separation distance, optical characteristics and functionalization. Nanoprobe structure is thus optimized to improve the sensitivity and selectivity, especially under two-photon excitation.
We will make use of two-photon luminescence of Au NPs and capability of single particle imaging to study the internalization of nanoprobes. Stability, as well as cell stress and toxicity will also be investigated. To demonstrate in-situ mRNA imaging at single cell level, we will initially evaluate our system by detecting tumour-related mRNAs, then study the expression of NFkB pathway in human macrophage-like U937 cells.

This project aims to develop novel nanoprobes to detect the spatial and temporal dynamic of RNA at single cell level. The immediate beneficiaries will be academics in diverse fields of research in bionanotechnology, healthcare, nanomaterials and photonics. Impact will be achieved by providing insights on the coupling of fluorescent protein and metal nanoparticle which will help developing new generation techniques for biological imaging and sensing, satisfy a need that spans numerous disciplines from fundamental research to technological studies, benefiting biosciences, pharmacy, medicine and nanomaterials. We will drive this impact agenda through communication, collaboration, and exploitation, all built on the experience we have gained during our well-established track record in knowledge exchange.

Potential internal beneficiaries
1, Centre for biophotonics hosts diverse research themes within the life sciences including drug delivery, infection and disease, and cancer research. Method and principle developed in this project can be transferred to imaging and sensing of other biomolecules; nanoprobes, combined TPL and energy transfer based imaging can be adapted to existing imaging facility in the Centre.
2, Centre for Industrial Control. Researchers at the Centre for Industrial control carry our mathematic modelling of a variety of cell signalling pathways. Data obtained from this project on transcription of NFkB genes can be directly used for mathematic modelling in Centre for Industrial control we will set up the collaboration through seminars and small group discussion
3, Cancer at Strathclyde (RICAS). Researchers at RICAS look at all aspects from cancer biology, to drug design and modelling. One of our aims is to detecting oncogene expression using the novel nanoprobes in various cancer cell-lines. The success in this aspect will direct our novel nanoprobes in early cancer diagnosis. We will hold seminars and small group discussion with RICAS to implement the technology.
Potential external beneficiaries
We will visit Professor Gad Frankel at Imperial College London who shown great interest in collaborating with us to use the novel nanoprobes to detect bacteria in complex matrixes, and to study pathogen-host interaction.
Potential beneficiaries in the UK and internal science community
Nanotechnologies have broader application in a wide scientific community worldwide. Through journal publications and conferences the new knowledge and techniques obtained from this project will reach the relevant disciplines. Additionally, research highlight and methods will be made available on university website. PI and CIs are fellows and members of a range of professional societies that provide a platform for dissemination. Applicants have broad research networks across physics, chemistry, biology, engineering and medicine. We will use meetings, workshops, training school, seminar, academic visit etc. to spread the research outcome.
Potential beneficiaries in high education
This project will facilitate final year projects and vacation projects. These projects will expose students to frontier research of nanotechnology and advanced laboratory techniques, thus enhance the training and learning activities. Researchers, especially ECR and students will benefit from the multidisciplinary nature of this project and combined experimental - theoretical approach.
Main future considerations
Food safety is one of the priorities areas of BBSRC, and it is a global challenge. Use of novel nanoprobes to detect food contamination by various microbes will be a new way of safety control. We will, in collaboration with Professor Frankel at Imperial, application in this area once we have demonstrated the principles outlined in the case report.