A new genetically-encoded aptamer platform for multi-colour RNA imaging

Many important processes in mammalian cells involve RNA. Of particular interest are those in which RNA molecules themselves act to catalyse events that affect a second RNA molecule. RNA molecules are often able to adopt a number of structures, and they can fluctuate between these either spontaneously (thermally-driven) or as a result of the actions of enzymes. A less well understood example is RNA splicing, in which large stretches of RNA are displaced from newly-transcribed RNA to form mRNA. The splicing machinery is RNA-based, and the RNA substrates are very long, sites are hard to recognise, and the use of these sites is often subject to complex tissue-specific regulation that may involve the formation of structures with the RNA. A good way of monitoring whether RNA undergoes changes in its structures or conformations is to specifically place fluorescent labels at two sites in the RNA. These labels are chosen such that, when they come into close proximity, they transfer the energy of fluorescence excitation from one to the other; this can be measured. This is a particularly good method for following the events on a single molecule, which is an essential approach for studying splicing.

The main drawback at present is that it is very difficult to introduce two labels at specific sites far inside a long RNA molecule. We propose to overcome this by genetically encoding RNA structures to bind to fluorescent tags. Having available a two-colour system to label RNA will provide a powerful new tool for RNA research as it will allow various RNA processing events to be directly compared rather than relying on fluorescence emission of a single fluorophore. Our inter-disciplinary approach is to exploit an artificial evolution technique known as SELEX to identify RNA structures (aptamers) that bind fluorophores that exhibit red emission. We will then incorporate these aptamers into long RNA molecules and investigate their potential as reporters of RNA biology. This will have a major impact in RNA research, and we will ensure both that the aptamers become commercially available and that the ability to follow RNA fluorescence is recognised as opening up new opportunities to search for drugs that affect RNA-based reactions.

This TRDF proposal seeks to establish a new tool to probe reactions involving RNA for use in single molecule spectroscopy and in living cells. Current methods for labelling RNA are either inconvenient and inefficient for long RNA molecules, such as those involved in regulated pre-mRNA splicing or lnc RNAs, such as Xist, or they produce high backgrounds of free fluorophore in vivo. A new approach has been developed recently in which aptamers have been developed that bind the chromophore of green fluorescent protein and thereby confer fluorescence activity. Such aptamers can be readily built into the RNA substrates. However, there are pressing reasons for developing a second set that produce fluorescence at longer wavelengths for studying reactions in vivo and in vitro. Apart from normal co-localization, FRET, etc. assays, the use of red and green aptamers encoded in a single gene would allow (for example) the time taken for transcription between the two sequences to be measured in vitro or in vivo, and for studies to be done on the conformation and organisation of pre-mRNA; their use in separate genes would allow work to be done on co-expression of adjacent genes. A suite of small molecule binding aptamers will be developed either to mimic the fluorescent properties of a representative red fluorescent protein or to bind to widely-used single molecule fluorophores. The fluorescent properties and binding specificity of the newly-developed "red" aptamers will be explored using bulk and single molecule assays in vitro and in cells. Finally, we will use the new and established aptamers in combination to demonstrate the advantages of the system.

Economic Impact

1. Investigate potential commercial exploitation of the research (Months 7-12)
This work is at the fundamental method development stages, which provides potential opportunities to explore the commercial potential of these imaging agents, most likely towards the final quarter of this 12 month project. Our collaborative team will work closely with UoS's Research & Knowledge Exchange Services (RKES), UoL's Technology Transfer Office and the University of Bonn's Technology Transfer Office to explore opportunities to secure intellectual property (IP). This will include surveying the IP potential of the proposed methods directed towards Synthetic Biology and imaging applications. Upon identification of relevant IP, UoS and UoL will work together to maximise its impact, engaging with commercial partners (e.g., GlaxoSmithKline) to identify, approach and secure industrial sponsors.

Synthetic Biology - We consider the development of novel aptamer modules that specifically bind to novel small molecule reporters the beginning of a platform technology in which these RNA aptamer sequences can be a highly sensitive reporting module that could be used for post-synthetic modification ranging from affinity probes, cross-linking agents and radiolabels. We will explore opportunities for commercialization with vendors who have a specific interest in DNA/RNA research (e.g., Link Technologies, IDT Technologies, Aptamer Group).

Diagnostic Platforms - The primary aim for this project is to expedite the utility of the emerging method development towards its application as a platform technology for RNA imaging. Once proof of concept has been established, the wider applicability of these emerging methods to be used as biosensing platforms will be explored, for example, through established collaborations with the Fraunhofer Institute based in the newly commissioned Technology Innovation Centre (UoS). These collaborations will facilitate further industrial engagement (e.g., Lumora, Roche Diagnostics) heading towards the Point-of-Care Medical Diagnostics sector projected to be worth $US 16.6 billion by 2016.

Societal Impact

2. Promote wider understanding of the impact of Synthetic Biology through public engagement activities.

GAB and SP will disseminate the latest developments to the wider public through our respective websites as well as through the popular press. The UoS has a long-established strategic partnership with the Glasgow Science Centre (Meet the Expert, GSC), the Huntarian Museum and various opportunities through the Glasgow Science Festival (GSF) and a Coffee House Lecture series. GAB and SP will present at the Meet the Expert over the duration of the project. GAB will deliver a Synthetic Biology seminar as part of the UoS's Coffee House Lecture series.

Academic Impact

3. Contribute to the economic competitiveness of the UK through enhancement of researcher career development.

SP and the UoL-based PDRA will be trained in a multi-disciplinary research environment, which spans synthetic organic chemistry, biochemistry and biophysics. In order to facilitate the upskilling, both PDRAs will be encouraged to attend courses designed to maximise their research performance (e.g. UoS Developing your Research Reputation).

Metrics

Success in terms of impact would be to form at least one new industrial partnership, and success in attracting funding that builds upon this research. Once an aptamer has been identified and shows desirable binding characteristics and specificity, this work will form the basis of our first publication. The aim is to have achieved these three goals by the end of this 12 month project.