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

Lead Research Organisation: University of Strathclyde
Department Name: Pure and Applied Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We have developed in-depth knowledge of how the Spinach aptamer folds and functions in vitro. We have also developed synthetic methods to prepare novel fluoruophores which can be used to develop a new generation of fluorogenic aptamers for multi-colour imaging. another outcome of this work was that nuclear extract arising from mammlian cells results in unfolding of the Spinach aptamer. therefore, a new generation of aptamers which are more stable in mammalian systems are required.
Exploitation Route We have recently been awarded a BBSRC Pathfinder award () which is being used to develop a new generation of fluorogenic RNA aptamers for imaging purposes.
Sectors Chemicals,Healthcare

 
Description This work has provided us with the scope and limitations of current fluoruogenic aptamer technology. whilst current Spinach aptamers do not work particularly well in RNA splicing assays, they are excellent tools to report on in vitro transcriptional assays. The way forward for this work is to understand how these aptamer systems can be used to prove RNA splicing. The major outcome of this work is the ability of the Spinach aptamer to be used as a reporter of transcriptional inhibition. we developed a real-time assay (currently being patented) where the fluoruogenic aptamer is a key reporter of trnascriptional inhibition. We are planning to widen the development of this technology in order for it to be used in vitro and in cells as a real-time tool to probe RNA biology.
First Year Of Impact 2017
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Title Developed new methods to report transcriptional inhibition in real-time 
Description This award was used to develop a new real-time assay which measures transcriptional inhibition. This assay uses the fluruogenic Spinach aptamer as a fluorescence readout. In the presence of a transcriptional inhibitor, the fluorescent readout is quenched. We have shown that this is a versatile assay which can be used to report a variety of different types of transcriptional inhibitors ranging from protein to DNA binding molecules. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact We are currently securing intellectual property for this method. Once the patent is granted, we will publish the results. 
 
Description Collaboration with Dr Ulrich Rant (TU Munich and now Dynamics Biosystems) 
Organisation Dynamic Biosensors
Country Germany 
Sector Private 
PI Contribution * The aim of this collaboration was to determine if the Dynamic Biosensors (switchSENSE) machine could be use to determine binding kinetics with modified nucleic acids. My team provided modified nucleic acids and nucleic acid binding molecules.
Collaborator Contribution The dynamic Biosensors team used their unique switchSENSE machine to establish a workflow for the determination of binding kinetics
Impact Aman, K.; Padroni, G.; Parkinson, J.A.; Welte, T.; Burley, G.A. "Structural and kinetic profiling of allosteric modulation of duplex DNA induced by DNA-binidng polyamide analogues" Chemistry - A European Journal. Early view publication [link] https://onlinelibrary.wiley.com/doi/full/10.1002/chem.201805338 Cover art: https://onlinelibrary.wiley.com/toc/15213765/0/0
Start Year 2016
 
Description Collaboration with Dr Ulrich Rant (TU Munich and now Dynamics Biosystems) 
Organisation Technical University of Munich
Department Walter Schottky Institute
Country Germany 
Sector Academic/University 
PI Contribution * The aim of this collaboration was to determine if the Dynamic Biosensors (switchSENSE) machine could be use to determine binding kinetics with modified nucleic acids. My team provided modified nucleic acids and nucleic acid binding molecules.
Collaborator Contribution The dynamic Biosensors team used their unique switchSENSE machine to establish a workflow for the determination of binding kinetics
Impact Aman, K.; Padroni, G.; Parkinson, J.A.; Welte, T.; Burley, G.A. "Structural and kinetic profiling of allosteric modulation of duplex DNA induced by DNA-binidng polyamide analogues" Chemistry - A European Journal. Early view publication [link] https://onlinelibrary.wiley.com/doi/full/10.1002/chem.201805338 Cover art: https://onlinelibrary.wiley.com/toc/15213765/0/0
Start Year 2016
 
Description Invited presentation to fluorescence conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact An invited presentation at the Dynamic Biosensors annual meeting. This was used to showcase the potential of switchSENSE technology to obtain high quality data on the binding kinetics of small molecule-nucleic acids and nucleic acid interactions. the presentation was delivered to a blend of industrialists and academic groups.
Year(s) Of Engagement Activity 2017
URL https://www.dynamic-biosensors.com/user-meeting/