An innovative approach to 'printing' functional protein microarrays from RNA microarrays.

Proteins are the fundamental building blocks of all living cells and are essential for the proper functioning of an organism. Understanding how proteins interact with each other, and with other biological molecules, lies at the heart of all biological research and has clear implications for scientific progress within both health and environmental fields. For example, new therapeutics and more efficient bioenergy generation both rely on understanding and exploiting protein interactions. It is therefore unsurprising that developing tools to study protein interactions is a key priority within the BBSRC strategic plan.

One of the most efficient ways of investigating protein interactions is to generate a single surface containing hundreds-to-thousands of proteins, which can all be tested for interactions in one step. A surface of this type is known as a functional protein microarray and can be used to conduct high throughput interaction studies. Considering the real world applications of functional protein microarrays, within the medical arena alone, they have the potential to underpin better health through their use in drug discovery, disease diagnosis and medical screening. Unfortunately, to date, the successful creation of functional protein microarrays has been particularly challenging and they have therefore failed to deliver the impact anticipated.

This application seeks funding for pilot research to demonstrate a novel concept for generating functional protein microarrays that overcomes the limitations of the current approaches. The concept involves using an array of protein precursors (RNA molecules) on a surface, known as an RNA microarray, to generate a corresponding protein microarray on a specially prepared facing surface. The experimental setup involves placing the two surfaces opposite each other in a sandwich arrangement, with a specific biological solution in-between that converts the RNA molecules into protein molecules. Using a novel chemistry step, the newly formed protein molecules in solution attach themselves to the specially prepared facing surface, forming the functional protein microarray. To prove the novel concept, this project will involve demonstrating each stage of the process in turn, before bringing it all together to create a functional protein microarray by effectively 'printing' it from the precursor RNA microarray.

This work is innovative, timely and multi-disciplinary, employing the latest advances in chemistry, to facilitate the attachment of the proteins to the microarray slide surface, as well as our recent, state-of-the-art, patented technology for generating the precursor RNA microarray. Importantly, this application is not about incremental further development of this RNA microarray technology, but is instead about exploiting it and proving a novel concept for a new tool in a completely separate field; specifically, for the generation of functional protein microarrays. The potential of this research is considerable, offering a step change in capability by creating functional protein microarrays with greater robustness, smaller spot sizes and unrestricted protein sizes, in a simple and efficient manner. This overcomes the key limitations of existing functional protein microarray technologies and unlocks the vast benefits originally forecast.

Proteomic research spans the biosciences; hence, the BBSRC strategic plan highlights the development of new tools to study proteomics as a key priority underpinning future research advancements. Functional protein microarrays have the potential to significantly benefit proteomic research, but have failed to deliver the anticipated impact due to well recognised limitations. To overcome these limitations, this application requests funding for pilot research to prove the novel concept of generating covalently-bound functional multi-protein microarrays from multi-RNA microarrays.

To demonstrate the concept it is proposed to combine our state-of-the-art, patented, multi-RNA microarray technology with a novel label that can covalently couple a protein to a microarray slide surface upon light activation. Specifically, the novel label is an azide which is incorporated into the protein via an amber suppressor tRNA containing an azide-modified amino acid. The azide can then couple, via 'click' chemistry, to a surface-immobilised dibenzocyclooctyne, generated from a light activated cyclopropenone-masked cyclooctyne. Experimentally, the multi-RNA microarray slide will be set up in a sandwich arrangement facing a second slide, onto which the proteins become immobilised, with an in vitro translation mix in between the two slides. The end result is that the multi-RNA slide 'prints' a functional protein microarray, with each protein covalently immobilised on the surface via its incorporated azide. This work will involve demonstrating each stage of the process in turn, before testing the overall concept. The potential of this research is considerable, offering a step change in capability by creating functional protein microarrays with greater robustness, smaller spot sizes and unrestricted protein sizes, in a simple and efficient manner. This overcomes the key limitations of existing functional protein microarray technologies and unlocks the vast benefits originally predicted.

This work seeks to prove the novel concept of generating covalently-bound functional multi-protein microarrays from multi-RNA microarrays. Creating functional protein microarrays via this approach would have the potential to significantly benefit proteomic research, a field that spans the breadth of the biosciences domain. Discussions with Portsmouth University Research and Innovation Services indicate that the potential impact of this work falls into four broad categories:

1) Commercialisation
Early steps towards commercialisation will be explored during this project, but a promising proof of concept will require some further investment to mature and strengthen the commercial position. It is anticipated that the primary beneficiaries from successful commercialisation and exploitation will be companies that already manufacture products and supply services for the biosciences array market within the UK and worldwide (e.g. Agilent). Adding this tool to their product line has the potential to create wealth and economic prosperity through increased turn-over, profit and exports, and creating and safeguarding jobs. More broadly, secondary beneficiaries will include those companies that support the biosciences array manufacturers, whether that is in supplying or servicing equipment or providing the specific consumables required.

2) Further development of the tool
Towards the end of this work, once encouraging proof of concept data has been obtained and commercialisation is being explored, it is likely that further development of the tool would be required. This could be completed independently, but would benefit from collaborative research with specific proteomics users. In this manner, the collaborators would also gain early access to the tool, allowing them to conduct research that would not previously have been practicable. Once robust data is generated and the commercial/IP position is secure, then it would be possible to freely publish and release information and data generated using this technology. Such publications would further support commercialisation by generating broad interest and consequently demand for its use. Collaborators are likely to be drawn from across the proteomics field, including academia (e.g. Cambridge Centre for Proteomics) as well as from industry, public sector organisations and the third sector. This not only provides immediate benefits to the knowledge economy by generating research outputs (e.g. publications, patents, further income generation), but these outputs also have the potential to be successfully exploited, generating further impact. This activity will also strengthen collaborative links and encourage placements between institutions, as well as resulting in a host of highly skilled individuals gaining a broader appreciation of the applications of the tool and a detailed understanding of its use.

3) Longer term broader employment of the tool across the research community
Once the tool has been commercialised (see 1) and used to publish exemplary research (see 2) the intent is that it would see broad use across the research community. The beneficiaries at this stage are all those operating within biosciences R&D, including academia, industry, public sector organisations and the third sector. Bringing together the benefits from (1) and (2), the widespread use of the tool will create wealth and economic prosperity for suppliers within the biosciences array market, support research outputs, the knowledge economy and the exploitation of research findings.

4) Public dissemination
Throughout this project it will be possible to engage with the general public and local schools to raise the profile of this project. This will encourage interest in biosciences research and UK innovation and provide a context to local bioscience education.