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

Lead Research Organisation: University of Portsmouth
Department Name: Inst of Biomedical and Biomolecular Sc

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description 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. 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. This project has demonstrated a novel approach for generating protein microarrays. Specifically, it involves using an array of protein precursors (RNA molecules) on a surface, known as an RNA microarray (created using our recent, state-of-the-art, patented technology), 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 molecular coupling step, the newly formed protein molecules in solution attach themselves to the specially prepared facing surface, forming the protein microarray.

This grant has not only demonstrated an efficient and effective means of generating protein microarrays, it has also exemplified a further utility of our novel RNA array technology (discovered and developed following earlier BBSRC investment).
Exploitation Route Creating protein microarrays from RNA microarrays has the potential to significantly benefit proteomic research, a field that spans the breadth of the biosciences domain. However, some further development of the method and demonstration of the broad utility of the approach would be required before being taking forward for commercialisation by industry. Ultimately, the method would be anticipated to be of use to others for studying protein interactions in a high throughput format.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings of this research project strength the exemplification of patents Callaghan AJ (2012) EU Patent EP2732047 and Callaghan AJ (2014) US Patent 20140087972.
 
Description DigiLab Global Equipment Provision
Amount £30,000 (GBP)
Organisation DigiLab Global 
Sector Private
Country United States
Start 11/2015 
 
Description ICURe (Innovation to Commercialise University Research)
Amount £30,000 (GBP)
Organisation Innovation to Commercialisation of University Research 
Sector Charity/Non Profit
Country Unknown
Start 12/2015 
End 04/2016
 
Title Protein Array from RNA Array 
Description As part of this research, a novel method has been developed which allows protein arrays to be generated from RNA arrays. Example utilities include: - generating bespoke functional protein arrays for proteomic studies - supporting high throughput approaches for exploring translation and mechanisms to specifically inhibit translation of potential targets, for example, for therapeutic purposes. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact A manuscript is being prepared so that the method can be used more widely by those in the field. 
 
Description Collaboration with DigiLab Global 
Organisation DigiLab Global
Country United States 
Sector Private 
PI Contribution Collaboration with DigiLab Global is allowing researchers on the project to access specific instrumentation. This is allowing them to explore and develop automated approaches within the context of our patented novel surface technology.
Collaborator Contribution DigiLab Global provided specific instrumentation to support the research as well as their unique expertise in relation to instrumentation support.
Impact This research is on-going. The disciplines involved include biochemistry and molecular biology coupled with instrumentation engineering expertise.
Start Year 2015
 
Description Collaboration with Prof. Denise Bazzolli at the Universidade Federal de Viçosa 
Organisation Federal University of Viçosa
Country Brazil 
Sector Academic/University 
PI Contribution My research team are undertaking molecular interaction studies guided by microbiology data provided by Prof. Bazzolli's team.
Collaborator Contribution Prof. Bazzolli's team are providing microbiology data and expertise.
Impact One of Prof. Bazzolli's team has secured funding to undertake a year-long placement in my research group to support this collaboration.
Start Year 2015
 
Description Collaboration with Prof. Paul Langford at Imperial College London 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team are conducting in vitro molecular interaction studies, and exploring the applicability of utilising our patented novel array technology, to explore cellular mechanisms within bacteria.
Collaborator Contribution Prof. Langford's team provide microbiology expertise and access to in vivo testing studies.
Impact A joint BBSRC grant was secured to support this collaborative research.
Start Year 2015
 
Title METHOD OF IMMOBILISING RNA ONTO A SURFACE 
Description A novel method to generate RNA arrays was developed. 
IP Reference WO2012156718 
Protection Patent application published
Year Protection Granted 2012
Licensed No
Impact - Discussions, under NDA, are on-going with interested companies. - Academic collaborations are supporting the further development of the technology.
 
Description Commercialisation discussions with Cambridge Protein Arrays 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact Presentation and discussions on 'Novel RNA Array Technology' to Cambridge Protein Arrays, December 2013.

Effective networking.
Year(s) Of Engagement Activity 2013
 
Description Commercialisation discussions with DigiLab Global. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation and discussions on the 'Novel RNA Array Technology' to DigiLab Global, January 2014. This lead to them visiting our labs and observing the RNA Array Technology process. DigiLab Global subsequently supported further research in this area by supplying equipment to explore automation approaches.

Effective networking.
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Commercialisation discussions with Horiba Scientific 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation and discussions on the 'Novel RNA Array Technology' to Horiba Scientific. The company funding our attendance at their site in France to undertake some collaborative experiments to explore possible opportunities.

Effective networking.
Year(s) Of Engagement Activity 2012
 
Description Commercialisation discussions with TTPLabtech 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Commercialisation discussions with TTPLabtech.
Year(s) Of Engagement Activity 2016
 
Description Maintaining an Active Online Presence 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My group has an active Twitter account with around 200 followers. We publish highlights from our research, outreach and engagement activities.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Patented Array Technology Website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact My group has a specific website highlighting our innovative patented array technology to potential commercial partners/business users.
Year(s) Of Engagement Activity 2016
 
Description Promoting PG study 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Presentation by members of my research team at various departmental events for undergraduates to promote engagement in postgraduate study. this involved the individuals highlighting their research work, including their day to day work, opportunities for collaboration and engagement as well as their outputs and impact.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Science Fairs 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Myself and my team have participated in supporting a number of Science Fairs in the region, engaging with attendees to promote science and the research we undertake.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Myself and my team regularly support University Open days. Activities can be many and varied, including giving talks, presenting posters, running hands-on laboratory demonstrations and engaging in question and answer sessions. There are usually a number of these events per year, with over 100 participants (schools and college students, sometimes accompanied by a parent/guardian) attending each event. Feedback from such events has highlighted our success in inspiring the next generation of scientists and has been specifically linked to an increase in the number of students applying to study Biochemistry over the last few years.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013