Enabling precision distance measurements in long RNAs
Lead Research Organisation:
University of Oxford
Department Name: Oxford Chemistry
Abstract
Technologies that explore the shape and motion of complex biomolecules such as ribonucleic acids (RNA) are fundamentally important for understanding the role of such biomolecules in human biology, or disease. The development of new analytical methods is of high importance, as this increases the range of tools available to the biologist for the precise investigation of biomolecule structure and behaviour.
This project seeks to develop a new technology for the characterisation of RNA - the use of biosynthesis to introduce unnatural functional groups into RNA that can be reacted with probes that contain 'stable free radicals', which are termed 'spin labels'. This work will offer a new and totally unexplored method for the analysis of 'long' RNA (that is, RNA which is longer than can reliably be prepared through chemical synthesis).
The key biosynthesis methodology is called 'Stepwise transcription', which uses the ability of certain RNA polymerase enzymes to accept unnatural nucleoside triphosphates (NTPs, the building blocks used by Nature to construct RNA) as an RNA chain is biosynthesised. These unnatural nucleosides contain a chemical functionality which is tolerated by the polymerase, but which enables post-biosynthesis 'labelling' of the RNA. In the case of our spin labels, this enables the use of electron paramagnetic resonance spectroscopy (EPR) to analyse the biomolecule. This technique has been applied with success for the analysis of small RNA and DNA motifs which can be prepared through chemical synthesis, but only very rarely to spin labelled long RNAs (>50 nucleotides) due to the current difficulties with their preparation. The EPR technique itself is highly informative, not only measuring distances, but also motion and orientation.
The project itself will involve the synthesis of NTPs that contain appropriate functionality to install the spin label post-biosynthesis. While many modified NTPs already exist (which we will use in this project as a starting point), to obtain the best information in the EPR experiments, we will need to design new, more rigidified labelling handles in the NTP. This is an exciting challenge, and one that has consequences beyond EPR (other probes could be installed on these same handles). We will also synthesize a range of spin labels to attach to the biosynthesized RNA. To test the methodology, we have identified a 71 nucleotide RNA motif that is known to undergo structural change under defined conditions. We will label this motif, and explore by EPR spectroscopy the change in its shape, thus establishing this new technology as a viable and easily applied method for RNA spin labelling.
This project seeks to develop a new technology for the characterisation of RNA - the use of biosynthesis to introduce unnatural functional groups into RNA that can be reacted with probes that contain 'stable free radicals', which are termed 'spin labels'. This work will offer a new and totally unexplored method for the analysis of 'long' RNA (that is, RNA which is longer than can reliably be prepared through chemical synthesis).
The key biosynthesis methodology is called 'Stepwise transcription', which uses the ability of certain RNA polymerase enzymes to accept unnatural nucleoside triphosphates (NTPs, the building blocks used by Nature to construct RNA) as an RNA chain is biosynthesised. These unnatural nucleosides contain a chemical functionality which is tolerated by the polymerase, but which enables post-biosynthesis 'labelling' of the RNA. In the case of our spin labels, this enables the use of electron paramagnetic resonance spectroscopy (EPR) to analyse the biomolecule. This technique has been applied with success for the analysis of small RNA and DNA motifs which can be prepared through chemical synthesis, but only very rarely to spin labelled long RNAs (>50 nucleotides) due to the current difficulties with their preparation. The EPR technique itself is highly informative, not only measuring distances, but also motion and orientation.
The project itself will involve the synthesis of NTPs that contain appropriate functionality to install the spin label post-biosynthesis. While many modified NTPs already exist (which we will use in this project as a starting point), to obtain the best information in the EPR experiments, we will need to design new, more rigidified labelling handles in the NTP. This is an exciting challenge, and one that has consequences beyond EPR (other probes could be installed on these same handles). We will also synthesize a range of spin labels to attach to the biosynthesized RNA. To test the methodology, we have identified a 71 nucleotide RNA motif that is known to undergo structural change under defined conditions. We will label this motif, and explore by EPR spectroscopy the change in its shape, thus establishing this new technology as a viable and easily applied method for RNA spin labelling.
Technical Summary
This project will establish the use of RNA polymerase (RNAP)-based site-selective modification of long RNAs (>50 nt) with functional groups that are amenable for the introduction of spin labels (suitable paramagnetic centres) using CuAAC (Click) chemistry. This enables the study of RNA conformation, and conformational change, using electron paramagnetic resonance (EPR) spectroscopy. This would represent the first time that biosynthesis has been used to prepare a spin-labelled nucleic acid; this technology will open up a wealth of possibilities to explore the dynamic behaviour of complex nucleic acids and their interactions with other biomolecules. Several key technology challenges will be studied:
1. The synthesis of appropriate modified nucleoside triphosphates (NTPs). Novel reagents will be required due to the need for conformationally-rigid attachment of the free radical on the nucleic acid.
2. The synthesis of appropriate nitroxide (and other) spin labels. We plan to explore a range of stable free radicals as structural probes, and paramagnetic metal ions; again, the synthesis of novel structures is likely to be required from a magnetic properties and solubility perspective.
3. Testing of RNAP-based incorporation of these NTPs into long RNA via stepwise transcription. This will require flexibility in linker design / label positioning.
4. Spin labelling of these structures with appropriate nitroxides, and evaluation of the labelled biomolecule. This is the climax of the project - demonstrating that indeed long RNA spin labelling can be achieved, and can give meaningful structural information by EPR methods such as continuous wave (CW) and double electron-electron resonance (DEER).
5. An additional aspect of the project will be use of state-of-the-art EPR spectrometers and methods.
To achieve these goals, this project thus involves the collaboration of a synthetic chemistry group (EA), an EPR group (JL/HEM), and a biosynthesis group (CP).
1. The synthesis of appropriate modified nucleoside triphosphates (NTPs). Novel reagents will be required due to the need for conformationally-rigid attachment of the free radical on the nucleic acid.
2. The synthesis of appropriate nitroxide (and other) spin labels. We plan to explore a range of stable free radicals as structural probes, and paramagnetic metal ions; again, the synthesis of novel structures is likely to be required from a magnetic properties and solubility perspective.
3. Testing of RNAP-based incorporation of these NTPs into long RNA via stepwise transcription. This will require flexibility in linker design / label positioning.
4. Spin labelling of these structures with appropriate nitroxides, and evaluation of the labelled biomolecule. This is the climax of the project - demonstrating that indeed long RNA spin labelling can be achieved, and can give meaningful structural information by EPR methods such as continuous wave (CW) and double electron-electron resonance (DEER).
5. An additional aspect of the project will be use of state-of-the-art EPR spectrometers and methods.
To achieve these goals, this project thus involves the collaboration of a synthetic chemistry group (EA), an EPR group (JL/HEM), and a biosynthesis group (CP).
Planned Impact
This project seeks to develop a new technology with the potential for significant impact in structural biology. The technology specifically involves the combination of stepwise transcription, bioorthogonal Click spin labelling, and EPR spectroscopy - all underpinned by synthetic chemistry, which will provide the nucleoside triphosphate and spin label building blocks that are the foundation of the proposal. The technology itself, and its constituent components, are expected to impact broadly on a range of other disciplines and beneficiaries.
Industrial impact: Analytical techniques such as FRET are commonly applied in industrial settings as a means of visualising biological processes. We expect the underused tool of EPR spectroscopy to become more widely adopted in the biomedical and agrochemical industry as a direct consequence of our work - this is particularly envisaged in light of the growing importance of RNA research in these fields. Preliminary conversations between JL and industrialists at both Merck and Syngenta have revealed significant interest in this method, and its potential as a new tool.
To achieve this impact, we will provide a highly credible proof-of-concept with the planned riboswitch studies, and will engage widely with the professional industry networks of our team. Further, Prof Tom Brown's company, ADTBio, offers a means to commercialise nucleoside building blocks, making the reagents of this technology readily available to commercial users. As the 2016 BBRSC Entrepreneur of the Year, Prof Brown is clearly an ideal collaborator to explore industrial impact. We will equally explore spin label commercialisation as a potential avenue to broaden impact, as EPR becomes more widely used in the scientific community. For example, labels that exhibit improved water solubility would be highly useful in other EPR contexts, such as protein spin labelling.
Economic and societal impact: The technology will equip molecular biologists with a new tool to explore RNA structure and function. This will provide information that can be used in the design of new therapies, and clear societal benefits will emerge for patient groups including improved therapies (and therefore quality of life). Economic benefits are also envisaged should any commercial applications be developed, for example in the collaboration with ADTBio as mentioned. The reputation of UK science will be enhanced by the ambition and scope of the project, and delivery and dissemination of the results. Societal impact is anticipated through our public engagement activities, as well as the use by others of the technology that arises from the project.
People impact: This project directly provides training in interdisciplinary science to the post-doctoral researcher, as well as building a wide network of contacts in different disciplines. Presentation, leadership, and project management skills, which form an essential part of the collaborative feedback loop, will also be developed. The technology developed will open up new funding and research opportunities for the project team of the PI and Co-Is, by identifying new project areas to apply the research to, or new collaborators.
We expect benefits to the post-doctoral researcher and collaborator team to be realised within the timeframe of the grant (e.g. new research / funding opportunities, reputation benefits). The societal and economic benefits will arise from the uptake of the technology by other end-users, and has a longer term benefit. A key aspect of will be regular (Skype and face-to-fact) meetings of the collaborators, in order to maximise communication of results, and optimise responsive project planning and execution.
Industrial impact: Analytical techniques such as FRET are commonly applied in industrial settings as a means of visualising biological processes. We expect the underused tool of EPR spectroscopy to become more widely adopted in the biomedical and agrochemical industry as a direct consequence of our work - this is particularly envisaged in light of the growing importance of RNA research in these fields. Preliminary conversations between JL and industrialists at both Merck and Syngenta have revealed significant interest in this method, and its potential as a new tool.
To achieve this impact, we will provide a highly credible proof-of-concept with the planned riboswitch studies, and will engage widely with the professional industry networks of our team. Further, Prof Tom Brown's company, ADTBio, offers a means to commercialise nucleoside building blocks, making the reagents of this technology readily available to commercial users. As the 2016 BBRSC Entrepreneur of the Year, Prof Brown is clearly an ideal collaborator to explore industrial impact. We will equally explore spin label commercialisation as a potential avenue to broaden impact, as EPR becomes more widely used in the scientific community. For example, labels that exhibit improved water solubility would be highly useful in other EPR contexts, such as protein spin labelling.
Economic and societal impact: The technology will equip molecular biologists with a new tool to explore RNA structure and function. This will provide information that can be used in the design of new therapies, and clear societal benefits will emerge for patient groups including improved therapies (and therefore quality of life). Economic benefits are also envisaged should any commercial applications be developed, for example in the collaboration with ADTBio as mentioned. The reputation of UK science will be enhanced by the ambition and scope of the project, and delivery and dissemination of the results. Societal impact is anticipated through our public engagement activities, as well as the use by others of the technology that arises from the project.
People impact: This project directly provides training in interdisciplinary science to the post-doctoral researcher, as well as building a wide network of contacts in different disciplines. Presentation, leadership, and project management skills, which form an essential part of the collaborative feedback loop, will also be developed. The technology developed will open up new funding and research opportunities for the project team of the PI and Co-Is, by identifying new project areas to apply the research to, or new collaborators.
We expect benefits to the post-doctoral researcher and collaborator team to be realised within the timeframe of the grant (e.g. new research / funding opportunities, reputation benefits). The societal and economic benefits will arise from the uptake of the technology by other end-users, and has a longer term benefit. A key aspect of will be regular (Skype and face-to-fact) meetings of the collaborators, in order to maximise communication of results, and optimise responsive project planning and execution.
Publications
Hardwick JS
(2020)
2'-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis.
in Nucleic acids research
| Description | This research has enabled us to develop synthetic routes to unnatural nucleic acids, which can be incorporated into RNA using biosynthetic methods. We have successfully shown synthesis of this modified RNA, and the attachment of a 'label' at a site which can be probed using EPR spectroscopy. This represents the first such example of biosynthesised 'long' spin labelled RNA. In further work, we were able to achieve the first examples of X-ray crystal structures of spin-labelled DNA, which provided a new level of insight into how 'spin labels' associate with the DNA duplex. We were also able to use molecular dynamics modelling to simulate the process of how this association takes place. Collectively, this work has provided an unprecedented level of insight into the localisation of spin labels in nucleic acid structures, and that binding of these labels to the duplex does not significantly affect duplex conformation. |
| Exploitation Route | We expect to use the preliminary results from this technology grant to submit a full responsive mode funding application. This will enable us to extensively map out the technology, with a view to dissemination though publications. Other researchers will be able to use the findings of this work in designing new modified nucleic acids with the linker strategy we have employed. |
| Sectors | Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology |