Nucleic acid nanostructures as tags for cryo-electron microscopy
<p>Cryo-electron microscopy (cryo-EM) is an indispensable tool for structural biologists, allowing the reconstruction of biomolecules in their native, hydrated state to nanometre, or sub-nanometre, resolution. Unfortunately in situ cryo-EM characterisation is currently limited by our ability t...
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2019
|
| _version_ | 1826300323312959488 |
|---|---|
| author | Silvester, E |
| author2 | Turberfield, A |
| author_facet | Turberfield, A Silvester, E |
| author_sort | Silvester, E |
| collection | OXFORD |
| description | <p>Cryo-electron microscopy (cryo-EM) is an indispensable tool for structural biologists, allowing the reconstruction of biomolecules in their native, hydrated state to nanometre, or sub-nanometre, resolution. Unfortunately in situ cryo-EM characterisation is currently limited by our ability to interpret micrographs and tomograms, as a low signal-to-noise ratio means different structures are usually difficult to distinguish. To overcome this limitation, microscopists often use cryo-EM tags: electron-dense, nano-scale objects with specific targeting properties. This thesis presents two related projects involving the use of nucleic acid nanostructures as cryo-EM tags.</p>
<p>In Project 1, I introduce functionalised DNA-origami nanostructures as tags. I begin with the cryo-EM characterisation of various nanostructures in cell lysate to assess their imaging properties in a biological context. I find that solid lattice-based nanostructures provide high contrast in this environment. By cryo-EM, the outline of nanostructures is often difficult to visualise, whereas the internal arrangement of helices results in a distinctive `stripy' effect. As a result, honeycomb-lattice origami nanostructures with different shapes can be difficult to distinguish, whereas nanostructures of different lattice types are easily identifiable. Based on this preliminary work, I present a signpost origami nanostructures for tagging, comprising a high-contrast, asymmetric `sign' for identification, and a six-helix bundle `post' for targeting. To functionalise nanostructures for targeting, I have characterised an RNA aptamer which binds green fluorescent protein (GFP) and quantified its binding affinity to GFP analogues. The aptamer is attached to a signpost nanostructure by hybridisation and in situ binding is demonstrated with a murine leukaemia virus test system. High-contrast, high-efficiency labelling of viral glycoproteins is observed. Electroporation of polyamine-stabilised nanostructures into E. coli is also shown for intracellular labelling applications.</p>
<p>In Project 2, I present methods for designing 3D RNA nanostructures for co-transcriptional folding. This provides the groundwork for the development of genetically-encoded cryo-EM tags. I firstly introduce a grid-like architecture, where an RNA nanostructure consists of a set of interacting units arranged on a square lattice. Each unit is a branched hairpin structure, with units connected by a path of single-stranded linkers and through kissing interactions between complementary hairpin arms. For this design, six different nanostructures are synthesised. Supermolecular assembly of nanostructures into finite super-structures is demonstrated, as is the incorporation of an aptamer sequence for GFP binding. Finally, I investigate an origami-like route toward RNA nanostructure design, by introducing two new assembly motifs for incorporation into an already-established 2D RNA origami architecture in an effort to build in 3D. My first origami-like design uses paranemic crossovers formed between bulge loops situated on adjacent sheets. I show that these nanostructures do not fold into the intended 3D structure and suggest explanations for this observation. In my second origami-like design, I introduce the combined bulge-loop/kissing-loop motif. Two-sheet nanostructures, held together through these motifs, have been designed. Although a more promising prospect than the previous design, the yield of well-folded structures is low. Based on these observations, the grid-like architecture will provide a more promising launching pad for the development of genetic tags.</p> |
| first_indexed | 2024-03-07T05:15:23Z |
| format | Thesis |
| id | oxford-uuid:dd011b46-68f6-438c-9720-63b84dc3cd5c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:15:23Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:dd011b46-68f6-438c-9720-63b84dc3cd5c2022-03-27T09:21:52ZNucleic acid nanostructures as tags for cryo-electron microscopyThesishttp://purl.org/coar/resource_type/c_db06uuid:dd011b46-68f6-438c-9720-63b84dc3cd5cEnglishORA Deposit2019Silvester, ETurberfield, A<p>Cryo-electron microscopy (cryo-EM) is an indispensable tool for structural biologists, allowing the reconstruction of biomolecules in their native, hydrated state to nanometre, or sub-nanometre, resolution. Unfortunately in situ cryo-EM characterisation is currently limited by our ability to interpret micrographs and tomograms, as a low signal-to-noise ratio means different structures are usually difficult to distinguish. To overcome this limitation, microscopists often use cryo-EM tags: electron-dense, nano-scale objects with specific targeting properties. This thesis presents two related projects involving the use of nucleic acid nanostructures as cryo-EM tags.</p> <p>In Project 1, I introduce functionalised DNA-origami nanostructures as tags. I begin with the cryo-EM characterisation of various nanostructures in cell lysate to assess their imaging properties in a biological context. I find that solid lattice-based nanostructures provide high contrast in this environment. By cryo-EM, the outline of nanostructures is often difficult to visualise, whereas the internal arrangement of helices results in a distinctive `stripy' effect. As a result, honeycomb-lattice origami nanostructures with different shapes can be difficult to distinguish, whereas nanostructures of different lattice types are easily identifiable. Based on this preliminary work, I present a signpost origami nanostructures for tagging, comprising a high-contrast, asymmetric `sign' for identification, and a six-helix bundle `post' for targeting. To functionalise nanostructures for targeting, I have characterised an RNA aptamer which binds green fluorescent protein (GFP) and quantified its binding affinity to GFP analogues. The aptamer is attached to a signpost nanostructure by hybridisation and in situ binding is demonstrated with a murine leukaemia virus test system. High-contrast, high-efficiency labelling of viral glycoproteins is observed. Electroporation of polyamine-stabilised nanostructures into E. coli is also shown for intracellular labelling applications.</p> <p>In Project 2, I present methods for designing 3D RNA nanostructures for co-transcriptional folding. This provides the groundwork for the development of genetically-encoded cryo-EM tags. I firstly introduce a grid-like architecture, where an RNA nanostructure consists of a set of interacting units arranged on a square lattice. Each unit is a branched hairpin structure, with units connected by a path of single-stranded linkers and through kissing interactions between complementary hairpin arms. For this design, six different nanostructures are synthesised. Supermolecular assembly of nanostructures into finite super-structures is demonstrated, as is the incorporation of an aptamer sequence for GFP binding. Finally, I investigate an origami-like route toward RNA nanostructure design, by introducing two new assembly motifs for incorporation into an already-established 2D RNA origami architecture in an effort to build in 3D. My first origami-like design uses paranemic crossovers formed between bulge loops situated on adjacent sheets. I show that these nanostructures do not fold into the intended 3D structure and suggest explanations for this observation. In my second origami-like design, I introduce the combined bulge-loop/kissing-loop motif. Two-sheet nanostructures, held together through these motifs, have been designed. Although a more promising prospect than the previous design, the yield of well-folded structures is low. Based on these observations, the grid-like architecture will provide a more promising launching pad for the development of genetic tags.</p> |
| spellingShingle | Silvester, E Nucleic acid nanostructures as tags for cryo-electron microscopy |
| title | Nucleic acid nanostructures as tags for cryo-electron microscopy |
| title_full | Nucleic acid nanostructures as tags for cryo-electron microscopy |
| title_fullStr | Nucleic acid nanostructures as tags for cryo-electron microscopy |
| title_full_unstemmed | Nucleic acid nanostructures as tags for cryo-electron microscopy |
| title_short | Nucleic acid nanostructures as tags for cryo-electron microscopy |
| title_sort | nucleic acid nanostructures as tags for cryo electron microscopy |
| work_keys_str_mv | AT silvestere nucleicacidnanostructuresastagsforcryoelectronmicroscopy |