{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/386584"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/386584","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"A General Platform for Ribosome Engineering","abstract":"Ribosomal protein synthesis provides a blueprint for the programmable and atomically precise as- sembly of molecular building blocks into large and complex molecular structures. Emerging research aims to reprogram the genetic code in vitro and in vivo to assemble non-canonical polymers with this approach. However, the properties of the natural ribosome limit existing work to close analogues of the canonical amino acids. This thesis develops a directed evolution technology to engineer ribosomes in vitro. The focus of this work is to create a general platform capable of identifying ribosome variants that have an expanded substrate scope for diverse non-canonical monomers (ncMs), though the advances described herein need not be limited to this application. The creation of such ribosomes will transform genetic code expansion and reprogramming in vitro and in vivo by permitting the encoded synthesis and evolution of diverse non-canonical polymers beyond polypeptides on the ribosome. Chapter 1 introduces genetic code reprogramming and describes existing work in the field of ribo- some engineering. In particular, I describe how the development of stapled orthogonal ribosomes - ribosomes where large and small subunits participate in a parallel and independent translation pathway - now permits the creation of highly modified ribosomes which may enable the discovery of new ri- bosomal function. I discuss existing approaches of discovering ribosomes with new functions in vitro and in vivo. I then consider how the work in this thesis, and in the field more broadly, may be extended towards creating general molecular assemblers capable of polymerizing near-arbitrary building blocks in a programmable fashion, towards a new type of nanotechnology and alternative forms of life. Chapter 2 addresses a core limitation of orthogonal ribosomes, their typically low activity and dy- namic range on orthogonal reporters. The chapter describes a general solution to this problem by ap- plying biophysical modelling and computational design algorithms to discover 5’ untranslated regions and synonymous codon substitutions in orthogonal mRNAs that are highly and exclusively translated by orthogonal ribosomes in vivo. We leverage one of the orthogonal mRNAs created to incorporate four distinct non-canonical amino acids into a protein using four distinct quadruplet codons for the first time in vivo. Chapter 3 describes STRIVE - stapled orthogonal ribosome in vitro evolution - an in vitro ribosome evolution platform. Importantly, STRIVE uses stapled orthogonal ribosomes, which will permit the creation of ribosome variants previously inaccessible, and is based on a reconstituted translation sys- tem, which will facilitate codon reassignment to near-arbitrary non-canonical monomers for ribosome selections. It is therefore an ideal platform for the directed evolution of ribosomes towards radically new functions. Chapter 4 addresses the challenge of achieving ribosome orthogonality in vitro, which is more difficult than in vivo because of the absence of competition. The chapter demonstrates that it is possible to artificially introduce such competition using a host ribosome-sequestering mRNA. The chapter further shows that this solution is compatible with the STRIVE set-up developed in chapter 3. Chapter 5 describes HiSTRIVE - a combination of the STRIVE platform with deep sequencing that permits the multiplexed assessment of diverse ribosome variants across translation conditions and allows for the direct discovery of ribosome variants with altered properties. The chapter then describes our initial application of HiSTRIVE to discover ribosomes that polymerize polyproline sequences in the absence of elongation factor P in vitro. Chapter 6 considers how to leverage STRIVE to allow for direct selections for the incorporation of non-canonical monomers. This requires high-fidelity reassignment of one or multiple codons. The chapter describes the creation of blank codons in STRIVE and shows how the blank codons can be reassigned using the flexizyme system. Chapter 7 concludes the thesis with a description of ongoing work to use HiSTRIVE to discover ribosome variants with altered polymerization properties under a reprogrammed genetic code. In par- ticular, β-amino acids are an important class of non-canonical monomers of great medical importance. The chapter describes the first direct selection of ribosome variants for β2R-Phe polymerization. Chapter 8 draws some overall conclusions about the work described here and presents an outlook to future research enabled by this thesis.","abstract_html":"Ribosomal protein synthesis provides a blueprint for the programmable and atomically precise as- sembly of molecular building blocks into large and complex molecular structures. Emerging research aims to reprogram the genetic code in vitro and in vivo to assemble non-canonical polymers with this approach. However, the properties of the natural ribosome limit existing work to close analogues of the canonical amino acids. This thesis develops a directed evolution technology to engineer ribosomes in vitro. The focus of this work is to create a general platform capable of identifying ribosome variants that have an expanded substrate scope for diverse non-canonical monomers (ncMs), though the advances described herein need not be limited to this application. The creation of such ribosomes will transform genetic code expansion and reprogramming in vitro and in vivo by permitting the encoded synthesis and evolution of diverse non-canonical polymers beyond polypeptides on the ribosome. Chapter 1 introduces genetic code reprogramming and describes existing work in the field of ribo- some engineering. In particular, I describe how the development of stapled orthogonal ribosomes - ribosomes where large and small subunits participate in a parallel and independent translation pathway - now permits the creation of highly modified ribosomes which may enable the discovery of new ri- bosomal function. I discuss existing approaches of discovering ribosomes with new functions in vitro and in vivo. I then consider how the work in this thesis, and in the field more broadly, may be extended towards creating general molecular assemblers capable of polymerizing near-arbitrary building blocks in a programmable fashion, towards a new type of nanotechnology and alternative forms of life. Chapter 2 addresses a core limitation of orthogonal ribosomes, their typically low activity and dy- namic range on orthogonal reporters. The chapter describes a general solution to this problem by ap- plying biophysical modelling and computational design algorithms to discover 5’ untranslated regions and synonymous codon substitutions in orthogonal mRNAs that are highly and exclusively translated by orthogonal ribosomes in vivo. We leverage one of the orthogonal mRNAs created to incorporate four distinct non-canonical amino acids into a protein using four distinct quadruplet codons for the first time in vivo. Chapter 3 describes STRIVE - stapled orthogonal ribosome in vitro evolution - an in vitro ribosome evolution platform. Importantly, STRIVE uses stapled orthogonal ribosomes, which will permit the creation of ribosome variants previously inaccessible, and is based on a reconstituted translation sys- tem, which will facilitate codon reassignment to near-arbitrary non-canonical monomers for ribosome selections. It is therefore an ideal platform for the directed evolution of ribosomes towards radically new functions. Chapter 4 addresses the challenge of achieving ribosome orthogonality in vitro, which is more difficult than in vivo because of the absence of competition. The chapter demonstrates that it is possible to artificially introduce such competition using a host ribosome-sequestering mRNA. The chapter further shows that this solution is compatible with the STRIVE set-up developed in chapter 3. Chapter 5 describes HiSTRIVE - a combination of the STRIVE platform with deep sequencing that permits the multiplexed assessment of diverse ribosome variants across translation conditions and allows for the direct discovery of ribosome variants with altered properties. The chapter then describes our initial application of HiSTRIVE to discover ribosomes that polymerize polyproline sequences in the absence of elongation factor P in vitro. Chapter 6 considers how to leverage STRIVE to allow for direct selections for the incorporation of non-canonical monomers. This requires high-fidelity reassignment of one or multiple codons. The chapter describes the creation of blank codons in STRIVE and shows how the blank codons can be reassigned using the flexizyme system. Chapter 7 concludes the thesis with a description of ongoing work to use HiSTRIVE to discover ribosome variants with altered polymerization properties under a reprogrammed genetic code. In par- ticular, β-amino acids are an important class of non-canonical monomers of great medical importance. The chapter describes the first direct selection of ribosome variants for β2R-Phe polymerization. Chapter 8 draws some overall conclusions about the work described here and presents an outlook to future research enabled by this thesis.","abstract_has_math":false,"creators":["Oehm, Sebastian"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Chin, Jason"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-09-30","date_published":"2023-09-30","updated_at":"2026-07-24T01:33:21Z","subjects":["genetic code expansion","ribosome engineering","synthetic biology"],"languages":[],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/847fc108-3427-4dff-9579-7d3ef12f195a/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.119732","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Chin, Jason"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Boehringer Ingelheim Fonds PhD Fellowship"]},{"key":"dc:creator","label":"Author","values":["Oehm, Sebastian"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-09-30"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/386584"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["genetic code expansion","ribosome engineering","synthetic biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/847fc108-3427-4dff-9579-7d3ef12f195a/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-08-11"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.119732"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/cf47e758-2330-4795-b0b6-74859eac183c/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Ribosomal protein synthesis provides a blueprint for the programmable and atomically precise as- sembly of molecular building blocks into large and complex molecular structures. 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In particular, I describe how the development of stapled orthogonal ribosomes - ribosomes where large and small subunits participate in a parallel and independent translation pathway - now permits the creation of highly modified ribosomes which may enable the discovery of new ri- bosomal function. I discuss existing approaches of discovering ribosomes with new functions in vitro and in vivo. I then consider how the work in this thesis, and in the field more broadly, may be extended towards creating general molecular assemblers capable of polymerizing near-arbitrary building blocks in a programmable fashion, towards a new type of nanotechnology and alternative forms of life. Chapter 2 addresses a core limitation of orthogonal ribosomes, their typically low activity and dy- namic range on orthogonal reporters. The chapter describes a general solution to this problem by ap- plying biophysical modelling and computational design algorithms to discover 5’ untranslated regions and synonymous codon substitutions in orthogonal mRNAs that are highly and exclusively translated by orthogonal ribosomes in vivo. We leverage one of the orthogonal mRNAs created to incorporate four distinct non-canonical amino acids into a protein using four distinct quadruplet codons for the first time in vivo. Chapter 3 describes STRIVE - stapled orthogonal ribosome in vitro evolution - an in vitro ribosome evolution platform. Importantly, STRIVE uses stapled orthogonal ribosomes, which will permit the creation of ribosome variants previously inaccessible, and is based on a reconstituted translation sys- tem, which will facilitate codon reassignment to near-arbitrary non-canonical monomers for ribosome selections. It is therefore an ideal platform for the directed evolution of ribosomes towards radically new functions. Chapter 4 addresses the challenge of achieving ribosome orthogonality in vitro, which is more difficult than in vivo because of the absence of competition. The chapter demonstrates that it is possible to artificially introduce such competition using a host ribosome-sequestering mRNA. The chapter further shows that this solution is compatible with the STRIVE set-up developed in chapter 3. Chapter 5 describes HiSTRIVE - a combination of the STRIVE platform with deep sequencing that permits the multiplexed assessment of diverse ribosome variants across translation conditions and allows for the direct discovery of ribosome variants with altered properties. The chapter then describes our initial application of HiSTRIVE to discover ribosomes that polymerize polyproline sequences in the absence of elongation factor P in vitro. Chapter 6 considers how to leverage STRIVE to allow for direct selections for the incorporation of non-canonical monomers. This requires high-fidelity reassignment of one or multiple codons. The chapter describes the creation of blank codons in STRIVE and shows how the blank codons can be reassigned using the flexizyme system. Chapter 7 concludes the thesis with a description of ongoing work to use HiSTRIVE to discover ribosome variants with altered polymerization properties under a reprogrammed genetic code. In par- ticular, β-amino acids are an important class of non-canonical monomers of great medical importance. The chapter describes the first direct selection of ribosome variants for β2R-Phe polymerization. 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However, the properties of the natural ribosome limit existing work to close analogues of the canonical amino acids. This thesis develops a directed evolution technology to engineer ribosomes in vitro. The focus of this work is to create a general platform capable of identifying ribosome variants that have an expanded substrate scope for diverse non-canonical monomers (ncMs), though the advances described herein need not be limited to this application. The creation of such ribosomes will transform genetic code expansion and reprogramming in vitro and in vivo by permitting the encoded synthesis and evolution of diverse non-canonical polymers beyond polypeptides on the ribosome. Chapter 1 introduces genetic code reprogramming and describes existing work in the field of ribo- some engineering. In particular, I describe how the development of stapled orthogonal ribosomes - ribosomes where large and small subunits participate in a parallel and independent translation pathway - now permits the creation of highly modified ribosomes which may enable the discovery of new ri- bosomal function. I discuss existing approaches of discovering ribosomes with new functions in vitro and in vivo. I then consider how the work in this thesis, and in the field more broadly, may be extended towards creating general molecular assemblers capable of polymerizing near-arbitrary building blocks in a programmable fashion, towards a new type of nanotechnology and alternative forms of life. Chapter 2 addresses a core limitation of orthogonal ribosomes, their typically low activity and dy- namic range on orthogonal reporters. The chapter describes a general solution to this problem by ap- plying biophysical modelling and computational design algorithms to discover 5’ untranslated regions and synonymous codon substitutions in orthogonal mRNAs that are highly and exclusively translated by orthogonal ribosomes in vivo. We leverage one of the orthogonal mRNAs created to incorporate four distinct non-canonical amino acids into a protein using four distinct quadruplet codons for the first time in vivo. Chapter 3 describes STRIVE - stapled orthogonal ribosome in vitro evolution - an in vitro ribosome evolution platform. Importantly, STRIVE uses stapled orthogonal ribosomes, which will permit the creation of ribosome variants previously inaccessible, and is based on a reconstituted translation sys- tem, which will facilitate codon reassignment to near-arbitrary non-canonical monomers for ribosome selections. It is therefore an ideal platform for the directed evolution of ribosomes towards radically new functions. Chapter 4 addresses the challenge of achieving ribosome orthogonality in vitro, which is more difficult than in vivo because of the absence of competition. The chapter demonstrates that it is possible to artificially introduce such competition using a host ribosome-sequestering mRNA. The chapter further shows that this solution is compatible with the STRIVE set-up developed in chapter 3. Chapter 5 describes HiSTRIVE - a combination of the STRIVE platform with deep sequencing that permits the multiplexed assessment of diverse ribosome variants across translation conditions and allows for the direct discovery of ribosome variants with altered properties. The chapter then describes our initial application of HiSTRIVE to discover ribosomes that polymerize polyproline sequences in the absence of elongation factor P in vitro. Chapter 6 considers how to leverage STRIVE to allow for direct selections for the incorporation of non-canonical monomers. This requires high-fidelity reassignment of one or multiple codons. The chapter describes the creation of blank codons in STRIVE and shows how the blank codons can be reassigned using the flexizyme system. Chapter 7 concludes the thesis with a description of ongoing work to use HiSTRIVE to discover ribosome variants with altered polymerization properties under a reprogrammed genetic code. In par- ticular, β-amino acids are an important class of non-canonical monomers of great medical importance. The chapter describes the first direct selection of ribosome variants for β2R-Phe polymerization. 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