{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/405964"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/405964","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"An Inverse Electron-Demand Diels-Alder Strategy for Mapping RNA Methylation","abstract":"Post-transcriptional RNA modifications, such as N6-methyladenosine (m6A), 2'-O-methylation (Nm), and 5-methylcytosine (m5C), are critical regulators of gene expression. Mapping these modifications at single-nucleotide resolution, however, remains a major challenge as current methods often suffer from low resolution or antibody-related biases. While bioorthogonal chemistry offers a powerful approach for RNA analysis, established reactions like the copper- catalysed azide-alkyne cycloaddition (CuAAC) can have disadvantages for modification mapping, including copper-mediated RNA degradation that can compromise data integrity. This highlights a critical need for further biocompatible reactions tailored for epitranscriptome analysis. To address this, this thesis details the development and systematic evaluation of a bioorthogonal strategy specifically designed to be advantageous for mapping RNA modifications. The foundational work was conducted in an isolated in vitro setting on synthetic oligonucleotides to investigate precisely how the installed chemical modifications impact key detection technologies, namely reverse transcription and nanopore sequencing. In this work, an inverse electron-demand Diels-Alder (IEDDA) reaction between allyl-modified nucleosides and a dipyridyl tetrazine was developed and characterised. This reaction uniquely generates a sterically bulky double-addition product for all nucleosides explored. To facilitate the creation of the necessary tools for this work, a highly efficient, single-step synthesis of the required allyl-modified phosphoramidites was also devised directly from a commercially available precursor. Using synthetic RNA oligonucleotides, it was demonstrated that the IEDDA adduct acts as an effective steric block that consistently induces full truncation of cDNA synthesis during reverse transcription, which could provide an unambiguous readout for high- resolution mapping with standard Illumina sequencing platforms. Furthermore, it was shown that the same modification produces a distinct and detectable signal during nanopore direct RNA sequencing, establishing the dual compatibility of the approach. Altogether, this thesis establishes a powerful and versatile antibody- and copper-free platform for epitranscriptome analysis, validated here in an isolated setting using synthetic oligonucleotides. The dual-readout methodology, which leverages a robust and biocompatible ligation, paves the way for transcriptome-wide mapping of multiple RNA modifications at site resolution. This successful in vitro proof-of-concept lays the groundwork for future applications in living cells. Ultimately, this chemical strategy could be combined with metabolic labelling to hijack the native RNA methylation machinery, allowing for the installation of a minimally perturbing allyl handle at endogenous modification sites for subsequent chemical ligation and transcriptome-wide analysis using Illumina or nanopore-based platforms.","abstract_html":"Post-transcriptional RNA modifications, such as N6-methyladenosine (m6A), 2'-O-methylation (Nm), and 5-methylcytosine (m5C), are critical regulators of gene expression. Mapping these modifications at single-nucleotide resolution, however, remains a major challenge as current methods often suffer from low resolution or antibody-related biases. While bioorthogonal chemistry offers a powerful approach for RNA analysis, established reactions like the copper- catalysed azide-alkyne cycloaddition (CuAAC) can have disadvantages for modification mapping, including copper-mediated RNA degradation that can compromise data integrity. This highlights a critical need for further biocompatible reactions tailored for epitranscriptome analysis. To address this, this thesis details the development and systematic evaluation of a bioorthogonal strategy specifically designed to be advantageous for mapping RNA modifications. The foundational work was conducted in an isolated in vitro setting on synthetic oligonucleotides to investigate precisely how the installed chemical modifications impact key detection technologies, namely reverse transcription and nanopore sequencing. In this work, an inverse electron-demand Diels-Alder (IEDDA) reaction between allyl-modified nucleosides and a dipyridyl tetrazine was developed and characterised. This reaction uniquely generates a sterically bulky double-addition product for all nucleosides explored. To facilitate the creation of the necessary tools for this work, a highly efficient, single-step synthesis of the required allyl-modified phosphoramidites was also devised directly from a commercially available precursor. Using synthetic RNA oligonucleotides, it was demonstrated that the IEDDA adduct acts as an effective steric block that consistently induces full truncation of cDNA synthesis during reverse transcription, which could provide an unambiguous readout for high- resolution mapping with standard Illumina sequencing platforms. Furthermore, it was shown that the same modification produces a distinct and detectable signal during nanopore direct RNA sequencing, establishing the dual compatibility of the approach. Altogether, this thesis establishes a powerful and versatile antibody- and copper-free platform for epitranscriptome analysis, validated here in an isolated setting using synthetic oligonucleotides. The dual-readout methodology, which leverages a robust and biocompatible ligation, paves the way for transcriptome-wide mapping of multiple RNA modifications at site resolution. This successful in vitro proof-of-concept lays the groundwork for future applications in living cells. Ultimately, this chemical strategy could be combined with metabolic labelling to hijack the native RNA methylation machinery, allowing for the installation of a minimally perturbing allyl handle at endogenous modification sites for subsequent chemical ligation and transcriptome-wide analysis using Illumina or nanopore-based platforms.","abstract_has_math":false,"creators":["Ivey, Galway"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Bernardes, Gonçalo","Blackwell, John Henry"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-10-30","date_published":"2025-10-30","updated_at":"2026-07-24T01:33:11Z","subjects":["Bioorthogonal","IEDDA","RNA Methylation","Metabolic Hijacking","Reverse Transcription","Nanopore"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/d5a4c2cd-f915-4d06-98d0-fbd3bbc07620/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.132092","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Bernardes, Gonçalo","Blackwell, John Henry"]},{"key":"dc:creator","label":"Author","values":["Ivey, Galway"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-10-30"]},{"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/1810/405964"]},{"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":["Bioorthogonal","IEDDA","RNA Methylation","Metabolic Hijacking","Reverse Transcription","Nanopore"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/d5a4c2cd-f915-4d06-98d0-fbd3bbc07620/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2027-07-13"]},{"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.132092"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/3a36b4db-ffff-4a43-88af-a1768fd94027/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Post-transcriptional RNA modifications, such as N6-methyladenosine (m6A), 2'-O-methylation (Nm), and 5-methylcytosine (m5C), are critical regulators of gene expression. Mapping these modifications at single-nucleotide resolution, however, remains a major challenge as current methods often suffer from low resolution or antibody-related biases. While bioorthogonal chemistry offers a powerful approach for RNA analysis, established reactions like the copper- catalysed azide-alkyne cycloaddition (CuAAC) can have disadvantages for modification mapping, including copper-mediated RNA degradation that can compromise data integrity. This highlights a critical need for further biocompatible reactions tailored for epitranscriptome analysis. To address this, this thesis details the development and systematic evaluation of a bioorthogonal strategy specifically designed to be advantageous for mapping RNA modifications. The foundational work was conducted in an isolated in vitro setting on synthetic oligonucleotides to investigate precisely how the installed chemical modifications impact key detection technologies, namely reverse transcription and nanopore sequencing. In this work, an inverse electron-demand Diels-Alder (IEDDA) reaction between allyl-modified nucleosides and a dipyridyl tetrazine was developed and characterised. This reaction uniquely generates a sterically bulky double-addition product for all nucleosides explored. To facilitate the creation of the necessary tools for this work, a highly efficient, single-step synthesis of the required allyl-modified phosphoramidites was also devised directly from a commercially available precursor. Using synthetic RNA oligonucleotides, it was demonstrated that the IEDDA adduct acts as an effective steric block that consistently induces full truncation of cDNA synthesis during reverse transcription, which could provide an unambiguous readout for high- resolution mapping with standard Illumina sequencing platforms. Furthermore, it was shown that the same modification produces a distinct and detectable signal during nanopore direct RNA sequencing, establishing the dual compatibility of the approach. Altogether, this thesis establishes a powerful and versatile antibody- and copper-free platform for epitranscriptome analysis, validated here in an isolated setting using synthetic oligonucleotides. The dual-readout methodology, which leverages a robust and biocompatible ligation, paves the way for transcriptome-wide mapping of multiple RNA modifications at site resolution. This successful in vitro proof-of-concept lays the groundwork for future applications in living cells. Ultimately, this chemical strategy could be combined with metabolic labelling to hijack the native RNA methylation machinery, allowing for the installation of a minimally perturbing allyl handle at endogenous modification sites for subsequent chemical ligation and transcriptome-wide analysis using Illumina or nanopore-based platforms."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["4ed0c4b6df6751655193a52e10c24ce9","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["An Inverse Electron-Demand Diels-Alder Strategy for Mapping RNA Methylation"]}]}],"canonical_facts":{"dc:contributor.advisor":["Bernardes, Gonçalo","Blackwell, John Henry"],"dc:creator":["Ivey, Galway"],"dc:date.issued":["2025-10-30"],"dc:description.abstract":["Post-transcriptional RNA modifications, such as N6-methyladenosine (m6A), 2'-O-methylation (Nm), and 5-methylcytosine (m5C), are critical regulators of gene expression. Mapping these modifications at single-nucleotide resolution, however, remains a major challenge as current methods often suffer from low resolution or antibody-related biases. While bioorthogonal chemistry offers a powerful approach for RNA analysis, established reactions like the copper- catalysed azide-alkyne cycloaddition (CuAAC) can have disadvantages for modification mapping, including copper-mediated RNA degradation that can compromise data integrity. This highlights a critical need for further biocompatible reactions tailored for epitranscriptome analysis. To address this, this thesis details the development and systematic evaluation of a bioorthogonal strategy specifically designed to be advantageous for mapping RNA modifications. The foundational work was conducted in an isolated in vitro setting on synthetic oligonucleotides to investigate precisely how the installed chemical modifications impact key detection technologies, namely reverse transcription and nanopore sequencing. In this work, an inverse electron-demand Diels-Alder (IEDDA) reaction between allyl-modified nucleosides and a dipyridyl tetrazine was developed and characterised. This reaction uniquely generates a sterically bulky double-addition product for all nucleosides explored. To facilitate the creation of the necessary tools for this work, a highly efficient, single-step synthesis of the required allyl-modified phosphoramidites was also devised directly from a commercially available precursor. Using synthetic RNA oligonucleotides, it was demonstrated that the IEDDA adduct acts as an effective steric block that consistently induces full truncation of cDNA synthesis during reverse transcription, which could provide an unambiguous readout for high- resolution mapping with standard Illumina sequencing platforms. Furthermore, it was shown that the same modification produces a distinct and detectable signal during nanopore direct RNA sequencing, establishing the dual compatibility of the approach. Altogether, this thesis establishes a powerful and versatile antibody- and copper-free platform for epitranscriptome analysis, validated here in an isolated setting using synthetic oligonucleotides. The dual-readout methodology, which leverages a robust and biocompatible ligation, paves the way for transcriptome-wide mapping of multiple RNA modifications at site resolution. This successful in vitro proof-of-concept lays the groundwork for future applications in living cells. Ultimately, this chemical strategy could be combined with metabolic labelling to hijack the native RNA methylation machinery, allowing for the installation of a minimally perturbing allyl handle at endogenous modification sites for subsequent chemical ligation and transcriptome-wide analysis using Illumina or nanopore-based platforms."],"dc:format.checksum.md5":["4ed0c4b6df6751655193a52e10c24ce9","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.132092"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/3a36b4db-ffff-4a43-88af-a1768fd94027/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/405964"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/d5a4c2cd-f915-4d06-98d0-fbd3bbc07620/download","http://purl.org/NET/rdflicense/allrightsreserved"],"dc:rights.embargodate":["2027-07-13"],"dc:rights.embargotype":["embargo"],"dc:subject":["Bioorthogonal","IEDDA","RNA Methylation","Metabolic Hijacking","Reverse Transcription","Nanopore"],"dc:title":["An Inverse Electron-Demand Diels-Alder Strategy for Mapping RNA Methylation"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T01:33:11Z"}