{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/397804"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/397804","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Development of synthetic RNA-targeting catalysts from biostable nucleic acid analogues","abstract":"RNAs are major biomolecular players in biology and disease, and are a rich source of novel drug targets for molecular medicine. However, therapeutic modulation of specific RNAs remains a significant technical challenge. Single-stranded nucleic acids can fold into complex three-dimensional structures capable of catalysis of a wide range of chemical reactions, particularly RNA processing, playing critical roles in biology. RNA endonuclease “ribozymes” and “DNAzymes”, either re-purposed from nature or evolved in vitro, have been an active area of research with the promise of applications as molecular tools for specific RNA knockdown. In principle, they could provide a rich source of specific, modular, ‘programmable’ single agents. However, key challenges remain, mainly due to the limitations of natural DNA and RNA backbones. To enhance the prospects of in vivo applications, modification of oligonucleotide catalysts with synthetic DNA and RNA analogues—known as XNAs—is being explored, as well as the development of catalysts entirely composed of XNAs, “XNAzymes”. Our aim here is to evolve, engineer, and test RNA-cleaving XNAzymes capable of targeting disease-associated RNA under physiological conditions. This approach seeks to overcome the limitations of other RNA knockdown methods, principally the lack of single-nucleotide specificity and off-target effects, and—in the case of ribozyme and DNAzyme catalysts in particular—poor biostability and activity on long, structured all-RNA targets under physiological conditions. To explore the possibility of using XNAzymes to specifically target microRNAs and other non-coding RNAs, we retargeted a previously characterised XNAzyme composed of 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (FANA). We demonstrated the effectiveness of this approach in generating novel catalysts with high specificity for individual non-coding RNAs within families of similar sequences in in vitro assays under quasi-physiological conditions; however, we noted that the FANA chemistry still faces issues of poor biostability and ability to recruit RNase H, which is likely to reduce specificity in vivo. To address these limitations, we concurrently attempted to evolve novel first-in-class XNAzymes composed of highly biostable chemistries with reduced or no ability to recruit RNase H: locked nucleic acids (LNA), 2′OMe-RNA, or combinations of both, targeting microRNA-122. Additionally, we sought to improve the activity of a previously selected 2′OMe-RNA catalyst (“R15/5-K”), targeting specifically the transcript of the KRAS oncogenic G12D variant (cG35A). Through re-selection, we enhanced its catalytic rate by four-fold (0.42 h⁻¹) and increased its maximum cleavage by 25% (cf. rate of KRAS transcript turnover is between 0.2 and 0.6 h⁻¹). We also characterised its capacity for allele-specific cleavage of long transcripts under near-physiological conditions, and its biostability in the presence of serum nucleases and cell lysates. As a step towards in vivo characterisation of this and other oligo catalysts, we have established assays to measure persistence and activity of XNAzymes inside cells and rigorously explored potential sources of errors and false positives in knockdown experiments, as well as strategies to avoid them. Taken together, we hope these results and general strategies will serve as a framework for development and evaluation of such catalysts and facilitate progress towards clinical applications.","abstract_html":"RNAs are major biomolecular players in biology and disease, and are a rich source of novel drug targets for molecular medicine. However, therapeutic modulation of specific RNAs remains a significant technical challenge. Single-stranded nucleic acids can fold into complex three-dimensional structures capable of catalysis of a wide range of chemical reactions, particularly RNA processing, playing critical roles in biology. RNA endonuclease “ribozymes” and “DNAzymes”, either re-purposed from nature or evolved in vitro, have been an active area of research with the promise of applications as molecular tools for specific RNA knockdown. In principle, they could provide a rich source of specific, modular, ‘programmable’ single agents. However, key challenges remain, mainly due to the limitations of natural DNA and RNA backbones. To enhance the prospects of in vivo applications, modification of oligonucleotide catalysts with synthetic DNA and RNA analogues—known as XNAs—is being explored, as well as the development of catalysts entirely composed of XNAs, “XNAzymes”. Our aim here is to evolve, engineer, and test RNA-cleaving XNAzymes capable of targeting disease-associated RNA under physiological conditions. This approach seeks to overcome the limitations of other RNA knockdown methods, principally the lack of single-nucleotide specificity and off-target effects, and—in the case of ribozyme and DNAzyme catalysts in particular—poor biostability and activity on long, structured all-RNA targets under physiological conditions. To explore the possibility of using XNAzymes to specifically target microRNAs and other non-coding RNAs, we retargeted a previously characterised XNAzyme composed of 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (FANA). We demonstrated the effectiveness of this approach in generating novel catalysts with high specificity for individual non-coding RNAs within families of similar sequences in in vitro assays under quasi-physiological conditions; however, we noted that the FANA chemistry still faces issues of poor biostability and ability to recruit RNase H, which is likely to reduce specificity in vivo. To address these limitations, we concurrently attempted to evolve novel first-in-class XNAzymes composed of highly biostable chemistries with reduced or no ability to recruit RNase H: locked nucleic acids (LNA), 2′OMe-RNA, or combinations of both, targeting microRNA-122. Additionally, we sought to improve the activity of a previously selected 2′OMe-RNA catalyst (“R15/5-K”), targeting specifically the transcript of the KRAS oncogenic G12D variant (cG35A). Through re-selection, we enhanced its catalytic rate by four-fold (0.42 h⁻¹) and increased its maximum cleavage by 25% (cf. rate of KRAS transcript turnover is between 0.2 and 0.6 h⁻¹). We also characterised its capacity for allele-specific cleavage of long transcripts under near-physiological conditions, and its biostability in the presence of serum nucleases and cell lysates. As a step towards in vivo characterisation of this and other oligo catalysts, we have established assays to measure persistence and activity of XNAzymes inside cells and rigorously explored potential sources of errors and false positives in knockdown experiments, as well as strategies to avoid them. Taken together, we hope these results and general strategies will serve as a framework for development and evaluation of such catalysts and facilitate progress towards clinical applications.","abstract_has_math":false,"creators":["Donde, Maria"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Taylor, Alex"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-05-27","date_published":"2025-05-27","updated_at":"2026-07-22T22:24:16Z","subjects":["Catalytic oligos","Nucleic acids","RNA knockdown","SELEX","XNA","XNAzyme"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/d9bd2527-c791-4c77-bdab-fb3c8162f447/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000342164719"],"render_values":[{"text":"0000-0003-4216-4719","href":"https://orcid.org/0000-0003-4216-4719","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.126840","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Taylor, Alex"]},{"key":"dc:creator","label":"Author","values":["Donde, Maria"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000342164719"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-05-27"]},{"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/1810/397804"]},{"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":["Catalytic oligos","Nucleic acids","RNA knockdown","SELEX","XNA","XNAzyme"]}]},{"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/d9bd2527-c791-4c77-bdab-fb3c8162f447/download","http://purl.org/NET/rdflicense/allrightsreserved"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2027-02-11"]},{"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.126840"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/94245a03-1b95-4b6b-a003-6cdf250919f4/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["RNAs are major biomolecular players in biology and disease, and are a rich source of novel drug targets for molecular medicine. 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Our aim here is to evolve, engineer, and test RNA-cleaving XNAzymes capable of targeting disease-associated RNA under physiological conditions. This approach seeks to overcome the limitations of other RNA knockdown methods, principally the lack of single-nucleotide specificity and off-target effects, and—in the case of ribozyme and DNAzyme catalysts in particular—poor biostability and activity on long, structured all-RNA targets under physiological conditions. To explore the possibility of using XNAzymes to specifically target microRNAs and other non-coding RNAs, we retargeted a previously characterised XNAzyme composed of 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (FANA). We demonstrated the effectiveness of this approach in generating novel catalysts with high specificity for individual non-coding RNAs within families of similar sequences in in vitro assays under quasi-physiological conditions; however, we noted that the FANA chemistry still faces issues of poor biostability and ability to recruit RNase H, which is likely to reduce specificity in vivo. To address these limitations, we concurrently attempted to evolve novel first-in-class XNAzymes composed of highly biostable chemistries with reduced or no ability to recruit RNase H: locked nucleic acids (LNA), 2′OMe-RNA, or combinations of both, targeting microRNA-122. Additionally, we sought to improve the activity of a previously selected 2′OMe-RNA catalyst (“R15/5-K”), targeting specifically the transcript of the KRAS oncogenic G12D variant (cG35A). Through re-selection, we enhanced its catalytic rate by four-fold (0.42 h⁻¹) and increased its maximum cleavage by 25% (cf. rate of KRAS transcript turnover is between 0.2 and 0.6 h⁻¹). We also characterised its capacity for allele-specific cleavage of long transcripts under near-physiological conditions, and its biostability in the presence of serum nucleases and cell lysates. As a step towards in vivo characterisation of this and other oligo catalysts, we have established assays to measure persistence and activity of XNAzymes inside cells and rigorously explored potential sources of errors and false positives in knockdown experiments, as well as strategies to avoid them. 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This approach seeks to overcome the limitations of other RNA knockdown methods, principally the lack of single-nucleotide specificity and off-target effects, and—in the case of ribozyme and DNAzyme catalysts in particular—poor biostability and activity on long, structured all-RNA targets under physiological conditions. To explore the possibility of using XNAzymes to specifically target microRNAs and other non-coding RNAs, we retargeted a previously characterised XNAzyme composed of 2′-deoxy-2′-fluoro-β-D-arabino nucleic acid (FANA). We demonstrated the effectiveness of this approach in generating novel catalysts with high specificity for individual non-coding RNAs within families of similar sequences in in vitro assays under quasi-physiological conditions; however, we noted that the FANA chemistry still faces issues of poor biostability and ability to recruit RNase H, which is likely to reduce specificity in vivo. To address these limitations, we concurrently attempted to evolve novel first-in-class XNAzymes composed of highly biostable chemistries with reduced or no ability to recruit RNase H: locked nucleic acids (LNA), 2′OMe-RNA, or combinations of both, targeting microRNA-122. Additionally, we sought to improve the activity of a previously selected 2′OMe-RNA catalyst (“R15/5-K”), targeting specifically the transcript of the KRAS oncogenic G12D variant (cG35A). Through re-selection, we enhanced its catalytic rate by four-fold (0.42 h⁻¹) and increased its maximum cleavage by 25% (cf. rate of KRAS transcript turnover is between 0.2 and 0.6 h⁻¹). We also characterised its capacity for allele-specific cleavage of long transcripts under near-physiological conditions, and its biostability in the presence of serum nucleases and cell lysates. As a step towards in vivo characterisation of this and other oligo catalysts, we have established assays to measure persistence and activity of XNAzymes inside cells and rigorously explored potential sources of errors and false positives in knockdown experiments, as well as strategies to avoid them. 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