Encoded synthesis and evolution of clinically approved 2'-modified ribonucleic acids

Xeno-nucleic acids (XNAs) are unnatural nucleic acids with altered sugar, phosphodiester backbone, or nucleobase components. 2'-modified nucleic acid chemistries, such as 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-2-methoxyethyl-RNA (MOE-RNA), have shown promise in clinical applications due to their enhanced properties like high antisense-binding affinity and increased biostability. However, conventional phosphor-amidite synthesis for these 2'-modified nucleic acids is limited in length, prevents parallel exploration of different sequences and modifications, and precludes evolution. This study aims to develop an encoded enzymatic synthesis approach using polymerase engineering to enable the efficient synthesis of 2'OMe-RNA and MOE-RNA. This approach opens up possibilities for the evolution and selection of modified aptamers and nucleic acid enzymes with therapeutic potential. First, I describe how structure-guided engineering on a thermophilic archaeal polymerase led to the identification and mutation of a two-residue nascent-strand steric gate near the active site. This modification alleviated steric clashes within the polymerase, enabling processive synthesis of 2'-modified nucleic acids, including mixed-chemistry 2'OMe-/MOE-RNA aptamers, and unlocking their evolution. Furthermore, a reverse transcriptase-free selection procedure was established for MOE-RNA aptamer selections, enabling re-selection of a 2'OMe-RNA VEGF aptamer in the MOE-RNA chemistry. Additionally, procedures for the de novo selection of Tau-binding RNA, 2'OMe-RNA, and MOE-RNA aptamers were established. These selection procedures involved pre-enrichment over four rounds, followed by deep screening using an Illumina HiSeq instrument. By advancing these aptamer selections further, this study lays the groundwork for the development of the first MOE-RNA aptamer and demonstrate the potential of encoded synthesis of 2'-modified nucleic acids in creating biostable aptamers using nucleic acid chemistries approved for human use.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Freund, Niklas

Advisor dc:contributor.advisor

• Holliger, Philipp

• aptamers
• directed evolution
• nucleic acid chemistry
• nucleic acid therapeutics
• polymerases
• SELEX
• xeno-nucleic acids

Licence

• https://www.repository.cam.ac.uk/bitstreams/1c72ef0e-9d34-44ea-9ab4-53c95a7fd89b/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.107154

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/366084