Abstract
N6-methyladenosine (\gls{m6a}) is one of the most common modifications found in messenger RNAs (mRNAs). The deposition of \gls{m6a} on mRNA plays a crucial role in regulating gene expression by influencing mRNA processing, structure, translation and stability. As a result, \gls{m6a} has been implicated in various diseases, including Huntington's disease (HD). This thesis focuses on targeting the differential \gls{m6a} pattern found in intron 1 of pathogenic RNA transcripts associated with HD. Our research had two primary objectives: first, we explored strategies leveraging the destabilising effect of \gls{m6a} on RNA duplex structures for selective RNA degradation using antisense oligonucleotides (ASOs), and second, we investigated how \gls{m6a} modulates RNA structure within intron 1 of HD pathogenic mRNA transcripts and its implication in small-molecule interactions.
To address the specific degradation of \gls{m6a}-modified HD pathogenic mRNA fragments, we tested various ASOs in RNaseH degradation assays to determine their ability to degrade methylated RNA over their unmethylated counterparts. On the structural front, we employed techniques such as NMR spectroscopy, circular dichroism (CD), and RNA secondary structure prediction to examine the influence of m6A on the conformation of a 42-nucleotide-long RNA fragment (\gls{htt42}) derived from Huntington's disease transcripts.
A significant development in this project was the establishment of a protocol for the large-scale production of labelled RNA, necessary for NMR spectroscopy. While traditionally performed in structural biology labs with specialized equipment for such processes, this work allows the successful production of milligram quantities of RNA in a molecular biology laboratory without specialized equipment. This represents a significant scale-up from the typical microgram quantities and is a crucial advancement towards obtaining NMR data on longer RNA fragments.
The NMR analysis presented in this thesis has allowed for the partial assignment of imino proton resonances and non-exchangeable protons in unstructured regions of \gls{htt42} RNA. Our NMR investigation of the methylated version of the \gls{htt42} RNA transcript constitutes one of the few solution NMR studies of longer RNA fragments containing \gls{m6a}. Our data analysis suggests the appearance of three additional base pairs in the presence of \gls{m6a}. These changes may reflect a shift in population stability or the formation of new base pairs.
Overall, this thesis outlines significant developments in RNA production for NMR studies and provides exploratory data that advance our understanding of \gls{m6a}'s role in RNA structure and binding interactions. Our results will help the further development of these projects by guiding molecular dynamics (MD) simulations for RNA structure prediction to perform computational screening of RNA-small molecule interactions and provide insights into ASO design strategies.
To address the specific degradation of \gls{m6a}-modified HD pathogenic mRNA fragments, we tested various ASOs in RNaseH degradation assays to determine their ability to degrade methylated RNA over their unmethylated counterparts. On the structural front, we employed techniques such as NMR spectroscopy, circular dichroism (CD), and RNA secondary structure prediction to examine the influence of m6A on the conformation of a 42-nucleotide-long RNA fragment (\gls{htt42}) derived from Huntington's disease transcripts.
A significant development in this project was the establishment of a protocol for the large-scale production of labelled RNA, necessary for NMR spectroscopy. While traditionally performed in structural biology labs with specialized equipment for such processes, this work allows the successful production of milligram quantities of RNA in a molecular biology laboratory without specialized equipment. This represents a significant scale-up from the typical microgram quantities and is a crucial advancement towards obtaining NMR data on longer RNA fragments.
The NMR analysis presented in this thesis has allowed for the partial assignment of imino proton resonances and non-exchangeable protons in unstructured regions of \gls{htt42} RNA. Our NMR investigation of the methylated version of the \gls{htt42} RNA transcript constitutes one of the few solution NMR studies of longer RNA fragments containing \gls{m6a}. Our data analysis suggests the appearance of three additional base pairs in the presence of \gls{m6a}. These changes may reflect a shift in population stability or the formation of new base pairs.
Overall, this thesis outlines significant developments in RNA production for NMR studies and provides exploratory data that advance our understanding of \gls{m6a}'s role in RNA structure and binding interactions. Our results will help the further development of these projects by guiding molecular dynamics (MD) simulations for RNA structure prediction to perform computational screening of RNA-small molecule interactions and provide insights into ASO design strategies.
| Original language | English |
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| Publication status | Published - Oct 2024 |
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