Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics
Messenger RNA (mRNA) therapeutics hold promise for treating diverse diseases but face delivery challenges, as effective cytosolic entry and subsequent translation are limited by endosomal entrapment. Nanoparticles aid mRNA transport across cell membranes, yet only a fraction of mRNA escapes endosome...
| Main Author: | |
|---|---|
| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2025
|
| Subjects: | |
| Online Access: | https://eprints.nottingham.ac.uk/81355/ |
| _version_ | 1848801318120980480 |
|---|---|
| author | Smart, Alfredo D. |
| author_facet | Smart, Alfredo D. |
| author_sort | Smart, Alfredo D. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Messenger RNA (mRNA) therapeutics hold promise for treating diverse diseases but face delivery challenges, as effective cytosolic entry and subsequent translation are limited by endosomal entrapment. Nanoparticles aid mRNA transport across cell membranes, yet only a fraction of mRNA escapes endosomes to reach the cytoplasm, limiting translational efficacy. Current methods to quantify delivered mRNA localisation lack precision, necessitating more quantitative approaches. This thesis addresses this gap by optimising the APEX2-RT-qPCR proximity biotinylation method to enable quantification of mRNA localisation in both the cytosol and endoplasmic reticulum (ER). Through this approach, the study lays groundwork for examining ER-associated localisation and translation, with preliminary investigation into the impact of signal peptides in directing delivered mRNA to the ER.
Stable HEK293T and A549 cell lines expressing APEX2 targeted to either the ER membrane (APEX2-ERM) or cytosol (APEX2-NES) were generated and APEX2 expression was characterised through RT-qPCR and western blotting. Structured illumination microscopy was used to evaluate the subcellular localisation of both the APEX2 epitope tag and APEX2-mediated labelling, with APEX2-ERM in HEK293T cells demonstrating the most effective localisation to its intended subcellular destination.
A refined streptavidin pulldown technique was also developed, using a biotinylated spike-in RNA to optimise the RNA pulldown and to normalise RTqPCR data from APEX2 RNA labelling experiments. This optimisation significantly advances the application of APEX2 with RT-qPCR, increasing data reproducibility across experiments.
In vitro transcribed (IVT) mRNAs encoding nanoluciferase with and without signal peptides were utilised to assess the extent to which APEX2-RT-qPCR could detect the localisation of delivered IVT mRNA to either the cytosol or the ER. APEX2-RT-qPCR data was most consistent when investigating ER localisation of signal peptide-containing mRNAs in the APEX2-ERM HEK293T cell line. The study then examined how substituting signal peptides in different coding regions affected both IVT mRNA localisation, as measured by APEX2-RT-qPCR, and translational output, as measured by functional assays. Overall, the PMEL SP led to greater mRNA localisation to the ER compared to the IL6 SP. Other methods were compared with APEX2-RTqPCR to investigate the localisation of both endogenous and signal peptide containing delivered mRNA across the cytosol and ER.
These findings highlight the utility of APEX2-RT-qPCR as a powerful tool for quantifying subcellular localisation of delivered mRNA to the ER, providing insights into how SPCS engineering may influence mRNA subcellular trafficking. This method, when combined with reporter assays, offers valuable potential for optimising mRNA therapeutic design by enabling quantitative assessments of localisation and translation output. |
| first_indexed | 2025-11-14T21:05:33Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-81355 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T21:05:33Z |
| publishDate | 2025 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-813552025-07-30T04:40:27Z https://eprints.nottingham.ac.uk/81355/ Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics Smart, Alfredo D. Messenger RNA (mRNA) therapeutics hold promise for treating diverse diseases but face delivery challenges, as effective cytosolic entry and subsequent translation are limited by endosomal entrapment. Nanoparticles aid mRNA transport across cell membranes, yet only a fraction of mRNA escapes endosomes to reach the cytoplasm, limiting translational efficacy. Current methods to quantify delivered mRNA localisation lack precision, necessitating more quantitative approaches. This thesis addresses this gap by optimising the APEX2-RT-qPCR proximity biotinylation method to enable quantification of mRNA localisation in both the cytosol and endoplasmic reticulum (ER). Through this approach, the study lays groundwork for examining ER-associated localisation and translation, with preliminary investigation into the impact of signal peptides in directing delivered mRNA to the ER. Stable HEK293T and A549 cell lines expressing APEX2 targeted to either the ER membrane (APEX2-ERM) or cytosol (APEX2-NES) were generated and APEX2 expression was characterised through RT-qPCR and western blotting. Structured illumination microscopy was used to evaluate the subcellular localisation of both the APEX2 epitope tag and APEX2-mediated labelling, with APEX2-ERM in HEK293T cells demonstrating the most effective localisation to its intended subcellular destination. A refined streptavidin pulldown technique was also developed, using a biotinylated spike-in RNA to optimise the RNA pulldown and to normalise RTqPCR data from APEX2 RNA labelling experiments. This optimisation significantly advances the application of APEX2 with RT-qPCR, increasing data reproducibility across experiments. In vitro transcribed (IVT) mRNAs encoding nanoluciferase with and without signal peptides were utilised to assess the extent to which APEX2-RT-qPCR could detect the localisation of delivered IVT mRNA to either the cytosol or the ER. APEX2-RT-qPCR data was most consistent when investigating ER localisation of signal peptide-containing mRNAs in the APEX2-ERM HEK293T cell line. The study then examined how substituting signal peptides in different coding regions affected both IVT mRNA localisation, as measured by APEX2-RT-qPCR, and translational output, as measured by functional assays. Overall, the PMEL SP led to greater mRNA localisation to the ER compared to the IL6 SP. Other methods were compared with APEX2-RTqPCR to investigate the localisation of both endogenous and signal peptide containing delivered mRNA across the cytosol and ER. These findings highlight the utility of APEX2-RT-qPCR as a powerful tool for quantifying subcellular localisation of delivered mRNA to the ER, providing insights into how SPCS engineering may influence mRNA subcellular trafficking. This method, when combined with reporter assays, offers valuable potential for optimising mRNA therapeutic design by enabling quantitative assessments of localisation and translation output. 2025-07-30 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/81355/1/SmartAlfredo14245040_corrections.pdf Smart, Alfredo D. (2025) Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics. PhD thesis, University of Nottingham. APEX2 proximity labelling proximity labelling APEX-RT-qPCR mRNA localisation mRNA trafficking mRNA therapeutics |
| spellingShingle | APEX2 proximity labelling proximity labelling APEX-RT-qPCR mRNA localisation mRNA trafficking mRNA therapeutics Smart, Alfredo D. Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics |
| title | Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics |
| title_full | Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics |
| title_fullStr | Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics |
| title_full_unstemmed | Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics |
| title_short | Molecular biology approaches to quantify subcellular localisation of mRNA therapeutics |
| title_sort | molecular biology approaches to quantify subcellular localisation of mrna therapeutics |
| topic | APEX2 proximity labelling proximity labelling APEX-RT-qPCR mRNA localisation mRNA trafficking mRNA therapeutics |
| url | https://eprints.nottingham.ac.uk/81355/ |