A proximity-labeling-based approach to directly detect mRNA delivery to specific subcellular locations

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Messenger RNA (mRNA) therapeutics offer significant potential but are hindered by delivery challenges, primarily endosomal entrapment limiting cytosolic access and translation.
• Current methods for assessing RNA delivery are often indirect, qualitative, or require labeled RNA, creating a need for direct, quantitative approaches.
• Understanding mRNA localization within the cell is crucial for optimizing therapeutic efficacy.

Purpose of the Study:

• To develop and validate a quantitative method for directly measuring mRNA delivery to specific subcellular locations.
• To investigate the efficiency of mRNA delivery to the cytoplasm versus the endoplasmic reticulum (ER).
• To explore the role of signal peptides in directing mRNA to the ER for enhanced translation.

Main Methods:

• Adaptation of the APEXseq proximity biotinylation technique for isolating mRNA at subcellular locations.
• Combination of APEX2 labeling with reverse-transcription quantitative PCR (RT-qPCR) to quantify mRNA in the cytoplasm and ER.
• Incorporation of a biotinylated spike-in RNA for data normalization and optimization of mRNA isolation.
• Integration with protein assays to assess the impact of signal peptides on mRNA localization and translation.

Main Results:

• The adapted APEXseq method provides a direct and quantitative measure of mRNA delivery to cellular compartments.
• mRNA delivery and subsequent translation were found to be most efficient in the endoplasmic reticulum compared to the cytoplasm.
• The study successfully utilized a spike-in RNA for improved methodological robustness and optimization.
• Initial investigations suggest signal peptides play a role in directing mRNA to the ER.

Conclusions:

• The developed APEXseq-based approach offers a powerful new tool for directly quantifying therapeutic mRNA delivery to specific subcellular sites.
• The endoplasmic reticulum is identified as a highly effective site for mRNA translation, suggesting strategic targeting could enhance therapeutic outcomes.
• This methodology facilitates future research into optimizing mRNA delivery and overcoming cellular barriers for therapeutic applications.