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

## TL;DR

The paper introduces a new method to directly measure mRNA delivery to specific parts of the cell, showing that mRNA is most effectively delivered to the endoplasmic reticulum.

## Contribution

A novel proximity-labeling method was developed to quantitatively assess mRNA delivery to subcellular sites, particularly the endoplasmic reticulum.

## Key findings

- mRNA delivery is most effective in the endoplasmic reticulum.
- Signal peptides influence mRNA delivery and translation at the endoplasmic reticulum.
- A biotinylated spike-in RNA improved data normalization and pull-down optimization.

## Abstract

Messenger RNA (mRNA) therapeutics show considerable promise but face delivery challenges, as effective cytosolic entry and subsequent translation are normally limited by endosomal entrapment. While various approaches have been used to investigate determinants of effective RNA delivery, these methods tend to be indirect, qualitative, or rely on labeled RNA. There is a need for quantitative approaches that can directly measure mRNA delivery to its functional sites within the cell. Here, we adapted the APEXseq approach for proximity biotinylation and isolation of mRNA at specific subcellular locations. We combined APEX2 labeling with reverse-transcription quantitative PCR to investigate mRNA delivery to the cytoplasm and endoplasmic reticulum, the two major sites of translation, and found it was most effective in the endoplasmic reticulum. We incorporated a biotinylated spike-in RNA to improve existing methodology by allowing normalization of data and optimization of mRNA pull-down conditions. Finally, we combined this method with protein assays to investigate the role of different signal peptides in mRNA delivery to, and translation at, the endoplasmic reticulum. This new approach shows promise as a tool for future investigation of productive delivery of therapeutic mRNA.

APEX2 proximity biotinylation was adapted to investigate delivery of in vitro transcribed mRNA to subcellular locations. This was used to compare the effects of different signal peptides on mRNA delivery to the endoplasmic reticulum, in parallel to protein synthesis, showing potential for future investigation of intracellular delivery of therapeutic mRNAs.