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Direct Counting of mRNA Copies Inside Individual Lipid Nanoparticles Using In Situ Lysis and Labeling.
Sadie Graves1,2,3, Martin Jasinski1,2, Erik Olsén1,4
1Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
Analytical Chemistry
|May 29, 2026
Summary
Accurately quantifying messenger RNA (mRNA) copies within lipid nanoparticles (LNPs) is crucial for therapeutic development. A new single-particle microscopy method directly counts mRNA molecules per LNP, overcoming limitations of existing techniques.
Area of Science:
- Biotechnology
- Nanomedicine
- Molecular Biology
Background:
- Optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutics is hindered by challenges in characterizing mRNA payload heterogeneity.
- Accurate quantification of mRNA copies per LNP is difficult with standard intensity-based methods due to dye interactions and labeling variability.
Purpose of the Study:
- To develop and validate a single-particle microscopy method for direct counting of mRNA copies within individual LNPs.
- To enable precise characterization of mRNA payload and LNP size for improved therapeutic formulation.
Main Methods:
- A novel single-particle microscopy technique was developed, confining LNPs in microwells for lysis and in situ mRNA staining.
- Direct fluorescence microscopy was used to count individual mRNA molecules released from each LNP.
- LNP size measurements were integrated with mRNA copy number quantification.
Main Results:
- The method accurately quantifies mRNA copies per LNP, independent of fluorescent dye properties.
- It allows characterization of LNPs formulated with unlabeled, therapeutic-grade mRNA.
- Application to Onpattro-based formulations revealed differences in mRNA copy number, particle size, and empty LNP fraction.
Conclusions:
- This direct counting method complements intensity-based approaches for mRNA quantification in LNPs.
- It provides a valuable tool for characterizing and screening clinically relevant LNP formulations.
- Enhanced characterization of mRNA-LNPs can accelerate the development of mRNA-based therapeutics.
