Issue:June 2025
PROTEIN BARCODING - Streamlining mRNA Therapeutic & Vaccine Development
INTRODUCTION
Nucleic acid-based vaccines and therapeutics, including mRNA-based platforms, represent a groundbreaking advance in healthcare. A critical determinant of therapeutic efficacy of these treatments is the delivery system, which ensures precise targeting to the intended tissues while also minimizing degradation and off-target effects.
Lipid nanoparticles (LNPs) have emerged as a powerful tool for delivery due to their low immunogenicity compared to viral vectors, along with their ability to encapsulate and protect nucleic acids and facilitate their entry into cells. LNPs are used in the COVID-19 mRNA vaccines, for siRNA delivery, and in numerous clinical trials with a range of payloads. However, the majority of these applications are in liver-focused disease areas simply because that is where LNPs accumulate in the body when administered systemically.1
TARGETING LNP DELIVERY
Even subtle changes in LNP lipid composition, ratios, particle size, or surface modifications can significantly alter how the LNP behaves in vivo and thus affect biodistribution, immunogenicity, and expression kinetics. As mRNA therapies move beyond the liver and into applications targeting other tissues, customized LNP formulations are essential for precise and effective delivery.
Directing mRNA to different tissues thus requires high-throughput screening of a vast number of mRNA-LNP formulations. In general, the LNP screening process involves formulating a library of different LNP compositions and administering them, often one at a time, in animal models. Tissues are collected and mRNA expression is measured, typically through protein output or reporter assays (Figure 1). This approach is labor-intensive, low-throughput, and expensive, as each formulation must be evaluated in separate animals or animal groups.
The specific steps in the workflow for creating and screening mRNA-LNPs vary depending on the specific assay requirements, but generally include the following:
Select, synthesize, and characterize the mRNA payload: Confirm the mRNA sequence after synthesis, determine purity, and characterize/optimize the 5’ cap and 3’ poly(A) tail.
Select and formulate lipid components: To achieve maximum efficacy, each LNP formulation must be optimized for specific biophysical characteristics such as shape, size, payload density, and surface charge.
Select the screening method: Select in vitro (2D or 3D cell culture) or in vivo (animal models) methods.
Select the screening readout: Fluorescence or next-generation sequencing, depending on the reporter used in the mRNA construct.
Encapsulate the mRNA into the LNP: The purified mRNA is combined with the lipid nanoparticle solution, allowing the mRNA to be encapsulated within the LNP’s hydrophobic core.
Quality control and characterize the final product: Perform analytical tests to assess the size, stability, encapsulation efficiency, and purity of the mRNA-LNP formulation.
Perform the screen: Based on the method and readout selected.
Draw conclusions: Identify the top candidates from the screen and feed the results back into mRNA and lipid formulations for further studies and optimization.
While different tools are available to support nanoparticle design, formulation, and quality control (steps 1-6), the screening step (step 7), still faces limitations with current methods, which include next-generation sequencing (NGS), mass spectrometry, flow cytometry, ELISA, and Western blotting.2-7 For example, using NGS for screening is efficient because it allows for the analysis of multiple mRNAs in a single animal. However, a significant limitation of this approach is that it measures delivery – ie, the presence of mRNA – rather than translation of the mRNA into the desired protein.5,6
This can lead to inaccurate results and erroneous candidate selection.
In contrast, screening that uses a protein-level readout, such as mass spectrometry (MS) or flow cytometry, provides evidence that the mRNA was both delivered and translated effectively.7,8
Limited tools are available for multiplexing at the protein level, which increases costs and reduces efficiency; in the case of flow cytometry, this also results in low sensitivity. Therefore, new tools that facilitate multiplex pooled screening with a direct protein-level readout could substantially improve mRNA-LNP screening and accelerate the development of new nucleic-acid-based therapeutic and vaccine candidates.
Newer techniques, such as protein barcoding, are now being employed to accelerate and scale up this process. This multiplexed approach enables the simultaneous evaluation of mRNA-LNP formulations in a single experiment, significantly accelerating the identification of lead candidates with optimal biodistribution and transfection profiles, at a reduced cost.
THE BASICS OF PROTEIN BARCODING
Protein barcodes are information-rich short stretches of amino acids that can be genetically added to the coding sequences of proteins. Proteins expressing unique barcodes can be co-expressed together, and desired proteins can be selected for protein engineering, mRNA translation, therapeutic delivery mechanisms, and many other protein-screening applications. Upon protein selection, distinct barcodes associated with each expressed protein can be directly identified and quantified with single-molecule resolution via next-gen protein sequencing (NGPS). This approach has the potential to transform drug discovery and development, similar to how DNA barcodes have advanced the field of genomics. Importantly, NGPS offers significant advantages over the use of MS for decoding protein barcodes. These advantages include a simple, user-friendly workflow on a benchtop instrument that distinguishes peptides based on recognition of specific amino acids rather than mass/charge ratio. Thus, protein barcodes for NGPS readout can be engineered to have highly similar physical properties while remaining distinguishable by their amino acid sequences.
By appending the coding sequences for protein barcodes to each mRNA coding sequence, expressed proteins will have unique tags, despite the proteins themselves having the same amino acid sequence. Sequencing the barcodes on the Platinum Pro® NGPS platform allows rapid identification of which mRNA had the highest expression, resulting in the most abundant protein. These sequencing results can also be used to determine the effectiveness of different lipid nanoparticle delivery systems.
ADVANTAGES FOR IN VIVO MRNA-LNP
While in vitro assays are essential for early screening, they can’t capture the full biological complexity of how mRNA-LNPs behave in the body. Once administered in vivo, LNPs interact with endogenous proteins that can coat the particle and significantly influence biodistribution, an effect that is both unpredictable and impossible to replicate in vitro. In vivo studies are therefore critical to truly understand delivery efficiency and tissue targeting. Protein barcoding offers a powerful solution by enabling multiplex-pooled screens with a direct protein-level readout, increasing the scale of screening while significantly reducing costs and the number of animals required for the all-important in vivo studies (Figure 2).
SIGNIFICANT REDUCTION IN IN VIVO MRNA-LNP SCREENING COSTS
As summarized in Table 1, a simulated study using multiplexing study with barcodes offers the potential to significantly improve the process economics of mRNA-LNP screening in animal models. By allowing multiple candidates to be tested simultaneously in a single animal, this approach maximizes data output per study and minimizes the number of animals needed. The result is a more efficient screening process that reduces both time and development costs, accelerating the path to identifying optimal delivery vehicles, and aligns with recent FDA plans to reduce animal testing in drug development.9
In vivo studies in mice using barcodes and NGPS can reduce costs by 71% and the number of animals required by a factor of eight. In studies with non-human primates (NHPs), the number of animals required can also be reduced eightfold, with an 86% reduction in costs.
PROTEIN BARCODES HELP UNLOCK THE FUTURE OF NUCLEIC ACID THERAPEUTICS
As mRNA and other nucleic-acid-based therapeutics and vaccines expand beyond liver-targeted applications, the need for precise, tissue-specific delivery becomes increasingly urgent. Protein barcoding offers a transformative leap forward in this effort, one that not only addresses the limitations of current screening technologies but also redefines the scale and efficiency of in vivo testing. By enabling multiplexed, protein-level readouts within a single animal, this approach dramatically reduces both time and cost while improving the quality of data generated.
More than just a technical advancement, protein barcoding is a catalyst for unlocking the full therapeutic potential of nucleic acid-based medicines. It empowers researchers to rapidly identify the most promising delivery systems, paving the way for safer, more effective treatments that reach beyond the liver and into a broader range of diseases and tissues. In short, protein barcoding doesn’t just streamline the development pipeline — it also opens new doors for innovation in genetic medicine.
*Cost estimates provided are for informational purposes only and are based on general assumptions regarding experimental design, throughput, and resource utilization. Actual savings will vary depending on multiple factors, including but not limited to: the specific workflow and protocols used, the scale and frequency of experiments, reagent and consumable costs, labor costs, institutional pricing and discount structures, equipment depreciation, and regulatory or compliance requirements. Quantum-Si makes no guarantees regarding specific cost reductions or financial outcomes. Customers should perform their own cost analysis based on their unique operational parameters.
REFERENCES
- Kulkarni, J.A., Witzigmann, D., Thomson, S.B., Chen, S., Leavitt, B.R., Cullis, P.R., and Van der Meel, R. (2021). The current landscape of nucleic acid therapeutics. Nat. Nanotechnol 16, 630–643. doi.org/10.1038/ s41565-021-00898-0
- Khirallah, J., and Xu, Q. (2023). A high-throughput approach of screening nanoparticles for targeted delivery of mRNA. Cell Reports Methods 3, 100572. doi. org/10.1016/j.crmeth.2023.100572.
- Guimaraes, P.P.G., Zhang, R., Spektor, R., Tan, M., Chung, A., Billingsley, M.M., El-Mayta, R., Riley, R.S., Wang, L., Wilson, J.M., and Mitchell, M.J. (2019). Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening. J Control Release 316, 404–417. doi.org/10.1016/j.jconrel.2019.10.028
- Cui, L., Pereira, S., Sonzini, S., van Pelt, S., Romanelli, S.M., Liang, L., Ulkoski, D., Krishnamurthy, V.R., Brannigan, E., Brankin, C., and Desai, A.S. (2022). Development of a high-throughput platform for screening lipid nanoparticles for mRNA delivery. Nanoscale 14, 1480- 1491. doi.org/10.1039/D1NR06858J.
- Gu, L., Li, C., Aach, J., Hill, D.E., Vidal, M., and Church, G.M. (2014). Multiplex single-molecule interaction profiling of DNA-barcoded proteins. Nature 515, 554– 557. doi.org/10.1038/nature13761.
- Biggs, B.W., Price, M.N., Lai, D., Escobedo, J., Fortanel, Y., Huang, Y.Y., Kim, K., Trotter, V.V., Kuehl, J.V., Lui, and L.M. (2024). High-throughput protein characterization by complementation using DNA barcoded fragment libraries. Mol Syst Biol 20, 1207–1229. doi:10.1038/ s44320-024-00068-z.
- Suzuki, Y., Miyazaki, T., Muto, H., Kubara, K., Mukai, Y., Watari, R., Sato, S., Kondo, K., Tsukumo, S.I., Yasutomo, K., Ito, M., and Tsukahara, K. (2022). Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates. Mol Ther Nucleic Acids 30, 226-240. doi: 10.1016/j. omtn.2022.09.017.
- Lowenthal, M.S., Antonishek, A.S., and Phinney, K.W., (2024). Quantification of mRNA in Lipid Nanoparticles Using Mass Spectrometry. Analytical Chemistry 96, 1214- 1222. doi.org/10.1021/acs.analchem.3c04406.
- https://www.fda.gov/news-events/press-announcements/fda-announces-plan-phase-out-animal-testing-requirement-monoclonal-antibodies-and-other-drugs.

Dr. Meredith Carpenter is Head of Scientific Affairs at Quantum-Si, where she manages external collaborations and publication strategy. Dr. Carpenter has over 10 years of experience in developing and deploying novel genomics and multi-omics tools in the biotech industry. Prior to Quantum-Si, she held roles as Director of Assay Development at Arc Bio and Senior Director of Strategic Alliances at Cantata Bio. She earned a BS in Biology from Emory University and a PhD in Molecular and Cell Biology from UC Berkeley, and she performed post-doctoral research in the Department of Genetics at Stanford University.
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