Roy, Olivia

Messenger RNA (mRNA) therapeutics have transformed vaccination and protein replacement strategies, yet efforts to improve their performance have focused largely on sequence engineering, nucleotide modification, and delivery vehicles. Here we show that mRNA function can be controlled by rational design of higher-order RNA architectures. We develop self-assembling mRNA origami (mRNA-OG), a class of unimolecular RNA nanostructures that encode protein-coding sequences within higher-order, programmable, and compact nucleic acid architectures. Using computational design and experimental validation, we demonstrate that mRNA-OG folds into well-defined nanostructures while remaining translationally competent in mammalian cells. Although folded mRNA-OG recruits ribosomes comparably to unfolded constructs, it produces lower protein output, indicating that RNA architecture can directly influence translational efficiency. The compact geometry of mRNA-OG also enhances encapsulation by cationic lipid delivery systems, suggesting a structural route to improved cargo packaging. Beyond its effects on translation, mRNA architecture modulates innate immune recognition. In primary human dendritic cells, folded and unfolded mRNA-OG elicit distinct cytokine programs and differential activation of stress-response pathways, including a modest induction of the integrated stress response that is not fully explained by canonical PKR signaling. Our results establish programmable structure as a new design parameter for mRNA therapeutics that can affect its functionality, delivery properties, and immune sensing.

Abstract Description Sequencing antigen-specific T cell receptors (TCR) remains a major technologic challenge, requiring labor- and cost-intensive single-cell approaches. We have developed a rapid and economical method for sequencing TCR mRNA from antigen stimulated peripheral blood mononuclear cells using targeted long-read nanopore sequencing with Oxford Nanopore Technologies’ (ONT) Minion sequencer. Targeted PCR amplification of TCR alpha and beta cDNA was performed with a template switching oligo (TSO). Dual unique molecular identifiers (UMI) were used for single cell, oligoclonal, or bulk cell sample specific sequence identification followed by nanopore sequencing. The reads were demultiplexed by UMIs and aligned to germline V,D, and J gene sequences from the IMGT database for identification. Error corrected sequences were then used to determine the non-templated complementarity determining region 3 (CDR3) sequence for the TCRs. From bulk PBMCs, TCR Beta Chain had 796 unique TRBV-TRBJ combination and TCR Alpha Chain had 2053 unique TRAV-TRAJ combinations. Human PBMC samples were spiked with 10% Jurkat cells and sequenced using this method for 12 hours resulting in 200K reads. After read demultiplexing, filtering (Q > 25), and alignment, it was found that 8.8% of TCR beta and 7.0% TCR alpha reads matched the Jurkat TCR sequence in the 10% spiked sample. This method provides a fast and cost-effective approach to generating antigen specific TCR sequences for downstream functional studies. Funding Sources Supported by NIH/NCI 5U01CA281660 Topic Categories Technological Innovations in Immunology (TECH)