Aug 2026· Journal of Visualized Experiments· Vol 234· 0 citations
Medicine
TL;DR
The versatility of the assay is demonstrated and a 4-fold reduction in 5' cap concentration for in vitro transcription results in equivalent translation in cellulo, illustrating how split luminescent tagging can be easily integrated into the mRNA therapeutic workflow, enabling monitoring of real-time mRNA-driven protein expression dynamics in cellulo.
Abstract
The swift vaccine development to combat COVID-19 illustrated the potential for messenger RNA (mRNA) therapeutics to transform drug development. Like mature mRNA, in vitro transcribed mRNA possesses the same elements including a 5' cap, untranslated regions (UTRs), coding sequence and a poly(A) tail. Previous work studying the effects these components have on mRNA translation has primarily utilized highly engineered reporter proteins which exhibit efficient translation and protein stability. With the structural elements of each mRNA differentially affecting their translation, it is imperative to identify the optimal design relevant to the therapeutic protein of interest (POI). To enable POI translation characterization, a split luciferase complementation system was employed. A short peptide tag (HiBiT), which can be fused to either terminus of the POI, associates with its complementary heterodimer (LgBiT) to reconstitute enzymatic activity in the presence of a cell-permeable substrate. To date, split luminescent tagging has been primarily used for high-throughput protein turnover studies. We have previously demonstrated how split luminescent tagging can be employed to enable high-throughput quantification of mRNA translation temporally in cellulo in HEK293 cells constitutively expressing the complementary heterodimer. Here, we further demonstrate the versatility of the assay and detail how this assay can be employed for optimizing in vitro transcription to reduce costs. The assay system can uniquely distinguish alterations in structural components whilst highlighting the effects of coding sequence optimization using non-engineered genes. Additionally, we demonstrate that a 4-fold reduction in 5' cap concentration for in vitro transcription results in equivalent translation in cellulo. These findings illustrate how split luminescent tagging can be easily integrated into the mRNA therapeutic workflow, enabling monitoring of real-time mRNA-driven protein expression dynamics in cellulo thereby offering a versatile method for the advancement of mRNA-based therapeutics.
mRNA therapeutics have demonstrated transformative potential in infectious disease, oncology and genetic disorders. Parallel to the rapid clinical advancement of mRNA therapies, research into in vitro transcription (IVT), the pivotal process of mRNA manufacturing, has intensified significantly. However, the majority of these studies omitted cap analog, an essential substrate for co-transcriptional capped IVT (co-IVT), likely to simplify experimental systems. Nevertheless, co-IVT remains the dominant industrial standard for the production of mRNA therapeutics. Here, we report an optimized co-IVT process that achieves a yield of more than 26 g/L while maintaining capping efficiency of over 99.5%, and achieving a 30-fold reduction in double-stranded RNA (dsRNA), the most critical process-related impurity. These performance gains substantially improve mRNA production efficiency and enhance mRNA quality, thereby accelerating the transition to commercial-scale manufacturing of mRNA therapeutics. Moreover, the performance was attained using either of two mechanistically distinct engineered T7 RNA polymerase (RNAP) mutants. Both mutants enabled robust mRNA synthesis under process conditions that have not been previously reported. These observations provide mechanistic insights into IVT and advance our fundamental understanding of this critical bio-manufacturing step.
Wei He, Kunkun Zhang, Guiying Ji et al.· bioRxiv· 0 citations
Efficient protein synthesis in eukaryotic cells typically requires a 5′ cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here, we describe a cell-free protocol to quantitatively assess cap-independent translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis, and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of translation activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key features • Stringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. • Evaluation of cap-independent translation suitable for testing combinations of IRES and CDS. • Translation analysis without radioactive labeling.
Max Cortot, Thorsten Stehlik, Aline Koch et al.· Bio-protocol· 0 citations
mRNA is a new medical modality as vaccine and raises hopes to become a protein replacement agent. Despite notable advances in manufacturing, stabilization and modification, its short half-life remains a major limitation. Circular RNA bears potential to solve this issue, as degradation of mRNA proceeds primarily from the 5' and 3' ends. However, circular RNAs normally lack a 5' cap and are less efficiently translated. We present a strategy to circularize mRNA covalently via click chemistry by modifying the 5' cap and the 3' poly(A) tail with bioorthogonal functional groups. The resulting Cyclic Click Cap-containing mRNA (CyCliCap-mRNA) is translationally muted by a photocleavable protecting group. Upon irradiation by light, the photocleavable protecting group is removed and the respective linear Cap0-mRNA released. We show that CyCliCap-mRNA can be activated for efficient translation at late timepoints (24-48 h post transfection) in HeLa cells, which is not possible with the respective linear mRNA. The light-mediated opening releases linear mRNA with a native Cap0 and thus overcomes the problem of inefficient translation of circular RNAs.
Bayram Terzi, Greta Charlotte Dahm, Kristoffer Heintz et al.· Angewandte Chemie· 0 citations
Viruses encode diverse regulatory elements, but their breadth and mechanisms remain poorly defined. To address this gap, we performed massively parallel reporter assays spanning ∼200,000 genomic segments from 297 vertebrate-infecting viral genera. We identified numerous viral elements that enhance RNA stability and translation through TENT4-mediated mixed tailing, distributed across 19 genera and grouped into six distinct subclasses, indicating extensive convergent evolution. We also found diverse TENT4-independent elements acting through alternative pathways. One such element, Pt1 from Potamipivirus, stabilizes linear mRNA to levels comparable to circular RNA, suggesting its potential for RNA therapeutics. Pt1 directly recruits canonical poly(A) polymerases (PAPγ/α)-previously thought to function exclusively in transcription-coupled nuclear pre-mRNA processing-to drive cytoplasmic polyadenylation. Together, these findings chart the rich landscape of viral regulation, extend the scope of poly(A)-tail biology, and establish the virome as a valuable source for uncovering host RNA regulatory mechanisms.
Jenny J Seo, Chemin Lee, Dongbin Lim et al.· Cell· 0 citations
A new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs), engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins is developed.
Synthetic mRNA can be used to reprogram biological systems and is increasingly used in vaccines, gene therapies and other advanced therapeutics. However, measuring mRNA abundance, molecular integrity and biological effects in complex samples remains challenging. Existing assays typically quantify short transcript regions or infer delivery from lipid or protein readouts. Here we present a long-read nanopore sequencing method that directly quantifies synthetic mRNA in complex cell and tissue samples. The approach enables absolute quantification of full-length synthetic mRNA, maps degradation at nucleotide resolution and simultaneously profiles associated host transcriptional responses. Applied to lipid nanoparticle (LNP) delivered mRNA in mice, the method revealed tissue-specific delivery and degradation patterns and uncovered a critical disconnect between mRNA accumulation and protein expression across organs. This approach enables integrated measurement of mRNA fate, integrity and biological responses, and will enable mechanistic studies of RNA delivery, stability, translation and innate immune recognition.
Victoria McLeod, D. Yuen, Moore Z. Chen et al.· bioRxiv· 0 citations