Measuring Live-Cell mRNA Translational Dynamics with Split Luminescent Tagging in HEK293 Cells

Summary

Here, we present a flexible, high-throughput-compatible method for measuring live mRNA translation dynamics in cellulo in HEK293s, using split luminescent tagging. This versatile assay distinguishes structural effects, supports in vitro transcription optimization, and streamlines mRNA therapeutic development.

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.

Introduction

Messenger RNA (mRNA) therapeutics and research applications have rapidly expanded in recent years, driven by the success of mRNA vaccines^1,2 and the growing recognition of RNA’s versatility as a programmable biomolecule. A central challenge in this field is the ability to quantitatively and dynamically assess mRNA translation efficiency in diverse contexts. The method described here – leveraging a split luminescent tagging system – provides a flexible, scalable platform for studying live mRNA translation in cellulo³. The overall goal of this method is to enable researchers to measure how sequence features, chemical modifications, and structural elements influence translation kinetics and subsequent protein turnover, thereby offering a powerful tool for optimizing mRNA constructs for therapeutic and basic science applications.

In vitro transcribed mRNA mirrors the essential architecture of mature transcripts, incorporating a 5′ cap, untranslated regions (UTRs), a coding sequence, and a poly(A) tail, while often including modified nucleotides to reduce immunogenicity^4,5. Extensive work has shown that each of these elements – alone and in combination – can be engineered to strongly influence translation efficiency and protein yield^6,7. Yet most prior studies have relied on highly optimized reporter proteins such as Green Fluorescent Protein (GFP)⁷, NanoLuc⁸, or the SARS-CoV-2 spike protein^8,9, which are engineered to translate with high efficiency and to produce very stable proteins, and therefore do not reflect the challenges faced in therapeutic contexts. Because therapeutic mRNAs encode diverse proteins of interest (POIs), each subject to unique structural constraints and cell-type-specific regulation, the design principles that maximize translation for one construct may not generalize to another, as previously reported³. Identifying the optimal configuration for a given therapeutic POI is therefore essential and requires methods that can sensitively and systematically evaluate how individual features of the transcript contribute to protein output.

Ribosome-focussed methods such as polysome profiling distinguish translation fractions through gradient centrifugation¹⁰, while ribosome profiling (Ribo-seq) provides nucleotide-level resolution of ribosome occupancy, initiation sites, and pausing¹¹. Like Ribo-seq, other sequencing-based approaches like High-Throughput Sequencing of RNA Isolated by Crosslinking Immunoprecipitation (HITS-CLIP) and Translating Ribosome Affinity Purification (TRAP) further characterize translation of RNA-binding protein–associated or ribosome-associated RNAs¹². Although powerful, these techniques require complex preparation, specialized equipment, and computational expertise, making them unsuitable for high-throughput therapeutic mRNA development. Nascent chain immunoprecipitation¹³ measures translation efficiency using FLAG-tagged proteins immunoprecipitated from ribosomes and quantified by Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). While adaptable to therapeutic mRNAs, the assay is labor-intensive, lacks temporal resolution, and is unsuitable for high-throughput applications. The SunTag system¹⁴ offers live-cell measurements of protein synthesis output and translational regulation from single mRNA molecules and works through the interaction of a single-chain variable fragment (scFv) fused to GFP with tandem repeats of the peptide epitope. This system enables dynamic measurements of translation and localization with single-mRNA resolution, and it has therefore been exploited to develop numerous nascent chain tracking systems^15,16. However, this approach is low throughput and requires the use of high-power microscopes.

While tagging of the nascent protein is unavoidable, the potential use of a small peptide tag was identified, derived from NanoLuc luciferase, to report on mRNA translation in live cells. The NanoLuciferase was engineered to be partitioned into a small, 11-amino acid (HiBiT) and a larger, 158-amino acid (LgBiT) subunit, which can rapidly dimerize and, in the presence of substrate, reconstitute a functional luciferase^17,18. There are several key advantages of the split luminescent tagging system for studies of mRNA dynamics. Firstly, the minimal size of the small subunit fused to the POI allows the encoded construct to remain as close as possible to the native protein of interest. As coding sequence length can directly affect translation ratios¹⁹, minimizing the tag length improves our prediction of translation dynamics of synthetic mRNAs. Crucially, this minimal tagging strategy enables the detection of differences in translation driven by synonymous coding sequence variation, allowing translational efficiency to be interrogated independently of protein sequence or stability³. For this reason, the use of larger tags, such as fluorescent proteins or full-length luciferases, is suboptimal. Secondly, the split luminescent tagging system employed here enables immediate signal generation. Its rapid and efficient in cellulo complementation reaction has a reported high affinity (K_D = 700 pM) and has been used to measure protein turnover modulation with readings as short as every 30 s¹⁸. While the original split luminescent tagging system primarily benchmarked against other split luciferase systems^17,18, the key advantage in this context is that signal generation is not limited by protein maturation. In contrast, fluorescent proteins require chromophore maturation, which can occur in the scale of minutes to hours²⁰ thereby limiting temporal resolution. Finally, the established split luminescent tagging system here employed overcomes limitations of split fluorescent protein systems, which typically require chromophore formation after complementation²¹, introducing an additional delay and limiting their suitability for capturing rapid, dynamic processes. Previous studies have demonstrated the utility of split luminescent tagging in protein turnover assays and degradation studies^22,23,24. Extending this established technology to translation kinetics represents a significant methodological advance. For example, while ribosome profiling can reveal ribosome density along transcripts, it cannot easily capture dynamic changes in translation initiation or elongation rates under different conditions. As previously illustrated³, this split luminescent tagging system can detect differences when altering cap structure, 5′ untranslated region (UTR) composition, codon optimization, nucleotide modifications, and poly(A) tail length, all of which are critical determinants of translation efficiency. This breadth of applicability makes the method uniquely powerful for dissecting the contributions of individual mRNA features to overall protein output. It is worth noting that, depending on the instrument used, both single-cell and whole-well data can be captured with this technology. Imaging platforms capable of acquiring luminescence-based images can empower this technology to be used to study single-cell translation dynamics. Here, we focus on a plate-reader, well-based application due to its medium-to-high-throughput nature and minimal requirement for data analysis.

Researchers considering this method should evaluate its suitability in light of their specific experimental objectives. The assay is particularly valuable in contexts where high-throughput screening of synthetic mRNA constructs is required, such as in mRNA-based therapeutic design pipelines. It is well-suited to comparative analyses of synthetic mRNA sequence features – including untranslated regions, codon usage, or nucleotide modifications – where subtle differences in translation efficiency can be detected³. Because the system is compatible with cell-free translation platforms, live-cell assays, and a timepoint-specific lytic assay, it offers flexibility for investigators who wish to tailor their experimental design to either mechanistic studies or applied research. At the same time, it is important to recognize the boundaries of the technique. Critically, the split luminescent tagging assay does not provide resolution of ribosome occupancy, a capability that remains unique to ribosome footprinting approaches, nor does it directly measure RNA stability or degradation, which may need to be assessed using complementary methods. However, due to the limitations of existing ribo-centric techniques, researchers are reliant on tag-based assays like the split luminescence system to investigate time-resolved mRNA-driven protein expression as a proxy for translational dynamics. Thus, while this system is a powerful tool for monitoring synthetic mRNA-driven protein expression, its appropriateness depends on whether the research question focuses on dynamic protein output rather than fine-grained ribosome positioning or RNA decay.

In summary, the split luminescent tagging system represents a powerful and versatile method for studying mRNA translation. Its overall goal is to provide researchers with a flexible, system-scalable, high-throughput, and capable of detecting differences in protein output arising from synthetic mRNA with varying structural and sequence elements³. The rationale for its development lies in overcoming the limitations of existing techniques, offering advantages in sensitivity, scalability, and temporal resolution. Positioned within the wider literature on RNA biology and therapeutics, this method provides a valuable addition to the experimental repertoire, enabling both fundamental insights and translational advances. By carefully considering the strengths and limitations of the assay, researchers can determine its appropriateness for their specific applications, thereby maximizing its impact in the rapidly evolving field of RNA science.

Protocol

A graphical depiction of the entire protocol is shown in Figure 1.

1. Template preparation for in vitro transcription (IVT)

NOTE: To prepare the DNA template for IVT, ensure the plasmid DNA contains an RNA polymerase promoter (generally T7 or SP6), 5’ and 3’ UTRs, and the open reading frame of the POI (Figure 1).

1. Linearize plasmid DNA using a restriction enzyme with a single-cutting site located after the 3’ UTR and assemble the reaction according to the manufacturer’s instructions. After incubation, clean up the restriction digest reaction using a spin column-based DNA purification kit. Quantify the DNA using a spectrophotometer.
2. To confirm linearization, prepare a 1% agarose TAE gel containing DNA stain Ethidium Bromide at 0.1 µg/mL. Mix 150 ng of digested DNA with water and loading dye. Load the agarose gel with the sample, uncut plasmid, and a DNA ladder, and run the agarose gel at 100 V for 40 min in a 1× Tris-acetic acid-EDTA (TAE) buffer. Visualize with a gel imager.
   CAUTION: Ethidium bromide solution is a potent mutagen and requires careful handling using appropriate personal protective equipment (PPE).
3. If the plasmid DNA does not have a poly(A) tail immediately after the 3’ UTR, introduce it using a PCR. Complete PCR according to the manufacturer’s instructions using a high-fidelity polymerase with appropriately designed primers. Ensure the amplicon includes the T7 promoter, open reading frame, and a poly(A) tail.
   NOTE: Capping technologies can have specific sequence requirements at the T7 promoter; therefore, these requirements can be satisfied by appropriate forward primer design. The reverse primer binds the end of the 3’ UTR and introduces a poly(A) tail (recommended length of at least 80 T). For high GC-containing constructs, step-up PCR and/or 3% DMSO are recommended.
4. To confirm successful PCR, prepare an agarose TAE gel containing Ethidium Bromide (see step 1.2). If the construct length is ≥1 kb or <1 kb, prepare a 1% or 2% gel, respectively. Mix 5 µL of the PCR reaction with water and loading dye. Load the agarose gel with both the sample and a DNA ladder, and run it at 100 V for 40 min in TAE buffer. Visualize with a gel imager.
5. After PCR size confirmation, clean up the PCR reaction using a spin column-based DNA purification kit. Elute in 15–20 µL of elution buffer (often provided with the DNA purification kit) or nuclease-free water to concentrate the DNA template for use in IVT. Quantify the DNA using a spectrophotometer.

2. In vitro transcription

1. Before starting, spray and wipe down the bench, gloves, and pipettes with an RNase decontamination solution. To improve yield and quality, perform this procedure in a sterile hood environment.
2. Thaw and assemble the components of a T7-based in vitro transcription (IVT) kit according to the manufacturer’s instructions.
   NOTE: Some manufacturers recommend the use of dithiothreitol (DTT, 0.1 M) to limit reaction oxidation and improve efficiency. This step is recommended when compatible with the kit. If using modified dNTPs, replace the equivalent unmodified nucleotide with the same amount of modified nucleotide. Evaluate the yield from a half reaction, as no substantial improvement in yield was observed from a full reaction. When synthesizing more than one mRNA, it is recommended to create a master mix to reduce pipetting errors and variability.
3. Pipette thoroughly and briefly mix, then incubate at 37 °C for 5 h with a heated lid to avoid condensation. Extend incubation to overnight if needed, as this has minimal effect on mRNA yield or integrity.
4. Add 1 µL of DNase I and pipette thoroughly. Incubate at 37 °C for 30 min.
5. Purification of in vitro transcribed (IVT) RNA
   1. Clean up the IVT reaction using a column-based RNA Cleanup kit as per the manufacturer’s instructions (different kits may lead to different levels of recovery and have varying binding capacities). For eluting, add 60 µL of nuclease-free water and incubate at room temperature for 5 min before centrifuging.
   2. Alternatively, use lithium chloride precipitation as a cheaper and more flexible approach.
      1. Add an equal volume of 7.5 M lithium chloride to the reaction, mix, and incubate at -20 °C overnight.
      2. Centrifuge at 18,300 × g for 20 min at 4 °C, then carefully remove supernatant without disrupting the RNA pellet.
      3. Wash with 950 µL of 70% ethanol and centrifuge at 18,300 × g for 5 min at 4 °C. Remove the supernatant, let the pellet dry for approximately 5 min at room temperature, and resuspend in nuclease-free water (60 µL is standard; reduce the volume depending on pellet size to increase concentration).
         CAUTION: Lithium Chloride may cause skin or eye irritation if proper PPE is not used. Wear gloves and eye protection when handling. And, ethanol is a highly flammable liquid and can cause eye irritation. Keep away from naked flames, and wear appropriate PPE, including eye protection.
6. Quantify the mRNA using a spectrophotometer. Ensure that the A260/A280 ratio for mRNA is ~2.0, and confirm that the A260/A230 ratio falls between 1.8 and 2.2.
7. To confirm successful IVT, prepare a 1% agarose TAE (non-denaturing) gel containing Ethidium Bromide. Mix 500 ng of mRNA with water and loading dye, and follow the manufacturer’s instructions for RNA ladder preparation and denaturing instructions. Load the agarose gel and run it at 80 V for 40 min in TAE buffer. Visualize with a gel imager.
8. For further confirmation of correct mRNA size, run the samples on a Bioanalyser.
9. To avoid freeze-thawing, aliquot mRNA into the volume required for the experiment. For example, use 1.2 pmol of mRNA per well in a 96-well plate. Therefore, aliquot the appropriate volume for 1.2 pmol × n replicates, then store at -80 °C.

3. HEK293 reverse transfection

NOTE: To achieve temporal measurements, a cell line constitutively expressing the complementary luciferase fragment is required. This can be achieved by creating a stable cell line through transfection of a plasmid expressing the required luciferase fragment and subsequent clonal selection. This allows the user to measure temporally resolved mRNA translational dynamics in any cellular background of choice. Alternatively, cell lines expressing the required luciferase fragment can be purchased. These experiments were conducted using HEK293 cells constitutively expressing the complementary heterodimer.

1. Before starting, thaw the mRNA on ice to avoid degradation.
2. Prepare transfection complexes. For a 96-well plate, 1.2 pmol of mRNA is required per well. If running in duplicates, make enough for 2.5 duplicates to allow for pipetting error. Therefore, combine 3 pmol of mRNA with Opti-MEM Reduced Serum Medium to a final volume of 50 µL (20 µL/well).
3. Add 0.5 µL of cationic lipid-based transfection reagent (0.2 µL/well), mix by pipetting up and down, and incubate at room temperature. After incubating for 5 min, mix by pipetting up and down, then add 20 µL per well to a 96-well white flat-bottom microplate. It is suggested that the transfection wells are placed in the center of the plate, leaving the edge wells clear.
4. Prepare HEK293 cells by removing media and washing with 2 mL of phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺. Remove the wash and add 500 µL of Trypsin-EDTA, then incubate at 37 °C for 3 min.
5. During cell dissociation, fill the edge wells of the 96-well plate with PBS to decrease media evaporation during cell culture. If all wells are required, the plate can be flooded with PBS in between the wells.
6. Add 4.5 mL of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1× L-glutamine and 10% fetal bovine serum (FBS) to collect cells. Perform cell count using a hemocytometer or automatic cell counter.
7. Transfer the required amount of cells (enough for 1 x 10⁵ cells to be seeded per well) plus 3 extra wells (for pipetting error) to a fresh tube and centrifuge at 500 × g for 2 min.
8. Remove supernatant and resuspend cells in the required volume of Leibovitz’s L-15 medium (phenol free) supplemented with 10% FBS and Endurazine Live Cell Substrate (1:200) to ensure a final concentration of 1 × 10⁶ cells/mL. Mix with a serological pipette to ensure thorough mixing of Endurazine. Pipette 100 µL (1 × 10⁵ cells) of resuspended cells per well.
   NOTE: If performing time course experiments running for 72 h, increase the volume to 200 µL to avoid evaporation.
9. Immediately post-transfection, place cells into a plate reader (luminometer) set at 37 °C. Take recordings at regular intervals with an integration time of 1 s. Due to the system's efficient complementation, the signal is detectable within minutes. Therefore, to get an accurate baseline reading, this step has to be executed promptly. These intervals are experiment- and instrument-dependent; however, in these experiments, measuring every 12 min for 18 h time course experiments, but only every 48 min for 72 h experiments, was found to be optimal. Luminescence does not require a filter for measurement; a better signal-to-noise ratio was observed when using a filter of 530–540 nm.
   NOTE: A Bioluminescence Resonance Energy Transfer (BRET) signal is produced from split-luciferase-tagged eGFP thus a filter at 530–540 nm will capture both a luminescent and fluorescent signal. To avoid capture of BRET, use a filter at 460–480 nm or alternatively, use a non-fluorescent protein such as MYL3 as a reference.

4. Data analysis

1. Generate fold changes in relative luminescence units (RLUs), by normalizing each condition’s signal to its first time point (t0, normalized to the average of the technical replicates), since the signal at the beginning of the assay is the background level.
2. Reduce variability between biological replicates by normalizing to a control condition.
3. Choose the statistical analysis for the assay, either single time point or area under the curve (AUC)—depending on the question being asked.
   1. For a single time point, use the time points where peak expression occurs (which is POI-dependent) along with the endpoint time. Plot the RLU calculated in step 4.1 at the specific time point (e.g., 240 min) and analyze it using an appropriate statistical test.
   2. Measure dynamics of protein translation and degradation by calculating the AUC for either a specific timeframe (i.e., t0 to 240 min) or for the length of the assay (i.e., t0 to endpoint). Normalize the AUC value for each biological replicate to the average of the reference mRNA, thereby yielding a measure of fold change in AUC that can be analyzed using appropriate statistical tests.

Results

There are several ways in which the cost-effectiveness of this protocol can be optimized. For example, the IVT reaction was miniaturized to half the volume recommended by the manufacturer, and it was found that the resulting yield satisfied the amount required for in cellulo experiments. It should be noted that sequence length and complexity impact IVT yield, so successful protocol optimization should yield at least 50 µg of mRNA per IVT reaction. Like IVT reaction kits, capping reagents also often cost a great ...

Discussion

The field of mRNA therapeutics has expanded rapidly in recent years, underscoring the importance of sequence design and optimization as a critical stage in the development pipeline. Significant efforts have focused on building computational tools to predict the most effective RNA structures and compositions, with sequence optimization receiving particular attention^8,9. While these tools are powerful, experimental validation of these designs is still an essential ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic Acid	Sigma-Aldrich	A6283	Component of TAE buffer
Agarose	Sigma-Aldrich	A9539	Gel electrophoresis
Agilent 2100 Bioanalyzer	Agilent	G2939B	Bioanalyser used for mRNA quality control
CLARIOstar Plus Luminometer	BMG Labtech	430-501S-F	Plate reader/luminometer used for measuring luminescence
CleanCap AG	Trilink Biotechnologies	N-7113	5' cap used for IVT
DMEM, high glucose, pyruvate	Thermo Fisher Scientific	41966029	Media used for culture of HEK293-LgBiT cells
DNase I	New England Biolabs	M0303	Degrades DNA template post-IVT reaction
Dulbecco's Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537	For cell washing and addition to plate to prevent evaporation
EDTA, pH 8.0, RNase-free	Thermo Fisher Scientific	AM9260	Component of TAE buffer
Ethidium Bromide	Sigma-Aldrich	E1510	Stain for gel electrophoresis
Fetal Bovine Serum (FBS)	Sigma-Aldrich	F7524	Supplement added to DMEM
HiScribe T7 High Yield RNA Synthesis Kit	New England Biolabs	E2040	In vitro transcription (IVT) kit
Leibovitz's L-15 Medium, no phenol red	Thermo Fisher Scientific	21083027	Media used for HEK293-LgBiT culture during luminescence measurements
L-glutamine	Thermo Fisher Scientific	25030-024	Supplement added to DMEM
Lipofectamine RNAiMAX	Thermo Fisher Scientific	13778150	Lipid-based transfection reagent
Lithium Chloride Precipitation Solution	Thermo Fisher Scientific	AM9480	Alternative approach for mRNA purification post-IVT
Monarch Spin PCR & DNA Cleanup Kit	New England Biolabs	T1130	Purification kit after plasmid linearization
NanoDrop One Microvolume UV-Vis Spectrophotometer	Thermo Fisher Scientific	ND-ONE-W	Spectrophotometer used for quantifying DNA and mRNA concentration
Nano-Glo Endurazine Live Cell Substrate	Promega	N2570	Substrate required for luminescence added to L-15 media
NucleoSpin RNA Cleanup Kit	Macherey-Nagel	12708612	Purifies mRNA post-IVT reaction
Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate	Thermo Fisher Scientific	136101	Plate used for HEK293-LgBiT plating and subsequent measurements
Opti-MEM Reduced Serum Medium	Thermo Fisher Scientific	31985062	Media used for transfection reaction
Phusion High-Fidelity PCR Kit	New England Biolabs	E0553	PCR kit
RiboRuler High Range RNA Ladder	Thermo Fisher Scientific	11883993	RNA ladder for gel electrophoresis
RNaseZap	Thermo Fisher Scientific	R2020	RNase decontamination spray
TRIZMA Base	Sigma-Aldrich	T6066	Component of TAE buffer
Trypsin-EDTA Solution	Sigma-Aldrich	T4174	Cell dissociation reagent
UltraPure DNase/RNase free Distilled Water	Thermo Fisher Scientific	10977035	Used for template preparation and for IVT