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 enabl....

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 incu....

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