Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

Expanding the RNA polymerase biocatalyst solution space for mRNA manufacture.

All mRNA products are currently manufactured in in vitro transcription (IVT) reactions that utilize single-subunit RNA polymerase (RNAP) biocatalysts. Although it is known that discrete polymerases exhibit highly variable bioproduction phenotypes, including different relative processivity rates and impurity generation profiles, only a handful of enzymes are generally available for mRNA biosynthesis. This limited RNAP toolbox restricts strategies to design and troubleshoot new mRNA manufacturing processes, which is particularly undesirable given the continuing diversification of mRNA product lines toward larger and more complex molecules. Herein, we describe development of a high-throughput RNAP screening platform, comprising complementary in silico and in vitro testing modules, that enables functional characterization of large enzyme libraries. Utilizing this system, we identified eight novel sequence-diverse RNAPs, with associated active cognate promoters, and subsequently validated their performance as recombinant enzymes in IVT-based mRNA production processes. By increasing the number of available characterized functional RNAPs by more than 130% and providing a platform to rapidly identify further potentially useful enzymes, this work significantly expands the RNAP biocatalyst solution space for mRNA manufacture, thereby enhancing the capability for application-specific and molecule-specific optimization of both product yield and quality.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.

Quality by design approach to improve quality and decrease cost of in vitro transcription of mRNA using design of experiments.

In vitro transcription (IVT) reaction is an RNA polymerase-catalyzed production of messenger RNA (mRNA) from DNA template, and the unit operation with highest cost of goods in the mRNA drug substance production process. To decrease the cost of mRNA production, reagents should be optimally utilized. Due to the catalytic, multicomponent nature of the IVT reaction, optimization is a multi-factorial problem, ideally suited to design-of-experiment approach for optimization and identification of design space. We derived a data-driven model of the IVT reaction and explored factors that drive process yield (in g/L), including impact of nucleoside triphosphate (NTP) concentration and Mg:NTP ratio on reaction yield and how to optimize the main cost drivers RNA polymerase and DNA template, while minimizing dsRNA formation, a critical quality attribute in mRNA products. We report a methodological approach to derive an optimum reaction design, with which cost efficiency of the reaction was improved by 44%. We demonstrate the validity of the model on mRNA construct of different lengths. Finally, we maximized the yield of the IVT reaction to 24.9 ± 1.5 g/L in batch, thus doubling the highest ever reported IVT yield.

SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.

Linearly Amplified Single-Stranded RNA-Derived Transcriptome Sequencing (LAST-seq).

Single-cell RNA sequencing (scRNA-seq) stands as a cutting-edge technology widely used in biological and biomedical research. Existing scRNA-seq methods rely on reverse transcription (RT) and second-strand synthesis (SSS) to convert RNA to cDNA before amplification. However, these methods often suffer from limited RT/SSS efficiency, which compromises the sensitivity of RNA detection. Here, we develop a new method, linearly amplified single-stranded RNA-derived transcriptome sequencing (LAST-seq), which directly amplifies the original single-stranded RNA without prior RT and SSS and offers high-sensitivity RNA detection and a low level of technical noise in single-cell transcriptome analysis. LAST-seq has been applied to quantify transcriptional bursting kinetics in human cells, advancing our understanding of chromatin organization's role in regulating gene expression. Key features • An RNase H/DNA polymerase-based strategy to attach the T7 promoter to single-stranded RNA. • T7 promoter mediated IVT on single stranded RNA template at single cell level.

Isolation of short RNAs with homogeneous 3'-ends using quaternary-amine anion exchange chromatography.

Visualizing RNA-protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single-nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein-binding partner.

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription.

SARS-CoV-2, the causative virus of the COVID-19 pandemic, follows SARS and MERS as recent zoonotic coronaviruses causing severe respiratory illness and death in humans. The recurrent impact of zoonotic coronaviruses demands a better understanding of their fundamental molecular biochemistry. Nucleoside modifications, which modulate many steps of the RNA lifecycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or anti-viral effect is unknown. Regardless, the viral RNA-dependent RNA polymerase will encounter these modifications as it transcribes through the viral genomic RNA. We investigated the functional consequences of nucleoside modification on the pre-steady state kinetics of SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted transcription system with modified RNA templates. Our findings show that N6-methyladenosine and 2'O-methyladenosine modifications slow the rate of viral transcription at magnitudes specific to each modification, which has the potential to impact SARS-CoV-2 genome maintenance.

Augmenting the landscape of chimeric antigen receptor T-cell therapy.

INTRODUCTION: The inception of recombinant DNA technology and live cell genomic alteration have paved the path for the excellence of cell and gene therapies and often provided the first curative treatment for many indications. The approval of the first Chimeric Antigen Receptor (CAR) T-cell therapy was one of the breakthrough innovations that became the headline in 2017. Currently, the therapy is primarily restricted to a few nations, and the market is growing at a CAGR (current annual growth rate) of 11.6% (2022-2032), as opposed to the established bio-therapeutic market at a CAGR of 15.9% (2023-2030). The limited technology democratization is attributed to its autologous nature, lack of awareness, therapy inclusion criteria, high infrastructure cost, trained personnel, complex manufacturing processes, regulatory challenges, recurrence of the disease, and long-term follow-ups. AREAS COVERED: This review discusses the vision and strategies focusing on the CAR T-cell therapy democratization with mitigation plans. Further, it also covers the strategies to leverage the mRNA-based CAR T platform for building an ecosystem to ensure availability, accessibility, and affordability to the community. EXPERT OPINION: mRNA-guided CAR T cell therapy is a rapidly growing area wherein a collaborative approach among the stakeholders is needed for its success.

An immuno-northern technique to measure the size of dsRNA byproducts in in vitro transcribed RNA.

Double-stranded RNA is an immunogenic byproduct present in RNA synthesized with in vitro transcription. dsRNA byproducts engage virus-sensing innate immunity receptors and cause inflammation. Removing dsRNA from in vitro transcribed messenger RNA (mRNA) reduces immunogenicity and improves protein translation. Levels of dsRNA are typically 0.1%-0.5% of total transcribed RNA. Because they form such a minor fraction of the total RNA in transcription reactions, it is difficult to confidently identify discrete bands on agarose gels that correspond to the dsRNA byproducts. Thus, the sizes of dsRNA byproducts are largely unknown. Total levels of dsRNA are typically assayed with dsRNA-specific antibodies in ELISA and immuno dot-blot assays. Here we report a dsRNA-specific immuno-northern blot technique that provides a clear picture of the dsRNA size distributions in transcribed RNA. This technique could complement existing dsRNA analytical methods in studies of dsRNA byproduct synthesis, dsRNA removal, and characterization of therapeutic RNA drug substances.

Optimizing in vitro expression balance of central dogma-related genes using parallel reaction monitoring.

The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.

Generation and Characterization of In Vitro Transcribed mRNA.

Here we describe the in vitro preparation of mRNA from DNA templates, including setting up the transcription reaction, mRNA capping, and mRNA labeling. We then describe methods used for mRNA characterization, including UV and fluorescence spectrophotometry, as well as gel electrophoresis. Moreover, characterization of the in vitro transcribed RNA using the Bioanalyzer instrument is described, allowing a higher resolution analysis of the target molecules. For the in vitro testing of the mRNA molecules, we include protocols for the transfection of various primary cell cultures and the confirmation of translation by intracellular staining and western blotting.

Quality by Digital Design for Developing Platform RNA Vaccine and Therapeutic Manufacturing Processes.

Quality by digital design (QbDD) utilizes data-driven, mechanistic, or hybrid models to define and optimize a manufacturing design space. It improves upon the QbD approach used extensively in the pharmaceutical industry. The computational models developed in this approach identify and quantify the relationship between the product's critical quality attributes (CQAs) and the critical process parameters (CPPs) of unit operations within the manufacturing process. This chapter discusses the QbDD approach in developing and optimizing unit operations such as in vitro transcription, tangential flow filtration, affinity chromatography, and lipid nanoparticle (LNP) formulation in mRNA vaccine manufacturing. QbDD can be an efficient framework for developing a production process for a disease-agnostic product that requires extensive experimental and model-based process-product interaction characterization during the early process development phase.

Mechanistic modeling of in vitro transcription incorporating effects of magnesium pyrophosphate crystallization.

The in vitro transcription (IVT) reaction used in the production of messenger RNA vaccines and therapies remains poorly quantitatively understood. Mechanistic modeling of IVT could inform reaction design, scale-up, and control. In this work, we develop a mechanistic model of IVT to include nucleation and growth of magnesium pyrophosphate crystals and subsequent agglomeration of crystals and DNA. To help generalize this model to different constructs, a novel quantitative description is included for the rate of transcription as a function of target sequence length, DNA concentration, and T7 RNA polymerase concentration. The model explains previously unexplained trends in IVT data and quantitatively predicts the effect of adding the pyrophosphatase enzyme to the reaction system. The model is validated on additional literature data showing an ability to predict transcription rates as a function of RNA sequence length.

The mRNA Vaccine Expressing Single and Fused Structural Proteins of Porcine Reproductive and Respiratory Syndrome Induces Strong Cellular and Humoral Immune Responses in BalB/C Mice.

PRRS is a viral disease that profoundly impacts the global swine industry, causing significant economic losses. The development of a novel and effective vaccine is crucial to halt the rapid transmission of this virus. There have been several vaccination attempts against PRRSV using both traditional and alternative vaccine design development approaches. Unfortunately, there is no currently available vaccine that can completely control this disease. Thus, our study aimed to develop an mRNA vaccine using the antigens expressed by single or fused PRRSV structural proteins. In this study, the nucleotide sequence of the immunogenic mRNA was determined by considering the antigenicity of structural proteins and the stability of spatial structure. Purified GP5 protein served as the detection antigen in the immunological evaluation. Furthermore, cellular mRNA expression was detected by immunofluorescence and western blotting. In a mice experiment, the Ab titer in serum and the activation of spleen lymphocytes triggered by the antigen were detected by ELISA and ICS, respectively. Our findings demonstrated that both mRNA vaccines can significantly stimulate cellular and humoral immune responses. More specifically, the GP5-mRNA exhibited an immunological response that was similar to that of the commercially available vaccine when administered in high doses. To conclude, our vaccine may show promising results against the wild-type virus in a natural host.

Development of a capillary zone electrophoresis method to monitor magnesium ion consumption during in vitro transcription for mRNA production.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.

Effect of in vitro transcription conditions on yield of high quality messenger and self-amplifying RNA.

Messenger RNA (mRNA) and self-amplifying RNA (saRNA) vaccines against SARS-CoV-2 produced using in vitro transcription (IVT) were clinically approved in 2020 and 2022, respectively. While the industrial production of mRNA using IVT has been extensively optimized, the optimal conditions for saRNA have been explored to a lesser extent. Most T7 polymerase IVT protocols have been specifically optimized for mRNA which is ∼5-10-fold smaller than saRNA and may have profound effects on both the quality and yield of longer transcripts. Here, we optimized IVT conditions for simultaneously increasing the yield of full-length transcripts and reducing dsRNA formation through Design of Experiments. Using a definitive screening approach, we found that the key parameters are temperature and magnesium in the outcome of RNA quality (% full length transcript) and yield in small scale synthesis. The most important parameter for reducing dsRNA formation for both mRNA and saRNA was Mg2+ concentration (10 mM). We observed that a lower temperature was vital for production of high quality saRNA transcripts. mRNA quality was optimal at higher Mg2+ concentration (>40 mM). High quality transcripts correspond to significantly reduced product yield for saRNA, but not for mRNA. The differences between mRNA and saRNA requirements for high quality product and the relationship between high quality large saRNA molecules and low temperature synthesis have not been reported previously. These findings are key for informing future IVT parameters design and optimization for smaller and larger RNA transcripts.

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development.

mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6 min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such that their consumption may be determined during IVT reactions. Furthermore, the HPLC method was used to rapidly assess the purification of the mRNA product, including removal of NTPs/Cap analogue and other contaminants after downstream purification, including solid phase extraction (SPE), oligo deoxythymidine (oligo-dT) affinity chromatography and tangential flow filtration (TFF). Using the developed method excellent precision was obtained with calibration curves for an external mRNA standard and NTPs giving correlation coefficients of 0.98 and 1.0 respectively. Intra- and inter-day studies on retention time stability of NTPs, showed a relative standard deviation ≤ 0.3% and ≤1.5% respectively. The mRNA retention time variability was ≤0.13%. This method was then utilised to monitor the progress of an IVT reaction for the production of Covid spike protein (C-Spike) mRNA to measure the increasing yield of mRNA alongside the consumption of NTPs during the reaction.

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.