Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

Gene-Specific H1 Eviction through a Transcriptional Activator→p300→NAP1→H1 Pathway.

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a gene-specific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B. An analysis in B cells confirms the strong dependency on NAP1-mediated H1 eviction for induction of the silent CD40 gene and further demonstrates that H1 eviction, seeded by activator-p300-NAP1-H1 interactions, is propagated over a CCCTC-binding factor (CTCF)-demarcated region through a distinct mechanism that also involves NAP1. Our results confirm direct transcriptional inhibition by H1 and establish a gene-specific H1 eviction mechanism through an activator→p300→NAP1→H1 pathway.

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.

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 life cycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or antiviral 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 N 6-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.

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.

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.

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.

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.

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts.

The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

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.

Recombinant T7 RNA polymerase production using ClearColi BL21(DE3) and animal-free media for in vitro transcription.

In vitro transcription (IVT) using T7 RNA polymerase (RNAP) is integral to RNA research, yet producing this enzyme in E. coli presents challenges regarding endotoxins and animal-sourced toxins. This study demonstrates the viable production and characterization of T7 RNAP using ClearColi BL21(DE3) (an endotoxin-free E. coli strain) and animal-free media. Compared to BL21(DE3) with animal-free medium, soluble T7 RNAP expression is ~50% lower in ClearColi BL21(DE3). Optimal soluble T7 RNAP expression in flask fermentation is achieved through the design of experiments (DoE). Specification and functional testing showed that the endotoxin-free T7 RNAP has comparable activity to conventional T7 RNAP. After Ni-NTA purification, endotoxin levels were approximately 109-fold lower than T7 RNAP from BL21(DE3) with animal-free medium. Furthermore, a full factorial DoE created an optimal IVT system that maximized mRNA yield from the endotoxin-free and animal-free T7 RNAP. This work addresses critical challenges in recombinant T7 RNAP production through innovative host and medium combinations, avoided endotoxin risks and animal-derived toxins. Together with an optimized IVT reaction system, this study represents a significant advance for safe and reliable reagent manufacturing and RNA therapeutics. KEY POINTS: • Optimized IVT system maximizes mRNA yields, enabling the synthesis of long RNAs. • Novel production method yields endotoxin-free and animal-free T7 RNAP. • The T7 RNAP has equivalent specifications and function to conventional T7 RNAP.

Microfluidic Bioreactor with Fibrous Micromixers for In Vitro mRNA Transcription.

A new type of microfluidic bioreactor with fibrous micromixers for the ingredient mixing and a long macrochannel for the in vitro transcription reaction was fabricated for the continuous production of mRNA. The diameter of the fibrous microchannels in the micromixers was tuned by using an electrospun microfibrous disc with different microfiber diameters. The micromixer with a larger diameter of fibrous microchannels exhibited a better mixing performance than the others. The mixing efficiency was increased to 0.95 while the mixture was passed through the micromixers, suggesting complete mixing. To demonstrate the continuous production of mRNA, the ingredients for in vitro transcription were introduced into the perfluoropolyether microfluidic bioreactor. The mRNA synthesized by the microfluidic bioreactor had the same sequence and in vitro/in vivo performances as those prepared by the bulk reaction. The continuous reaction in the microfluidic bioreactor with efficient mixing performance can be used as a powerful platform for various microfluidic reactions.

Diversity of σ66-Specific Promoters Contributes to Regulation of Developmental Gene Expression in Chlamydia trachomatis.

Promoter recognition by the RNA polymerase (RNAP) holoenzyme is a key step in gene regulation. In Chlamydia trachomatis, a medically important obligate intracellular bacterium, σ66 allows the RNAP to initiate promoter-specific transcription throughout the chlamydial developmental cycle. Here, we investigated the intrinsic properties of σ66-specific promoters with emphasis on their role in the developmental gene expression of C. trachomatis. First, we examined whether promoters that contain a 5'-T(-15)G(-14)-3' (TG) motif upstream from the -10 element appear more often than others in genes that are preferentially expressed during the early, middle, or late stages of the C. trachomatis developmental cycle. We then determined the critical genetic elements that are required for transcription initiation in vitro. We also assessed the activity of promoters in the presence of Scc4, which can directly interact with σ66RNAP. Finally, we evaluated the promoter-specific dynamics during C. trachomatis infection using a reporter assay. These results reveal that the TG motif is an important determinant in certain early or late promoters. The TG promoters that have the -35 element are recognized by σ66RNAP and Scc4 differently from those lacking the -35 element. Based on these properties, the σ66-specific promoters can fall into three classes. Architectural diversity, behavioral plasticity, and the specific interplays between promoters and the σ66RNAP likely contribute to developmental gene transcription in C. trachomatis. IMPORTANCE Meticulous promoter elucidation is required to understand the foundations of transcription initiation. However, knowledge of promoter-specific transcription remains limited in C. trachomatis. This work underscores the structural and functional plasticity of σ66-specific promoters that are regulated by σ66RNAP, as well as their importance in the developmental gene regulation of C. trachomatis.

Sulisobenzone is a potent inhibitor of the global transcription factor Cra.

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.

Scalable multimodal weak anion exchange chromatographic purification for stable mRNA drug substance.

Messenger RNA (mRNA) has emerged as a modality with immense therapeutic potential. Recent innovations in production process of mRNA call for procedures to isolate pure mRNA drug substance (DS) with high yield, high capacity, scalability, and compatibility with GMP production systems. Novel RNA modalities, such as circular RNA (circRNA), have further driven the need for non-affinity capture possibilities which are already widely used in the biopharmaceutical industry, for example, in monoclonal antibody processing. The principle that multimodal ion exchange/hydrogen bonding chromatography can be used to separate mRNA from in vitro transcription components has recently been demonstrated. Here, we apply and refine this approach to be suitable for scalable purification of multiple mRNA constructs with sufficient yields, purity, and stability, for use in mRNA production process. Binding capacity of the PrimaS-modified monolithic chromatographic column for mRNA enabled up to 7 mg/mL product isolation in a single chromatographic run, with 98% recovery and room temperature stability of the eGFP mRNA demonstrated for up to 28 days. This approach is independent of construct size or the presence of polyadenylic acid tail and is applicable for capture of a wide variety of RNAs, including mRNA, self-amplifying RNA, circRNA, and with optimization also smaller RNAs such as transfer RNA and others.

Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring.

The COVID-19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti-reverse cap analog (ARCA)-capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed-batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

Click-iT trinucleotide cap analog: Synthesis, mRNA translation, and detection.

The first example of the synthesis of a new trinucleotide cap analog containing propargyl group such as m7,3'-O-propargylG(5')PPP(5')AmpG is reported. The effect of the propargyl group in trinucleotide analog with a standard trinucleotide cap analog (GAG), m7G(5')ppp(5')AmpG was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured A549 lung carcinoma epithelial cells. The new propargyl cap analog is a substrate for T7 RNA polymerase. Notably, the mRNA capped with the propargyl cap is translated âˆ¼ 1.3 times more efficiently than the mRNA capped with the GAG cap. The most characteristic feature of the new propargyl cap analog is that the presence of the propargyl group allows further modification of the mRNA by chemical ligation of an azide-containing fluorescent-labeled substrate to the mRNA via click chemistry.

Mesoporous Silica Particle as an RNA Adsorbent for Facile Purification of In Vitro-Transcribed RNA.

Messenger RNA vaccines against SARS-CoV-2 hold great promise for the treatment of a wide range of diseases by using mRNA as a tool for generating vaccination antigens as well as therapeutic proteins in vivo. Increasing interest in mRNA preparation warrants reliable methods for in vitro transcription (IVT) of mRNA, which must entail the elimination of surplus side products such as immunogenic double-stranded RNA (dsRNA). We developed a facile method for the removal of dsRNA from in vitro transcribed RNA with mesoporous silica particles as RNA adsorbents. Various polyamines were tested for the facilitation of RNA adsorption onto mesoporous silica particles in the chromatography. Among the polyamines tested for RNA adsorption, spermidine showed a superior capability of RNA binding to the silica matrix. Mesoporous silica-adsorbed RNA was readily desorbed with elution buffer containing either salt, EDTA, or urea, possibly by disrupting electrostatic interaction and hydrogen bonding between RNA and the silica matrix. Purification of IVT RNA was enabled with the adsorption of RNA to mesoporous silica in a spermidine-containing buffer and subsequent elution with EDTA. By differing EDTA concentration in the eluting buffer, we demonstrated that at least 80% of the dsRNA can be removed from the mesoporous silica-adsorbed RNA. When compared with the cellulose-based removal of dsRNA from IVT RNA, the mesoporous silica-based purification of IVT RNA using spermidine and EDTA in binding and elution, respectively, exhibited more effective removal of dsRNA contaminants from IVT RNA. Thus, mRNA purification with mesoporous silica particles as RNA adsorbents is applicable for the facile preparation of nonimmunogenic RNA suitable for in vivo uses.

Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription.

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

A robust and versatile method for production and purification of large-scale RNA samples for structural biology.

Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.