T7 RNA Polymerase Research Articles

Electrochemiluminescence Biosensor for Ascorbic Acid Based on Target Transformation of Cell-Free RNA Transcription System and Duplex-Specific Nuclease-Assisted Recycling Amplification.

A cell-free RNA transcription system had been coupled with electrochemiluminescence (ECL) detection technology for the first time to develop an ascorbic acid (AA, acting as a model target) biosensor. The biosensor is composed of single-stranded DNA (ssDNA) sequences modified with alkynyl and azido groups, respectively, alongside an incomplete gene circuit framework. The addition of target AA and copper ions will cause the linkage of the two ssDNA sequences through a click chemistry reaction. This results in the subsequent reconstruction of a complete gene circuit. The reconstituted gene circuit, in conjunction with the T7 RNA polymerase, drives the transcription of substantial quantities of RNA. ssDNA labeled with ferrocene (Fc) (Fc-DNA) had been immobilized on a tris(2,2'-bipyridyl) ruthenium(II) chloride hexahydrate-doped SiO2 nanoparticle (Ru@SiO2 NPs) modified electrode first. The quenching effect of Fc on Ru@SiO2 causes the low ECL detected. The transcribed RNA sequence assisted double-stranded specific nuclease (DSN) to cut the ssDNA-Fc and the ECL of the system was enhanced. Optimal experimental conditions reveal that the ECL signal exhibits a linear correlation with the logarithmic concentration of AA, spanning a detection range from 100 nM to 1 mM, with a detection limit of 45 nM. This innovative methodology expands the utility of a cell-free RNA transcription system within the realm of biosensing applications.

Exploring the Impact of In Vitro-Transcribed mRNA Impurities on Cellular Responses.

Advances in mRNA technology have enabled mRNA-based therapies to enter a new era of medicine. Such therapies benefit from a single, standardized in vitro transcription (IVT) manufacturing process applicable to a wide range of targets. This process includes several downstream purification steps, which aim to eliminate impurities that potentially affect safety and efficacy. However, it is not fully understood which impurities are the most critical; hence, some efforts are still needed to establish the correlation between RNA impurities and their role in limiting therapeutic efficacy. To study this relationship, we produced in vitro-transcribed mRNAs using several bacteriophage T7 RNA polymerases, including one wild-type and four engineered variants. Important attributes of the mRNA such as integrity, purity, and functional activity were then measured using advanced physicochemical and cellular assays. For impurities including abortive transcripts, mRNAs containing partial poly(A) tails, and double-stranded (ds)RNA byproducts, structure-function relationships have been established by tracking cellular responses (i.e., protein expression, reactogenicity) in multiple cell models. By varying the T7 RNA polymerase, different levels of sense-antisense dsRNA byproducts were measured by mass photometry, contributing directly to immunological reactogenicity in bone marrow-derived dendritic cells. T7 RNA polymerase differences with regard to short (<20 nucleotides) 3'-loopback dsRNA byproducts were also further investigated using native mass spectrometry by precisely resolving these impurities at the nucleotide level. Overall, this study highlights the importance of developing sensitive and advanced analytical methods to characterize IVT mRNA impurities and understand their interaction with cellular machinery in order to ensure quality control of RNA-based therapies.

Light-Activated Gene Expression System Using a Caging-Group-Free Photoactivatable Dye.

Optical regulation of transcription using chemical compounds is an effective strategy to manipulate gene expression spatiotemporally. Conventional caging approaches with photoremovable protecting groups may require intense UV-light exposure and release potentially toxic byproducts. To address these problems, here we developed a light-mediated transcriptional regulation system by combining a caging-group-free photoactivatable dye PaX560 and a multidrug-binding transcriptional regulator QacR. The cationic dye generated from PaX560 through traceless photoconversion bound QacR and reduced its repressor function, resulting in transcriptional activation. Importantly, this system allowed transcriptional activation with a large dynamic range under mild visible light exposure and simultaneous detection of the state of the photoactivated effector. This module was integrated into the T7 RNA polymerase expression system to demonstrate light-activated transcription in vitro and in living cells.

High-Throughput Algorithmic Optimization of In Vitro Transcription for SARS-CoV-2 mRNA Vaccine Production.

The in vitro transcription (IVT) of messenger ribonucleic acid (mRNA) from the linearized deoxyribonucleic acid (DNA) template of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) was optimized for total mRNA yield and purity (by percent intact mRNA) utilizing machine learning in conjunction with automated, high-throughput liquid handling technology. An iterative Bayesian optimization approach successfully optimized 11 critical process parameters in 42 reactions across 5 experimental rounds. Once the optimized conditions were achieved, an automated, high-throughput screen was conducted to evaluate commercially available T7 RNA polymerases for rate and quality of mRNA production. Final conditions showed a 12% yield improvement and a 50% reduction in reaction time, while simultaneously significantly decreasing (up to 44% reduction) the use of expensive reagents. This novel platform offers a powerful new approach for optimizing IVT reactions for mRNA production.

Purification of T7 RNA polymerase for large scale production of mRNA vaccines and therapeutics

The COVID-19 Pandemic has revolutionized the field of mRNA vaccine technology, highlighting its utility and rapidly expanding potential, propelling it to the forefront of future vaccines & therapeutics for cryptic diseases. T7 RNA Polymerase (T7 RNAP) is a high-fidelity enzyme used for the synthesis of single-stranded mRNA from DNA templates using In vitro transcription (IVT). T7 RNAP is now the mainstay for the development of mRNA vaccines and therapeutics, accounting for 34 % of the overall raw material cost for mRNA manufacturing. Although small-scale purification processes for T7 RNAP exist, scalable process platforms and comprehensive analytical characterization are still lacking. Moreover, to address global raw material supply chain issues and to facilitate the efficient and large-scale production of mRNA vaccines & therapeutics, here we report a three-step purification process platform, yielding ∼ 85–100 mg/L of T7 RNAP from E. coli fermentation harvest. Also, to demonstrate its high-level purity and structural integrity, we have characterized it using orthogonal analytical techniques. Furthermore, when evaluated in an IVT system, this highly pure T7 RNAP showed activity that was comparable to the commercial T7 RNAP in producing integral RNA (10 kb) from a linearized plasmid DNA template. Collectively, these findings establish a purification process platform for the commercial production of T7 RNAP enzyme to support the future development of mRNA-based vaccines and therapeutics.

Electrochemiluminescence biosensor for MMP-2 determinationusing CRISPR/Cas13a and EXPAR amplification: a novel approach for anti-aging research.

Matrix metalloproteinase-2 (MMP-2) plays a pivotal role in anti-aging research. Developing advanced detection platforms for MMP-2 with high specificity, sensitivity, and accessibility is crucial. This study introduces a novel electrochemiluminescence (ECL) biosensor for MMP-2 determination, leveraging the CRISPR/Cas13a system and Exponential Amplification Reaction (EXPAR). The biosensor operates by utilizing the T7 RNA polymerase to transcribe RNA from a DNA template upon MMP-2 interaction. This RNA activates Cas13a, leading to signal amplification and ECL detection. The incorporation of the "photoswitch" molecule [Ru(phen)2dppz]2+ streamlines the process by eliminating the need for extensive electrode modification and cleaning. Under optimized conditions, the biosensor achieved an impressive detection limit of 12.8 aM for MMP-2. The platform demonstrated excellent selectivity, reproducibility, and stability, making it highly suitable for detecting MMP-2 in complex biological samples. This innovative approach shows great potential for applications in molecular diagnostics and anti-aging research.

Effective Synthesis of mRNA during In Vitro Transcription with Fewer Impurities Produced.

The remarkable efficacy of COVID-19 vaccines has established mRNA as a highly promising biomedical technology. However, the adequate application of mRNA therapeutics necessitates additional measures to mitigate the inherent immunogenicity, which is predominantly caused by dsRNA. As a byproduct of the in vitro transcription of mRNA, dsRNA was reported to be originated through several distinct mechanisms, including the extension of 3' loop-back hairpins, the extension of hybridized abortive transcripts, and promoter-independent transcription. The intricate mechanisms involved pose a dilemma as the reduction in dsRNA results in a concomitant decrease in other critical quality attributes of mRNA. Here, we demonstrate that the promoter binding motifs of T7 RNA polymerase directly impact the production of promoter-independent transcription-based dsRNA. Specifically, the G753A mutation significantly reduces the formation of dsRNA byproducts, which can further combine with modified nucleotides to enhance the effectiveness of dsRNA mitigation and with previously reported high-integrity mutation K389A to minimize side effects. Accordingly, the present study reports a cost-effective approach to synthesize high-purity, less immunostimulatory mRNA by using an engineered T7 RNA polymerase mutant.

A cell-free bacteriophage synthesis system for directed evolution

Efficient phage production has always been an urgent need in fields such as drug discovery, disease treatment, and gene evolution. To meet this demand, we constructed a robust cell-free synthesis system for generating M13 phage by simplifying its genome, enabling a three-times faster efficiency compared with the traditional method in vivo. We further developed a cell-free directed evolution system in droplets, comprising a modified helper plasmid (ΔPS-ΔgIII-ΔgVI) and the simplified M13 genome-carrying gene mutation library. This system was greatly improved when coupled with fluorescence-activated droplet sorting (FADS). We successfully evolved the T7 RNA polymerase (RNAP), achieving a twofold higher activity to read through the T7 terminator. Moreover, we evolved the tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity to read through the stop codon UAG.

Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes.

T7 RNA polymerase has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5' methyl guanosine caps on T7 RNAP derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.

Synthesis and purification of site-specifically modified T7 RNA polymerase for bioorthogonal conjugation applications

Synthesis and purification of site-specifically modified T7 RNA polymerase for bioorthogonal conjugation applications

Bidirectional Polymerization-Transcription Amplification-Encoded Dual-Color Fluorescent Biosensor for Label-Free and Primer-Free Detection of Multiple piRNAs.

PIWI-interacting RNAs (piRNAs) are a type of endogenous noncoding RNAs with a length of 24-31 nucleotides, and they can specifically bind with PIWI proteins to form the piRNA/PIWI complexes for regulating multiple physiological and pathological processes. Herein, we develop a bidirectional polymerization-transcription amplification-encoded dual-color fluorescent biosensor for label-free and primer-free measurements of multiple piRNAs. The designed hairpin probe contains a palindromic tail, and it can serve as the target recognition unit, polymerization primer, and transcription template. In the presence of target piRNAs, the hairpin probes are opened to expose a palindromic sequence that can trigger bidirectional polymerization and transcription reaction with the assistance of KF polymerase and T7 RNA polymerase for the production of numerous RNA aptamers. The aptamers subsequently bind with the corresponding fluorophores (DFHBI-1T/MG) to form the RNA aptamer-fluorophore complexes for the generation of enhanced fluorescence signals. This biosensor can sensitively detect piR-36026 with a limit of detection (LOD) of 82.08 aM and piR-36743 with a LOD of 44.44 aM. Moreover, it can quantify cellular piRNAs with single-cell sensitivity and distinguish cancer cells from normal cells. Furthermore, it has the capability of distinguishing the expression of piRNAs in the tissues of breast cancer patients and healthy individuals. By simply altering the target recognition site of the hairpin probe, this biosensor can be extended to detect various piRNAs, offering a powerful platform for piRNA-related clinical diagnostics and therapeutics.

Electrochemical detection of MMP-2 with enhanced sensitivity: Utilizing T7 RNA polymerase and CRISPR-Cas12a for neuronal studies

This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.

T7 RNA polymerase catalyzed transcription of the epimerizable DNA lesion, Fapy•dG and 8-oxo-2′-deoxyguanosine

Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2′-deoxyguanosine) are major products of 2’-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to dG. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G or U. Fitting of reaction data indicate that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast or slow reacting base pairs vary less than 2-fold as a function of the nucleotide opposite the lesion. Promoter dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side-by-side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

MutaT7GDE: A Single Chimera for the Targeted, Balanced, Efficient, and Processive Installation of All Possible Transition Mutations In Vivo.

Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.

One-tube target amplification-free CRISPR-Cas13 autocatalytic system

Clustered regularly interspaced short palindromic repeats (CRISPR) diagnostics have shown great promise for nucleic acid testing. However, CRISPR/Cas13a-based RNA sensing is hindered by the absence of accurate, fast and sensitive assays. Current Cas-based methods generally involve two separate steps: target-amplification and Cas-detection. This two-step method complicates detection, causes cross-contamination, and is difficult to deploy. Therefore, it is necessary to develop a facile one-step Cas13a-based RNA detection assay. We present a one-tube target amplification-free CRISPR-Cas13a autocatalytic circuit system that combines the Cas13a nuclease, T4 polynucleotide kinase, Bsu DNA polymerase and T7 RNA polymerase with a universally designed combinatorial DNA probe to create a one-pot assay for isothermal RNA detection. In this assay, after target RNA recognition, the cis-cleavage and trans-cleavage activities of Cas13 are activated, which leads to the initiation of a Cas13a autocatalytic reaction, resulting in an exponentially amplifying detection signal, thus enabling the detection of approximately 23.01 (95 % CI:16.95-41.23) copies/µL RNA in 45 min. A facile, easy-to-deploy, and one-tube Cas13a-based autocatalytic reaction system was constructed for rapid and highly sensitive RNA detection for the first time. This Cas13a-based autocatalytic system offers not only a versatile platform for directly detecting RNA targets but also a route toward point-of-care nucleic acid testing.

Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts.

Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 μg/mL luciferase in batch reactions and 60 ± 4 μg/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Emissive Guanosine Analog Applicable for Real-Time Live Cell Imaging.

A new emissive guanosine analog CF3thG, constructed by a single trifluoromethylation step from the previously reported thG, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3thG suggests that the electronic effects of the trifluoromethyl group dominate its behavior and demonstrates its susceptibility to microenvironmental polarity changes. In vitro transcription initiations using T7 RNA polymerase, initiated with CF3thG, result in highly emissive 5'-labeled RNA transcripts, demonstrating the tolerance of the enzyme toward the analog. Viability assays with HEK293T cells displayed no detrimental effects at tested concentrations, indicating the safety of the analog for cellular applications. Live cell imaging of the free emissive guanosine analog using confocal microscopy was facilitated by its red-shifted absorption and emission and adequate brightness. Real-time live cell imaging demonstrated the release of the guanosine analog from HEK293T cells at concentration-gradient conditions, which was suppressed by the addition of guanosine.

Advances in evolved T7 RNA polymerases for expanding the frontiers of enzymatic nucleic acid synthesis.

In vitro RNA synthesis technologies are crucial in developing therapeutic RNA drugs, such as mRNA vaccines and RNA interference (RNAi) therapies. Enzymatic RNA synthesis, recognized for its sustainability and efficiency, enables the production of extensive RNA sequences under mild conditions. Among the enzymes utilized, T7 RNA polymerase is distinguished by its exceptional catalytic efficiency, enabling the precise and rapid transcription of RNA from DNA templates by recognizing the specific T7 promoter sequence. With the advancement in clinical applications of RNA-based drugs, there is an increasing demand for the synthesis of chemically modified RNAs that are stable and resistant to nuclease degradation. To this end, researchers have applied directed evolution to broaden the enzyme's substrate scope, enhancing its compatibility with non-canonical substrates and reducing the formation of by-products. This review summarizes the progress in engineering T7 RNA polymerase for these purposes and explores prospective developments in the field.