PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

Correlating Transcription Initiation and Conformational Changes by a Single-Subunit RNA Polymerase with Near Base-Pair Resolution.

We provide a comprehensive analysis of transcription in real time by T7 RNA Polymerase (RNAP) using single-molecule fluorescence resonance energy transfer by monitoring the entire life history of transcription initiation, including stepwise RNA synthesis with near base-pair resolution, abortive cycling, and transition into elongation. Kinetically branching pathways were observed for abortive initiation with an RNAP either recycling on the same promoter or exchanging with another RNAP from solution. We detected fast and slow populations of RNAP in their transition into elongation, consistent with the efficient and delayed promoter release, respectively, observed in ensemble studies. Real-time monitoring of abortive cycling using three-probe analysis showed that the initiation events are stochastically branched into productive and failed transcription. The abortive products are generated primarily from initiation events that fail to progress to elongation, and a majority of the productive events transit to elongation without making abortive products.

A heat-inducible expression system for external control of gene expression in plastids.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western.

Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

ASIA: An automated stress-inducible adaptor for enhanced stress protein expression in engineered Escherichia coli.

Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a potent technique for protein expression in broad cells, but the energy requirements associated with this method impede the growth, leading to cell lysis when dealing with toxic and stress proteins. A Lemo21(DE3) strain denoted as L21 offers a solution by fine-tuning T7RNAP levels under rhamnose to induce T7 lysozyme (LysY) and enhance the protein production, but it requires optimization of inducer concentration, cultural temperature, and condition, even the types of carbon sources. Herein, we construct an automated stress-inducible adaptor (ASIA) employing different stress-inducible promoters from Escherichia coli. The ASIA system is designed to automatically regulate LysY expression in response to stress signals, thereby suppressing T7RNAP and amplifying the overexpression of stress protein cutinase ICCM. This approach fine-tunes T7RNAP levels and outperforms L21 in various temperatures and carbon source conditions. The ASIAhtp strain maintains ICCM yield at 91.6 mg/g-DCW even in the limiting carbon source at 1 g/L, which is 12-fold higher in protein productivity compared to using L21. ASIA as a versatile and robust tool for enhancing overexpression of stress proteins in E. coli is expected to address more difficult proteins in the future.

A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus.

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

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.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

Targeted C-to-T and A-to-G dual mutagenesis system for RhtA transporter in vivo evolution.

T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling in vivo C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G in vivo mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e). The C-to-T and A-to-G mutagenesis frequencies of T7-DualMuta were 4.02 × 10-3 and 1.20 × 10-2, respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to in vivo directed evolution of L-homoserine transporter RhtA, resulting in efficient variants that carried the four types of base conversions by T7-DualMuta. The evolved variants greatly increased the host growth rates at L-homoserine concentrations of 8 g/L, which was not previously achieved, and demonstrated the great in vivo evolution capacity. The novel molecular device T7-DualMuta efficiently provides both C/G-to-T/A and A/T-to-G/C mutagenesis on target regions, making it useful for various applications and research in Enzymology and Synthetic Biology studies. It also represents an important expansion of the base editing toolbox.ImportanceA T7-DualMuta system for simultaneous C-to-T and A-to-G in vivo mutagenesis was created. The mutagenesis frequency was 4.02 × 107 fold higher than the spontaneous mutation, which was reported to be approximately 10-10 bases per nucleotide per generation. This mutant system can be utilized for various applications and research in Enzymology and Synthetic Biology studies.

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110.

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.

Protein expression systems combining a flavonoid-inducible promoter and T7 RNA polymerase in Bacillus subtilis.

Recombinant protein production must be tightly controlled when overproduction adversely affects the host bacteria. We developed a flavonoid-inducible T7 expression system in Bacillus subtilis using the qdoI promoter to control the T7 RNA polymerase gene (T7 pol). Using the egfp reporter gene controlled by the T7 promoter in a multicopy plasmid, we confirmed that this expression system is tightly regulated by flavonoids, such as quercetin and fisetin. Altering the qdoI promoter for T7 pol control to its hybrid derivative increased the expression level by 6.6-fold at maximum values upon induction. However, faint expression leakage was observed under a noninducing condition. Therefore, the two expression systems with the original qdoI promoter and the hybrid construct can be used selectively, depending on the high control accuracy or production yield required.

High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA.

High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities.

Allosteric Probe Recognition Initiated Cascade Transcription Amplification for Sensitive Analysis of Exosomes.

Sensitive and accurate analysis of exosomes is important for many biological processes including as a biomarker for cerebral venous thrombosis (CVT) diagnosis and therapy. Herein, we established a sensitive and specific exosome detection approach based on target recognition initiated cascade signal amplification. In this method, an allosteric probe was designed with a hairpin structure for specific recognition of the exosome followed by signal amplification. After the cascade signal amplification process, spinach RNA sequences bind to DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one) to produce enhanced fluorescence signal (approximate 2000 fold than that of inactive DFHBI). Compared with former proposed exosome detection techniques, this method exhibited a comparable detection range, but with an easy-to-design toolbox. Therefore, we believe that the proposed approach holds great potential for exosome based early diagnosis and prognosis of disease.

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription.

RNA research and applications are underpinned by in vitro transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out in vitro RNA synthesis at low temperatures (4-25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3'-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for in vivo use.

PKR activation by noncanonical ligands: a 5'-triphosphate requirement versus antisense contamination.

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5' terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5'-phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5'-triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5' triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.

Isothermal detection of lncRNA using T7 RNA polymerase mediated amplification coupled with fluorescence-based sensor.

In this study, the isothermal detection of a cervical cancer-associated long non-coding RNA (lncRNA), namely, lncRNA-ATB, was performed for the first time with high selectivity and sensitivity via a T7 RNA polymerase transcription-mediated amplification system combined with a graphene oxide (GO) fluorescence-based sensor. Specific lncRNA primers with the T7 promoter overhang were designed and further had with the efficient amplification ability of T7 RNA polymerase. This detection platform distinguished the target lncRNA-ATB from other lncRNAs. In addition, the super fluorescence quenching ability of GO resulted in the development of a switch on/off fluorescence sensor. The resulting platform was able to detect target lncRNAs from samples of cervical cancer cell lines (HeLa) and human sera with high selectivity and a low detection limit of 1.96 pg. Therefore, the assay developed in this study demonstrated a high potential as an alternative tool for lncRNA quantification in clinical diagnosis.

Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production in Escherichia coli BL21 (DE3) by suppressing autolysis.

Escherichia coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, severe cell autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. Analysis of membrane structure and messenger RNA expression analysis showed that cells could underwent a form of programmed cell death (PCD) during the autolysis period. However, blocking three known PCD pathways in BL21 (DE3) did not completely alleviate autolysis completely. Consequently, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in the improved host BL21 (DE3-lac1G), which resistant to autolysis. As a consequence, the GDH activity at 43 h was greatly increased from 37.5 to 452.0 U/ml. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/ml/h at 43 h, compared to only 3 U/ml/h achieved using BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of 10 other enzymes. The engineered E. coli strain constructed in this study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows great potential for industrial applications.

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production.

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

Optimization of a T7-RNA polymerase system in Synechococcus sp. PCC 7002 mirrors the protein overproduction phenotype from E. coli BL21(DE3).

With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO2 conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research. KEY POINTS: • Promoter driving T7-RNA polymerase was optimized. • Up to 60-fold dynamic range in expression, depending on CO2 conditions. • Two-thirds of total protein is T7-RNA polymerase dependent.