MOLECULES
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2024
·Vol. 29
·期 11
·2461
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IF 4.2
Wei He,Xinya Zhang,Yangxiaoyu Zou,Ji Li,Chong Wang,Yucai He,Qiuheng Jin,Jianren Ye
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.
研究领域:
分子生物学
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生物技术
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疫苗学
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Yuanxun Gong,Jiayi Zhang,Huiying Liang,Yanling Wang,Zichun Song,Jihua Wei,Qianli Tang,Kai Zhang,Xianjiu Liao
Matrix metalloproteinases (MMPs) are critical for the degradation of extracellular matrix components, which is essential for processes such as tissue remodeling, wound healing, and angiogenesis. MMP-2, in particular, degrades type IV collagen and is associated with diseases such as cancer and cardiovascular conditions, making it a significant biomarker. This study presents a novel electrochemical biosensor for the detection of MMP-2. The biosensor combines the specificity of a programmable RNA detection system with the amplification capabilities of T7 RNA polymerase and CRISPR/Cas12a. In the absence of MMP-2, Peptide Nucleic Acid (PNA) adsorbs on the DNA template, preventing RNA amplification and resulting in a low electrochemical signal. When MMP-2 is present, it cleaves a specific peptide, releasing the DNA template and enabling RNA synthesis. This activation leads to the cleavage of single-stranded DNA probes, increasing the electrochemical signal. The biosensor demonstrates high sensitivity with a detection limit of 0.496 fM for MMP-2 and excellent specificity, effectively distinguishing MMP-2 from other proteins. It successfully detected MMP-2 in biological samples, such as liver cancer cell supernatants, indicating its potential for clinical diagnostics. This innovative biosensor offers a powerful tool for the early detection and monitoring of diseases associated with MMP-2 activity.
研究领域:
生物医学工程
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分子诊断
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纳米技术
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DOI
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2024
·
IF 4.3
He Wei,Zhang Xinya,Zou Yangxiaoyu,Li Ji,Chang Le,He Yu-Cai,Jin Qiuheng,Ye Jianren
Introduction: Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature.Methods: To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used.Results: The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.
研究领域:
分子生物学
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生物化学
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合成生物学
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DOI
PubMed
高影响因子
MOLECULES
·
2024
·Vol. 29
·期 19
·4713
·
IF 4.2
Wei He,Qi Geng,Guiying Ji,Ji Li,Dan Wang,Yucai He,Qiuheng Jin,Jianren Ye
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.
研究领域:
分子生物学
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生物技术
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药学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2024
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IF 3.7
Qing Wang,Jiajia You,Yichen Li,Jie Zhang,Yi Wang,Meijuan Xu,Zhiming Rao
In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT74) can increase the mutant frequency of the target gene by 1.12 × 104-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT74 system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT74 to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.
研究领域:
合成生物学
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分子遗传学
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DOI
PubMed
高影响因子
Viruses-Basel
·
2019
·Vol. 11
·期 4
·314
·
IF 3.811
Gaobo Zhang,Jian Yang,Fujun Qin,Congrui Xu,Jia Wang,Chengfeng Lei,Jia Hu,Xiulian Sun
Dendrolimus punctatuscypovirus (DpCPV), belonging to the genusCypoviruswithin the familyReoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed avp80-knockoutAutographa californicamultiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of aHindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing anegfpgene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infectSpodopetra exigualarvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.Keywords:DpCPV;reverse genetics system;vp80-knockout;foreign protein
研究领域:
病毒学
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分子生物学
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昆虫学
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生物技术
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DOI
PubMed
高影响因子
TALANTA
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2019
·
IF 4.916
Zengjin Li,Hui Guo,Fangyuan Xu,Wei Tang,Xinrui Duan
We reported a new strategy for sensitive monitoring in vitro RNA synthesis in real time based on fluorescence resonance energy transfer (FRET) from water-soluble conjugated polymer poly (9, 9-bis (6′-N, N, N,-trimethylammonium) hexyl) fluorene-co-alt-1,4-phenylene) bromide (PFP) to fluorogenic RNA aptamer/fluorophore (Spanich2/DFHBI and Broccoli/DFHBI) system. In this strategy, RNA of interest was transcribed accompanied by the Spanich2 or Broccoli. Then the 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) bound to the RNA aptamer sequence and thereby induced a fluorescence signal. PFP was used as the fluorescence energy donor, and Spanich2/DFHBI was the fluorescence energy acceptor. The fluorescence signal of Spanich2/DFHBI was amplified by light-harvesting and fluorescence amplification ability of PFP via FRET. And the limit of detection (LOD) (0.29 nM) was near 10-fold lower than that of RNA aptamer/DFHBI (LOD is 2.8 nM) alone by measuring the FRET ratio, which greatly reduced the variation of background signals. Most importantly, the addition of PFP did not interfere with RNA transcription in vitro , so this method was successfully applied to sensitively monitor RNA transcription and effect of T7 RNA polymerase inhibitor in real time, supplying a sensitive and simple method to study the modulation and inhibitor of RNA polymerase in vitro .
研究领域:
生物化学
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分子生物学
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分析化学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Zhongheng Wei,Huini Luo,Danlu Huang,Peixin Wei,Jiayi Zhang,Jihua Wei,Qianli Tang,Longjian Huang,Kai Zhang,Xianjiu Liao
Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5′-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti 3 C 2 /AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10 -9 U μL -1 and a wide dynamic range (1 × 10 -8 to 1 × 10 -5 U μL -1 ), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.
研究领域:
生物分析化学
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分子诊断
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癌症研究
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酶检测
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生物传感器与生物电子学
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Weiying Liu,Xiaolu Sui,Yunpeng Xu,Tingfei Xie,Yanzi Zhang,Aisha Zhang,Zhenyu Lin,Jihong Chen
MicroRNA-21 (miRNA-21) can serve as an early biomarker for acute kidney injury (AKI), it is important to develop sensitive and selective detection methods for miRNA-21 detection. In this study, an electrochemiluminescence (ECL) biosensor for miRNA-21which synergistically integrates T7 RNA polymerase-driven transcriptional amplification with CRISPR/Cas13a-mediated collateral cleavage and magnetic separation strategy had been designed. T7 RNA polymerase-driven transcriptional amplification has the characters of low requirements for conditions and high amplification efficiency. A cascaded signal amplification strategy which coupled enzymatic target recycling with CRISPR/Cas13a-mediated exponential signal amplification was proposed to achieve high detection sensitivity. And T7 promoter specificity and CRISPR/Cas13a sequence targeting had been adopted to ensure high specificity of the developed system. Furthermore, magnetic bead separation had been adopted to reduce the background signal. The parameters which may affected the performance of the biosensor had been optimized. Under optimized conditions, the ECL response of the system has a linear relationship with the logarithmic value of miRNA-21 concentration in the range of 1.0 fM to 1.0 nM, with a calculated limit of detection(LOD) of 0.33 fM (S/N = 3). The proposed methodology has been adopted to detect the target in clinical urine specimens with satisfied results.
研究领域:
生物化学
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分子生物学
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分析化学
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生物医学工程
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临床诊断
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产品关联
DOI
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
·
2023
·
IF 13.6
Jihua Wei,Zichun Song,Jiuying Cui,Yuanxun Gong,Qianli Tang,Kai Zhang,Xinlei Song,Xianjiu Liao
In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti 3 C 2 T x -compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.
研究领域:
生物技术
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分子生物学
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病毒学
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诊断技术
·
纳米技术
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产品关联
DOI
PubMed
高影响因子
METABOLIC ENGINEERING
·
2018
·
IF 7.674
Shubin Li,Tao Sun,Chunxiao Xu,Lei Chen,Weiwen Zhang
The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973) has been considered a good chassis candidate for “microbial cell factory” as it can perform oxygenic photosynthesis and its doubling time can be as short as 1.9 h. However, the limited genetic tools currently restrict its further research and application efforts using synthetic biology approaches. In this study, a series of genetic tools were systematically developed and optimized for Synechococcus 2973. First, the introduction of Tfp pilus assembly protein encoding gene pilN into Synechococcus 2973 successfully recovered its natural transformability, which greatly simplified the DNA transformation process. Second, a series of promoters with different strengths were evaluated and the super-strong promoters including P cpc560 from Synechocystis sp. PCC 6803, native P psbA2 and P psbA3 of Synechococcus 2973 were found with the highest activity of β -galactosidase among those evaluated by miller values. Some promoters related to photosystems (i.e., P psbA2 , P psbA3 , P 6803psbA2 and P cpc560 ) were also demonstrated to be induced by high intensity of light. Third, three lactose induction systems were evaluated, among which P lac combined with lacI q showed the best application prospect with great induction capacity, low leakage and middle induced expression. Fourth, the translational on riboswitch theoE* , the transcriptional off riboswitches theo/yitJ and xpt(C74U)/metE and an artificial inducing system combining theoE* with T7 RNA polymerase were successfully developed and characterized in Synechococcus 2973. Finally, by using T7 induction system to control the expression of both small RNA and chaperone Hfq, a small RNA regulatory tool was developed and optimized to be a strictly inducible off system for gene regulation in Synechococcus 2973. The work here presented valuable genetic toolboxes necessary for metabolic engineering and synthetic biology research in Synechococcus 2973, which will facilitate the future application of the fast growing cyanobacterial chassis.
研究领域:
合成生物学
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代谢工程
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微生物学
·
遗传学
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产品关联
DOI
PubMed
高影响因子
Frontiers in Microbiology
·
2021
·
IF 5.64
Xu Jiaqi,Dong Feng,Wu Meixian,Tao Rongsheng,Yang Junjie,Wu Mianbin,Jiang Yu,Yang Sheng,Yang Lirong
Efficient and novel recombinant protein expression systems can further reduce the production cost of enzymes. Vibrio natriegens is the fastest growing free-living bacterium with a doubling time of less than 10 min, which makes it highly attractive as a protein expression host. Here, 196 pET plasmids with different genes of interest (GOIs) were electroporated into the V. natriegens strain VnDX, which carries an integrated T7 RNA polymerase expression cassette. As a result, 65 and 75% of the tested GOIs obtained soluble expression in V. natriegens and Escherichia coli, respectively, 20 GOIs of which showed better expression in the former. Furthermore, we have adapted a consensus “what to try first” protocol for V. natriegens based on Terrific Broth medium. Six sampled GOIs encoding biocatalysts enzymes thus achieved 50–128% higher catalytic efficiency under the optimized expression conditions. Our study demonstrated V. natriegens as a pET-compatible expression host with a spectrum of highly expressed GOIs distinct from E. coli and an easy-to-use consensus protocol, solving the problem that some GOIs cannot be expressed well in E. coli.
研究领域:
生物技术
·
分子生物学
·
蛋白质工程
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DOI
PubMed
高影响因子
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