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
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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
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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
MOLECULAR CELL
·
2024
·
IF 14.5
Huihui An,Yifan Hong,Yeek Teck Goh,Casslynn W.Q. Koh,Shahzina Kanwal,Yi Zhang,Zhaoqi Lu,Phoebe M.L. Yap,Suat Peng Neo,Chun-Ming Wong,Alice S.T. Wong,Yang Yu,Jessica Sook Yuin Ho,Jayantha Gunaratne,Wee Siong Sho Goh
N 6 ,2′-O-dimethyladenosine (m 6 Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m 6 Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m 6 Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m 6 Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m 6 Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m 6 Am-modified RNAs. m 6 Am’s unique relationship with PCF11 means m 6 Am function is enhanced when PCF11 is reduced, which occurs during all- trans -retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m 6 Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m 6 Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m 6 Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma’s therapeutic response.
研究领域:
分子生物学
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癌症生物学
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转录组学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
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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
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
Virology Journal
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2025
·Vol. 22
·期 1
·1-14
·
IF 3.8
Liu Xianglan,Hu Jiaming,Wu Jiarui,Tian Yiru,Wang Jinbo,Wu Chunyan,Chen Qingfeng,Krall Leonard,He Yanping,Lu Qun
Background Human norovirus (HNV) is the predominant pathogen causing outbreaks of acute gastroenteritis globally. Despite significant efforts to combat norovirus infections, there is currently no FDA approved vaccine or antiviral drug available. Consequently, the development of effective antiviral agents is of critical importance. Methods and results In this study, a series of 41 5-cyano-2-thiacetyl aromatic pyrimidinone compounds were designed and synthesized. A cell viability-based screening for anti-murine norovirus (MNV) compounds was conducted, revealing that compound 29 (hereafter used as Co-29) exhibited antiviral activity against MNV. Co-29 demonstrated effective inhibition of MNV CW3 RNA replication, exhibiting an EC 50 of 58.22 μM. An RdRp enzyme activity assay indicated that Co-29 directly inhibits RdRp activity to both MNV and HNV. Molecular docking studies suggested that Co-29 interacts with the palm region of RdRp via hydrogen bonding with specific residues, which are conserved in RdRps across MNV and HNV norovirus variants. Conclusions In conclusion, our study suggests that the newly synthesized Co-29 may serve as a potential antiviral candidate or lead compound for future studies.
研究领域:
病毒学
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药物化学
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分子生物学
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DOI
PubMed
高影响因子
Frontiers in Microbiology
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2020
·
IF 4.236
Hu Ao,Li Jing,Tang Wei,Liu Ge,Zhang Haiwei,Liu Chunlan,Chen Xulin
Influenza virus RNA-dependent RNA polymerase (vRdRp) does not have capping activity and relies on the capped RNAs produced by the host RNA polymerase II (RNAPII). The viral polymerases process the capped RNAs to produce short capped RNA fragments that are used as primers to initiate the transcription of viral mRNAs. This process, known as cap-snatching, can be targeted by antiviral therapeutics. Here, anthralin was identified as an inhibitor against influenza a virus (IAV) infection by targeting the cap-snatching activity of the viral polymerase. Anthralin, an FDA-approved drug used in the treatment of psoriasis, shows antiviral activity against IAV infection in vitro and in vivo. Importantly, anthralin significantly reduces weight loss, lung injury, and mortality caused by IAV infection in mice. The mechanism of action study revealed that anthralin inhibits the cap-binding function of PB2 subunit and endonuclease activity of PA. As a result, viral mRNA transcription is blocked, leading to the decreases in viral RNA replication and viral protein expression. In conclusion, anthralin has been demonstrated to have the potential of an alternative antiviral against influenza virus infection. Also, targeting the captive pocket structure that includes the N-terminus of PA endonuclease domain and the C-terminal of PB2 cap-binding domain of IAV RdRp may be an excellent strategy for developing anti-influenza drugs.
研究领域:
病毒学
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药理学
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分子生物学
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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
GENOME BIOLOGY
·
2022
·Vol. 23
·期 1
·1-37
·
IF 17.906
Jiang Yongpeng,Huang Jie,Tian Kai,Yi Xiao,Zheng Haonan,Zhu Yi,Guo Tiannan,Ji Xiong
Background Mammalian cells have three types of RNA polymerases (Pols), Pol I, II, and III. However, the extent to which these polymerases are cross-regulated and the underlying mechanisms remain unclear.Results We employ genome-wide profiling after acute depletion of Pol I, Pol II, or Pol III to assess cross-regulatory effects between these Pols. We find that these enzymes mainly affect the transcription of their own target genes, while certain genes are transcribed by the other polymerases. Importantly, the most active type of crosstalk is exemplified by the fact that Pol III depletion affects Pol II transcription. Pol II genes with transcription changes upon Pol III depletion are enriched in diverse cellular functions, and Pol III binding sites are found near their promoters. However, these Pol III binding sites do not correspond to transfer RNAs. Moreover, we demonstrate that Pol III regulates Pol II transcription and chromatin binding of the facilitates chromatin transcription (FACT) complex to alter local chromatin structures, which in turn affects the Pol II transcription rate.Conclusion sOur results support a model suggesting that RNA polymerases show cross-regulatory effects: Pol III affects local chromatin structures and the FACT-Pol II axis to regulate the Pol II transcription rate at certain gene loci. This study provides a new perspective for understanding the dysregulation of Pol III in various tissues affected by developmental diseases.
研究领域:
分子生物学
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基因组学
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转录调控
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DOI
PubMed
高影响因子
DRUG DEVELOPMENT RESEARCH
·
2019
·Vol. 80
·期 6
·750-757
·
IF 1.742
Chang Zheng,Wen Hou,Jun Liu,Xiao-Fang Xu,Jing Lin,Ping-Hua Sun,Wei-Min Chen
The prevalence of multidrug resistance among clinically significant bacterial pathogens underlines a critical need for the development of new classes of antibacterial agents with novel structural scaffolds. Cajaninstilbene acid (CSA), which is isolated from pigeonpea leaves, has shown potent antibacterial activity. In this study, a series of 2-hydroxyl-4-methoxyl-3-(3-methylbut-2-en-1-yl)-6-(4-phenylbenzoylamino)benzoic acid derivatives derived from CSA were designed and synthesized, and their antibacterial activities were evaluated. Several synthesized compounds exhibit better antibacterial activity than CSA against Staphylococcus aureus , Staphylococcus epidermidis , and two strains of methicillin-resistant S. aureus . Meanwhile, the results of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays illustrate the good selectivity between bacteria and normal cells of the most active compounds 6u and 6v . Furthermore, well combinations with bacterial RNA polymerase of 6u arising from docking study imply the possible mechanism of antibacterial activity of these synthetic compounds.
研究领域:
药物化学
·
微生物学
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合成化学
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DOI
PubMed
高影响因子
Heliyon
·
2023
·
IF 4
Hang-fei Chen,Zhang-ping Li,Qi Wu,Chun Yu,Jing-Yi Yan,Yong-feng Bai,Sheng-mei Zhu,Mao-xiang Qian,Ming Liu,Li-feng Xu,Zheng Peng,Feng Zhang
Hyperactivation of ribosome biosynthesis (RiBi) is a hallmark of cancer, and targeting ribosome biogenesis has emerged as a potential therapeutic strategy. The depletion of TAF1B , a major component of selectivity factor 1 ( SL1 ), disrupts the pre-initiation complex, preventing RNA polymerase I from binding ribosomal DNA and inhibiting the hyperactivation of RiBi. Here, we investigate the role of TAF1B , in regulating RiBi and proliferation in stomach adenocarcinoma (STAD). We disclosed that the overexpression of TAF1B correlates with poor prognosis in STAD, and found that knocking down TAF1B effectively inhibits STAD cell proliferation and survival in vitro and in vivo. TAF1B knockdown may also induce nucleolar stress, and promote c-MYC degradation in STAD cells. Furthermore, we demonstrate that TAF1B depletion impairs rRNA gene transcription and processing, leading to reduced ribosome biogenesis. Collectively, our findings suggest that TAF1B may serve as a potential therapeutic target for STAD and highlight the importance of RiBi in cancer progression.
研究领域:
癌症生物学
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分子生物学
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细胞生物学
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DOI
PubMed
高影响因子
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