Analytical Methods
·
2018
·Vol. 10
·期 47
·5629-5633
·
IF 2.073
Hui Guo,Mengyue Fan,Zengjin Li,Wei Tang,Xinrui Duan
RNAs play critical roles in most cellular and developmental processes. The RNA in vitro synthesis quantification method with low background without requiring translation is highly desirable. In this study, we have described a ratiometric RNA aptamer–fluorophore complex, Spinach2/DFHBI–CM, for quantifying the in vitro synthesis of RNA. DFHBI–CM exhibits bright fluorescence upon excitation at 337 nm with or without RNA, and displays the characteristic emission band of triazolyl-coumarin centered at 420 nm. When DFHBI–CM is combined with RNA aptamer Spinach2 to generate Spinach2/DFHBI–CM fluorescence complexes, under the excitation wavelength of 447 nm, the fluorescence intensity at 502 nm is significantly enhanced. By comparing the emission intensity at 420 nm and 502 nm, ratiometric fluorescence measurement is achieved. Based on this principle, DFHBI–CM was applied as a fluorophore in RNA aptamer–fluorophore complex for ratiometrically detecting and quantifying RNA in vitro synthesis. Our design has the following advantages: (1) ratiometric fluorescence can minimize the environmental inferring factors and thus, more accurate and effective detection can be achieved; (2) compared with run-off transcription detection of RNA, this ratiometric fluorescent probe reports immediately the quantity of RNA synthesized in vitro.
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
高影响因子
Scientific Reports
·
2025
·Vol. 15
·期 1
·1-12
·
IF 3.9
Tang Zhouzhou,Li Zhihao,Wu Guofeng,Li Jianjun,Tan Jianye,Zhu Lixin
Osteosarcoma (OS) is the most common primary malignant bone tumor. Recent evidence suggests that the novel long noncoding RNA DHRS4 antisense RNA 1 (DHRS4-AS1) serves an important role in cancer progression and metastasis. However, its function and molecular mechanism in OS remain largely unknown. In the present study, DHRS4-AS1 expression was detected in OS cells by quantitative PCR. Gain- and loss-of-function experiments were conducted to study the effects of DHRS4-AS1 on the proliferation and apoptosis of OS cells. The potential mechanism of DHRS4-AS1 was examined through bioinformatics analysis and rescue experiments. DHRS4-AS1 was downregulated in OS cell lines. DHRS4-AS1 depletion promoted proliferation and inhibited apoptosis in OS cells, whereas DHRS4-AS1 overexpression had the opposite effects. Further research suggested that DHRS4-AS1 inhibited OS progression by regulating the microRNA-362-5p/aminopeptidase puromycin sensitive axis. The present findings suggested that DHRS4-AS1 may serve as a potential therapeutic target for OS.
研究领域:
肿瘤学
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分子生物学
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医学遗传学
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DOI
PubMed
高影响因子
ANALYST
·
2017
·Vol. 142
·期 24
·4843-4851
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IF 3.885
Ying-Xu Chen,Wen-Jing Zhang,Ke-Jing Huang,Mingbo Zheng,Ya-Cen Mao
In this work, we report an ultrasensitive electrochemical biosensor for microRNA-21 (miRNA-21) detection by using a competitive RNA–RNA hybridization configuration. A biotinylated miRNA of the self-same sequence with the target miRNA is mixed with the samples, and allowed competition with the target miRNA for a thiolated RNA probe immobilized onto a tungsten diselenide (WSe2) nanosheet modified electrode. Thereafter the current response is obtained by forming the hybridized biotinylated miRNA with streptavidin–horseradish peroxidase (HRP) conjugates to catalyze the H2O2 + hydroquinone (HQ) system. Benefiting from the high specific surface area of WSe2 nanosheets, the competitive hybridization configuration and the signal amplification of the H2O2 + HQ detection system, the proposed assay exhibits a wide linear range of 0.0001–100 pM towards target miRNA with a detection limit of 0.06 fM (S/N = 3), and shows excellent discrimination ability for base-mismatched miRNA sequences. Therefore, the designed platform has promising prospects for the detection of miRNA in biomedical research and early clinical diagnosis.
研究领域:
生物化学
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纳米技术
·
生物医学工程
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分子诊断
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产品关联
DOI
PubMed
高影响因子
Nature Protocols
·
2024
·
IF 13.1
Chen Xingyu,Wang Liu,Xie Jiahao,Nowak Jakub S.,Luo Bingnan,Zhang Chong,Jia Guowen,Zou Jian,Huang Dingming,Glatt Sebastian,Yang Yang,Su Zhaoming
RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure–function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.
研究领域:
分子生物学
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结构生物学
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冷冻电子显微镜
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RNA生物化学
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DOI
PubMed
高影响因子
STAR Protocols
·
2022
·
IF 0
Qiaojie Liu,Jiqin Wu,Peng Gong
Summary Nucleotide/nucleoside analogs (NAs) are important compounds used in antiviral drug development. To understand the action mode of NA drugs, we present an enzymology protocol to initially evaluate the intervention mechanism of the NTP forms of NAs on a coronaviral RNA-dependent RNA polymerase (RdRP). We describe the preparation of SARS-CoV-2 RdRP proteins and RNA constructs, followed by a primer-dependent RdRP assay to assess NTP forms of NAs. Two representative NA drugs, sofosbuvir and remdesivir, are used for demonstration of this protocol. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021) .
研究领域:
病毒学
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分子生物学
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药理学
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DOI
PubMed
NUCLEIC ACIDS RESEARCH
·
2020
·Vol. 48
·期 20
·11773-11784
·
IF 11.502
Lin Jiao,Liu Yan,Lai Peidong,Ye Huixia,Xu Liang
A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.
研究领域:
合成生物学
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分子遗传学
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基因工程
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DOI
PubMed
高影响因子
Cell Stem Cell
·
2024
·
IF 19.8
Xue Han,Linqing Xing,Yantao Hong,Xuechun Zhang,Bo Hao,J. Yuyang Lu,Mengyuan Huang,Zuhui Wang,Shaoqian Ma,Ge Zhan,Tong Li,Xiaowen Hao,Yibing Tao,Guanwen Li,Shuqin Zhou,Zheng Zheng,Wen Shao,Yitian Zeng,Dacheng Ma,Wenhao Zhang,Zhen Xie,Haiteng Deng,Jiangwei Yan,Wulan Deng,Xiaohua Shen
Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.
研究领域:
分子生物学
·
干细胞研究
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细胞衰老机制
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DOI
PubMed
高影响因子
Nature Communications
·
2018
·Vol. 9
·期 1
·1-15
·
IF 12.353
Usman Waqas Muhammad,Pham Tin Chanh,Kwok Yuk Yan,Vu Luyen Tien,Ma Victor,Peng Boya,Chan Yuen San,Wei Likun,Chin Siew Mei,Azad Ajijur,He Alex Bai-Liang,Leung Anskar Y. H.,Yang Mengsu,Shyh-Chang Ng,Cho William C.,Shi Jiahai,Le Minh T. N.
Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA. Here, we describe and validate a new strategy to generate large-scale amounts of RBC-derived EVs for the delivery of RNA drugs, including antisense oligonucleotides, Cas9 mRNA, and guide RNAs. RNA drug delivery with RBCEVs shows highly robust microRNA inhibition and CRISPR–Cas9 genome editing in both human cells and xenograft mouse models, with no observable cytotoxicity.
研究领域:
药理学
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分子生物学
·
基因治疗
·
生物技术
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产品关联
DOI
PubMed
高影响因子
Nature Communications
·
2024
·Vol. 15
·期 1
·1-14
·
IF 14.7
Yin Kun,Xu Yiling,Guo Ye,Zheng Zhong,Lin Xinrui,Zhao Meijuan,Dong He,Liang Dianyi,Zhu Zhi,Zheng Junhua,Lin Shichao,Song Jia,Yang Chaoyong
A fundamental objective of genomics is to track variations in gene expression program. While metabolic RNA labeling-based single-cell RNA sequencing offers insights into temporal biological processes, its limited applicability only to in vitro models challenges the study of in vivo gene expression dynamics. Herein, we introduce Dyna-vivo-seq, a strategy that enables time-resolved dynamic transcription profiling in vivo at the single-cell level by examining new and old RNAs. The new RNAs can offer an additional dimension to reveal cellular heterogeneity. Leveraging new RNAs, we discern two distinct high and low metabolic labeling populations among proximal tubular (PT) cells. Furthermore, we identify 90 rapidly responding transcription factors during the acute kidney injury in female mice, highlighting that high metabolic labeling PT cells exhibit heightened susceptibility to injury. Dyna-vivo-seq provides a powerful tool for the characterization of dynamic transcriptome at the single-cell level in living organism and holds great promise for biomedical applications.
研究领域:
基因组学
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分子生物学
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转录组学
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单细胞分析
·
肾脏研究
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产品关联
DOI
PubMed
高影响因子
ACS Applied Nano Materials
·
2024
·
IF 5.3
Meng-Fan Feng,Li-Miao Qin,Bo Sun,Xianghui Yu,Yuqing Wu
Circular RNA (circRNA) has emerged as a promising class of therapeutics and mRNA vaccines. While possessing greater stability and functionality akin to linear mRNA, circRNA still necessitates an efficient and adaptable delivery system to surmount the primary challenge of cellular uptake and membrane penetration. Inspired by the high invasiveness of the virus, in this study, the virus-like nanoparticles (VLNPs) of human papillomavirus (HPV) have been constructed as robust carriers for circRNA encapsulation and transfer through their coassembly monitoring. Notably, the functionalization of a transmembrane peptide (L17E) on the outer surface of VLNPs has led to the development of a more efficient nanocarrier, which has significantly enhanced both the transmembrane delivery and endosomal escape of circRNA. Validation studies employing VLNPs-L17E-packaged circRNA across various cell types have demonstrated notable improvements in target protein expression, ranging from 1.48- to 2.04-fold. These findings underscore the potential of HPV-VLNPs-L17E as a carrier for circRNA delivery, offering promising prospects for RNA therapy applications in the future.
研究领域:
分子生物学
·
纳米医学
·
治疗递送系统
·
基因工程
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产品关联
DOI
高影响因子
Jove-Journal of Visualized Experiments
·
2022
·
IF 1.424
Xiaofang Liu,Xiang Zhou,Lifeng Zhou,Jiafu Hu,Kai Guo
The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5′-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 µg/mL) in the soaking buffer for 24 h in the dark at 25 °C.The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.
研究领域:
分子生物学
·
植物病理学
·
线虫学
·
遗传学
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产品关联
DOI
PubMed
高影响因子
Nature Protocols
·
2025
·
IF 16
Xu Ziye,Lyu Yuexiao,Chen Haide,Chen Yu,Wang Yongcheng
Formalin-fixed paraffin-embedded (FFPE) samples represent a vast and valuable resource of patient material, often linked to extensive clinical history and follow-up data. However, achieving single-cell or single-nucleus RNA (sc/snRNA) profiling in these archived tissues remains challenging. To address this, we have developed snRandom-seq, a droplet- and random primer–based single-nucleus total RNA sequencing technology specifically designed for FFPE tissues. This method captures total RNAs by using random primers and demonstrates a low doublet rate (0.3%), increased RNA coverage and enhanced detection of non-coding and nascent RNAs compared to state-of-the-art high-throughput sc/snRNA-seq technologies. This protocol provides a comprehensive guide to isolating single nuclei from FFPE samples; performing in situ DNA blocking, reverse transcription and dA tailing reactions; barcoding single-nucleus droplets; and preparing sequencing libraries. The entire snRandom-seq process can be completed in 4 d. This platform serves as a powerful tool for snRNA-seq of clinical specimens, with broad applications in studying complex biological systems.
研究领域:
分子生物学
·
医学
·
生物信息学
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产品关联
DOI
PubMed
高影响因子
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