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Engineering Novel Molecular Beacon Constructs to Study Intracellular rna dynamics and Localization

Genomics, Proteomics & Bioinformatics 2017年第5期15卷 279-286页

作者： Zhao Ma Xiaotian Wu Christopher J.Krueger Antony K.Chen Department of Biomedical Engineering College of Engineering Peking University Beijing 100871 China Wallace H Coulter Department of Biomedical Engineering Georgia Institute of Technology Atlanta GA 30332 USA

With numerous advancements in novel biochemical techniques, our knowledge of the role of rnas in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding rna processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single rna transcripts in living cells with high spatial and temporal resolution. In this review, we discuss tech- niques that have shown great promise for single rna imaging, followed by highlights in our recent work in the development of molecular beacons （MBs）, a class of nanoscale oligonucleotide-probes, for detecting individual rna transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of rna functions and activities.

关键词： Y-O-methyl rna Phosphorothioate Molecular beacon rna dynamics Single-molecule rnaimaging

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a^(6)A-seq:N^(6)-allyladenosine-based cellular messenger rna metabolic labelling and sequencing

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Fundamental Research 2023年第5期3卷 657-664页

作者： Xiao Shu Chenyang Huang Tengwei Li Jie Cao Jianzhao Liu MOE Key Laboratory of Macromolecular Synthesis and Functionalization Department of Polymer Science and EngineeringZhejiang UniversityZheda Road 38Hangzhou 310027China Life Sciences Institute Zhejiang University866 Yuhangtang RdHangzhou 310058China

The integration of rna metabolic labelling by nucleoside analogues with high-throughput rna sequencing has been harnessed to study rna *** immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled *** we developed an a^(6)A-seq method,which takes advantage of N^(6)-allyladenosine(a^(6)A)metabolic labelling on cellular mrnas and profiles them in an immunoprecipitation-free and mutation-based manner.a^(6)A plays a role as a chemical sequencing tag in that the iodination of a^(6)A in mrnas results in 1,N^(6)-cyclized adenosine(cyc-A),which induces base misincorporation during rna reverse transcription,thus making a^(6)A-labelled mrnas detectable by sequencing.A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine.a^(6)A-seq was utilized to study cellular gene expression changes under a methionine-free stress *** with regular rna-seq,a^(6)A-seq could more sensitively detect the change of mrna production over a time *** experiment of a^(6)Acontaining mrna immunoprecipitation followed by qPCR successfully validated the high-throughput a^(6)A-seq ***,our results show a^(6)A-seq is an effective tool to study rna dynamics.

关键词： rna sequencing Adenosine analogue Metabolic labelling Base mutation Nascent rna rna dynamics rna tag

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Dynamic imaging of rna in living cells by CRISPR-Cas13 systems

Dynamic imaging of RNA in living cells by CRISPR-Cas13 syste...

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中国生物化学与分子生物学会2019年全国学术会议暨学会成立四十周年

作者： Liang-Zhong Yang Yang Wang Si-Qi Li Run-Wen Yao Huang Wu Gordon G.Carmichael Ling-Ling Chen State Key Laboratory of Molecular Biology Shanghai Key Laboratory of Molecular Andrology CAS Center for Excellence in Molecular Cell Science Shanghai Institute of Biochemistry and Cell Biology University of Chinese Academy of Sciences Chinese Academy of Sciences Department of Genetics and Developmental Biology University of Connecticut Stem Cell Institute University of Connecticut Health Center School of Life Science and Technology ShanghaiTech University

Visualizing the location and dynamics of rnas, particularly long noncoding rnas(lncrnas), in live cells is key to understanding function, but currently available tools are limited. Here, we identify two endonuclease-deficient, singlecomponent programmable rna guided rna-targeting Cas13 rnases(dCas13) that allow efficient lncrna labeling in live cells, even using single guide rnas with spacers of 20-27 nt in length. Compared to traditional aptamer-based strategies(such as the MS2-MCP system), the optimized dCas13 rna labeling system achieves a higher labeling efficiency and does not require genetic manipulation. Combination of two different CRISPR-dCas13 systems or CRISPR-dCas13 and MS2-MCP allows dual-color rna tracking in single live cells and the study of the dynamics of the lncrna NEAT1-associated paraspeckle nuclear bodies. Collectively, these results demonstrate that the CRISPR-dCas13 rna labeling system is a userfriendly and efficient tool to visualize rnas in live cells.

关键词： CRISPR-Cas13 NEAT1 nuclear stress bodies paraspeckle dynamics rna microscopy rna dynamics SatⅢ

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