MAM、FS、MGMはNIEHS(プロジェクト4、P42ES027704、PI、ルシン)によって資金提供されています。MAM、FS、RMM、MGM、PKSは、CPRITが資金を提供するGCC先端顕微鏡・画像情報学センター(RP170719)によってサポートされています。イメージングは、NIH(DK56338、CA125123)、CPRIT(RP150578)、ダン・L・ダンカン総合がんセンター、ジョン・S・ダン・ガルフ・コースト化学ゲノミクス・コンソーシアムからの資金提供を受けて、ベイラー医科大学の統合顕微鏡コアによってもサポートされています。

<ol>
	<li>Russell, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression:+Transcription.">Gene Expression: Transcription.</a> iGenetics: A Mendelian Approach. , 331-352 (2006).</li><li>Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variety+in+the+level+of+gene+control+in+eukaryotic+cells.">Variety in the level of gene control in eukaryotic cells.</a> Nature. 297, 365-371 (1982).</li><li>K&#246;hler, A., Hurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exporting+RNA+from+the+nucleus+to+the+cytoplasm.">Exporting RNA from the nucleus to the cytoplasm.</a> Nature Reviews Molecular Cell Biology. 8, 761-773 (2007).</li><li>Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C., Teichmann, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Technology+and+Biology+of+Single-Cell+RNA+Sequencing.">The Technology and Biology of Single-Cell RNA Sequencing.</a> Molecular Cell. 58, 610-620 (2015).</li><li>Shah, S., Lubeck, E., Zhou, W., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+Transcription+Profiling+of+Single+Cells+Reveals+Spatial+Organization+of+Cells+in+the+Mouse+Hippocampus.">In situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus.</a> Neuron. 92, 342-357 (2016).</li><li>Halpern, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bursty+Gene+Expression+in+the+Intact+Mammalian+Liver.">Bursty Gene Expression in the Intact Mammalian Liver.</a> Molecular Cell. 58, 147-156 (2015).</li><li>Stossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-induced+transcription+at+individual+alleles+is+independent+of+receptor+level+and+active+conformation+but+can+be+modulated+by+coactivators+activity.">Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity.</a> Nucleic Acids Research. 48, 1800-1810 (2020).</li><li>Coassin, S. R., Orjalo, A. V., Semaan, S. J., Johansson, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Detection+of+Nuclear+and+Cytoplasmic+RNA+Variants+Utilizing+Stellaris+RNA+Fluorescence+In+situ+Hybridization+in+Adherent+Cells.">Simultaneous Detection of Nuclear and Cytoplasmic RNA Variants Utilizing Stellaris RNA Fluorescence In situ Hybridization in Adherent Cells.</a> Methods in Molecular Biology. 1211, 189-199 (2014).</li><li>Gall, J. G., Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+detection+of+RNA-DNA+hybrid+molecules+in+cytological+preparations.">Formation and detection of RNA-DNA hybrid molecules in cytological preparations.</a> Proceedings of the National Academy of the Sciences of the United States of America. 63, 378-383 (1969).</li><li>Bauman, J. G., Wiegant, J., Borst, P., van Duijn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+fluorescence+microscopical+localization+of+specific+DNA+sequences+by+in+situ+hybridization+of+fluorochrome+labelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labelled RNA.</a> Experimental Cell Research. 128, 485-490 (1980).</li><li>Singer, R. H., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+gene+expression+visualized+in+chicken+muscle+tissue+culture+by+using+in+situ+hybridization+with+biotinated+nucleotide+analog.">Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with biotinated nucleotide analog.</a> Proceedings of the National Academy of the Sciences of the United States of America. 79, 7331-7335 (1982).</li><li>Femino, A. M., Fay, F. S., Fogarty, K., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+single+RNA+transcripts+in+situ.">Visualization of single RNA transcripts in situ.</a> Science. 280, 585-590 (1998).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nature Methods. 5, 877-879 (2008).</li><li>Orjalo, A. V., Johansson, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stellaris+RNA+Fluorescence+In+situ+Hybridization+for+the+Simultaneous+Detection+of+Immature+and+Mature+Long+Noncoding+RNAs+in+Adherent+Cells.">Stellaris RNA Fluorescence In situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells.</a> Methods in Molecular Biology. 1402, 119-134 (2016).</li><li>Johnson, C. V., Singer, R. H., Lawrence, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Fluorescent+Detection+of+Nuclear+RNA+and+DNA:+Implications+for+Genome+Organization.">Chapter 3 Fluorescent Detection of Nuclear RNA and DNA: Implications for Genome Organization.</a> Methods in Cell Biology. 35, 73-99 (1991).</li><li>Levsky, J. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+Past,+present+and+future.">Fluorescence in situ hybridization: Past, present and future.</a> Journal of Cell Science. 116, 2833-2838 (2003).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+A+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14, 22-29 (2012).</li><li>Stossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+estrogenic+mechanisms+of+bisphenol+A+analogs+through+high+throughput+microscopy-based+contextual+assays.">Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays.</a> Chemistry and Biology. 21, 743-753 (2014).</li><li>Szafran, A. T., Stossi, F., Mancini, M. G., Walker, C. L., Mancini, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+properties+of+non-estrogenic+substituted+bisphenol+analogs+using+high+throughput+microscopy+and+image+analysis.">Characterizing properties of non-estrogenic substituted bisphenol analogs using high throughput microscopy and image analysis.</a> PLoS One. 12, 1-19 (2017).</li><li>Rae, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GREB1+is+a+critical+regulator+of+hormone+dependent+breast+cancer+growth.">GREB1 is a critical regulator of hormone dependent breast cancer growth.</a> Breast Cancer Research and Treatment. 92, 141-149 (2005).</li><li>Heldring, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptors:+How+do+they+signal+and+what+are+their+targets.">Estrogen receptors: How do they signal and what are their targets.</a> Physiological Reviews. 87, 905-931 (2007).</li><li>Hah, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+extensive,+and+transient+transcriptional+response+to+estrogen+signaling+in+breast+cancer+cells.">A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells.</a> Cell. 145, 622-634 (2011).</li><li>Kadam, P., Bhalerao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size+calculation.">Sample size calculation.</a> International Journal of Ayurveda Research. 1, 55-57 (2010).</li><li>Kocanova, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+estrogen-responsive+genes+does+not+require+their+nuclear+co-localization.">Activation of estrogen-responsive genes does not require their nuclear co-localization.</a> PLoS Genetics. 6, (2010).</li><li>Shaffer, S. M., Wu, M. T., Levesque, M. J., Raj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbo+FISH:+A+Method+for+Rapid+Single+Molecule+RNA+FISH.">Turbo FISH: A Method for Rapid Single Molecule RNA FISH.</a> PLoS One. 8, (2013).</li><li>Kim, S. O., Kim, J., Okajima, T., Cho, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+paraformaldehyde-treated+individual+cells+investigated+by+atomic+force+microscopy+and+scanning+ion+conductance+microscopy.">Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy.</a> Nano Convergence. 4, (2017).</li><li>Shieh, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+ribonucleoside+vanadyl+complex+(RVC)+for+developing+a+multifunctional+tissue+preservative+solution.">Application of ribonucleoside vanadyl complex (RVC) for developing a multifunctional tissue preservative solution.</a> PLoS One. 13, 1-14 (2018).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+precision+in+quantitative+fluorescence+microscopy.">Accuracy and precision in quantitative fluorescence microscopy.</a> Journal of Cell Biology. 185, 1135-1148 (2009).</li><li>Vinegoni, C., Feruglio, P. F., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dynamic+range+fluorescence+imaging.">High dynamic range fluorescence imaging.</a> IEEE Journal of Selected Topics in Quantum Electronics. 25, (2019).</li><li>Tam, R., Shopland, L. S., Johnson, C. V., McNeil, J. A., Lawrence, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+RNA+FISH+for+visualizing+gene+expression+and+nuclear+architecture.">Application of RNA FISH for visualizing gene expression and nuclear architecture.</a> FISH: A Practical Approach. , 93-117 (2002).</li><li>Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I., North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial:+guidance+for+quantitative+confocal+microscopy.">Tutorial: guidance for quantitative confocal microscopy.</a> Nature Protocols. , (2020).</li><li>Trcek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-mRNA+counting+using+fluorescent+in+situ+hybridization+in+budding+yeast.">Single-mRNA counting using fluorescent in situ hybridization in budding yeast.</a> Nature Protocols. 7, 408-419 (2012).</li><li>Kwon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+in+situ+hybridization:+Quantitative+imaging+of+single+RNA+molecules.">Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules.</a> BMB Reports. 46, 65-72 (2013).</li><li>Sibarita, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvolution+microscopy.">Deconvolution microscopy.</a> Advcances in Biochemical Engineering/Biotechnology. 95, 201-243 (2005).</li><li>Takei, Y., Shah, S., Harvey, S., Qi, L. S., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Dynamic+Imaging+of+Genomic+Loci+by+Combined+CRISPR+Imaging+and+DNA+Sequential+FISH.">Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH.</a> Biophysical Journal. 112, 1773-1776 (2017).</li><li>Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Imaging+with+Multiplexed+Error-Robust+Fluorescence+in+situ+Hybridization+(MERFISH).">RNA Imaging with Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH).</a> Methods in Enzymology. 572, 1-49 (2016).</li><li>Shah, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+Spatial+Genomics+of+the+Nascent+Transcriptome+by+Intron+seqFISH.">Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH.</a> Cell. 174, 363-376 (2018).</li><li>Xia, C., Babcock, H. P., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+detection+of+RNA+using+MERFISH+and+branched+DNA+amplification.">Multiplexed detection of RNA using MERFISH and branched DNA amplification.</a> Scientific Reports. 9, 1-13 (2019).</li><li>Lubeck, E., Coskun, A. F., Zhiyentayev, T., Ahmad, M., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+in+situ+RNA+profiling+by+sequential+hybridization.">Single-cell in situ RNA profiling by sequential hybridization.</a> Nature Methods. 11, 360-361 (2014).</li><li>Wang, G., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+imaging+of+high-density+libraries+of+RNAs+with+MERFISH+and+expansion+microscopy.">Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy.</a> Scientific Reports. 8, (2018).</li><li>Wassie, A. T., Zhao, Y., Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+microscopy:+principles+and+uses+in+biological+research.">Expansion microscopy: principles and uses in biological research.</a> Nature Methods. 16, 33-41 (2019).</li><li>Zimmerman, S. G., Peters, N. C., Altaras, A. E., Berg, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+RNA+ISH,+RNA+FISH+and+protein-RNA+double+labeling+(IF/FISH)+in+Drosophila+ovaries.">Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.</a> Nature Protocols. 8, 2158-2179 (2013).</li><li>Kochan, J., Wawro, M., Kasza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+mRNA+and+protein+in+single+cells+using+immunofluorescence-combined+single-molecule+RNA+FISH.">Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.</a> Biotechniques. 59, 209-221 (2015).</li></ol>

著者らは開示するものは何もない。

記載されている smFISH の方法論は、以前に公開されたプロトコル7、12、14に基づいています。このプロトコルでは、ウェットラボ(播種密度、固定時間、透過性、プローブ濃度を含む)からイメージングおよび画像解析に最適化された重要なステップを説明し、遺伝子転写の単一細胞分析を行うことに興味を持つ研究所に完全...

遺伝子の転写と翻訳は、DNA配列からRNA、タンパク質1に移り、生物学の中心的な教義に関与する2つの主要なプロセスです。本研究では、転写中のメッセンジャーRNA(mRNA)の産生に焦点を当てます。新生RNA分子には、非コードイントロンとコーディングエキソンの両方が含まれます。イントロンは、mRNAが細胞質に移動してリボソーム2,3に翻訳する前に共転写される。
従来、mRNAの測定は、RT-qPCRまたはノーザンブロッティングなどの古典的なアッセイを用いて細胞のバルク集団(数百〜数百万)で行われます。非常に強力であるが、これらの方法は、細胞間変動(表現型ヘテロジェネリティ)、転写活性対立遺伝子の数の同定、そしておそらくより重要なことに、空間情報を欠いているという洞察を提供しないことから、限定的である。最近では、単一細胞RNAシーケンシング(RNAseq)を可能にするゲノムワイド技術は、イメージング法とシーケンシング4との間のギャップを埋め始めた。しかし、RNAseqは比較的低い検出効率に苦しんでおり、処理手順は空間情報5を完全に失う結果となる。単一細胞RNAEqは、同一性細胞の集団の中で表現型ヘテロジニアリティに関する洞察を提供することができるが、その現場でのRNA蛍光(RNA-FISH)は、空間レベルでの標的遺伝子発現のより完全な探索を促進し、かつアレル単位6,7に基づく。
RNA FISHは、固定細胞内の標的RNA分子の検出と局在化を可能にする技術です。従来とは異なり、骨状の方法、現在の最先端のRNA-FISHは、標的RNA配列と相補的である市販の核酸プローブを利用し、これらのプローブはWatson-Crick塩基対合体8によってそれらの標的にハイブリダイズする。初期のシチュハイブリダイゼーション技術は、1969年にGallとPardueによって確立された同様のプロトコルを含み、サンプルとして標的RNA9に特異的にハイブリダイズした核酸プローブで処理された。もともとアッセイは放射性または色分け検出を使用して行われました。しかし、1980年代には、DNA FISHおよび後のRNA FISHへの蛍光ハイブリダイゼーション(FISH)プロトコルの開発は、より敏感な検出への経路を開き、10,11を多重化する可能性を秘めている。
RNA FISHのさらなる発展は、単一RNA分子(smFISH)12,13を検出し定量する能力をもたれている。直接検出は、プローブ自体がフルオロフォアで標識される方法です。smFISHの課題は、蛍光シグナルを明確で回折制限されたスポットとして検出するのに十分なハイブリダイジングプローブ/ターゲットが必要な点です。この問題に対処するために、1つは、標的RNA8、12、14の様々な領域に相補的な短い一本鎖DNAオリゴヌクレオチドのプローブセットを使用することができる。複数のプローブの結合は、局所的な蛍光色素密度を増加させ、蛍光顕微鏡による明確で高強度のスポットとしてRNAを可視化します。この方法は、プローブセットプール内のオリゴヌクレオチドのオフターゲット結合は、真のシグナルとスプリアスオリゴヌクレオチド結合を区別するためにスポットサイズと強度を定量的に解析することによって真のRNAスポットシグナルと区別できるため有利であり、したがって偽陽性12を低減することによってより正確な分析を促進する。
mRNAを検出する代替方法は、古典的なBACクローンベースのニック翻訳法と、最近開発された分岐DNAシステムです。BACクローンのニック翻訳法では、標識されるDNAは酵素的にニックされ、新しいヌクレオチドが露出した3'末端に追加されます。DNAポリメラーゼIの活性は、所望のプローブ15,16に至る標識ヌクレオチドを付加する。分岐DNA技術は、RNA標的と対にハイブリダイズするオリゴヌクレオチドプローブを含み、これらのプローブは複数のシグナル増幅分子を利用して増幅される。この方法は、信号対雑音比を大幅に改善することができますが、蛍光スポットは大きく異なるサイズと強度17であるため、増幅は正確な定量を歪めることができます。
このプロトコルのモデルシステムとして、ガルベストンベイ/ヒューストンシップチャンネルにおける内分泌破壊化学物質の影響を定量化するためにNIEHSスーパーファンド研究プログラムの参加の一環として完全に採用され、エストロゲン受容体(ER)陽性乳癌細胞株MCF-7をERアゴニスト、17β-エストラジオール(E2)7,18,19で一晩投与した。E2は、原型的なER標的遺伝子を含む多くのER標的遺伝子を調節する、GREB17、20、21、22。ここで説明する smFISH プロトコルは、GREB1 を対象とする 2 つのスペクトル分離プローブ セットのセットを使用します。エキソンにハイブリダイズする1つとイントロンに他の1つを、成熟したRNAと新生RNA7の両方の測定を可能にする。次に、画像smFISH標識サンプルにデコンボリューションと相まって蛍光顕微鏡を使用し、画像分析ルーチンを適用して細胞当たりの活性対立胞および成熟RNAの数を定量化する。

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17&#946; Estradiol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2257</td>
			<td style="width:167px;">CAS Number 50-28-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambion SSC (20X) Buffer, Rnase-free</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AM9770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT10017CV</td>
			<td>Manufactured by Invitrogen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8417</td>
			<td>Without sodium pyruvate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran Sulfate, 50% Solution Sterile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3730-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline Ca++/Mg++</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21030CV</td>
			<td>Manufactured by Calbiochem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">07-678-005</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">353047</td>
			<td>Manufactured by Decon Laboratories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum (FBS), 500 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-753-2978</td>
			<td>Manufactured by Gemini Bio Products</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hyclone Water, Molecular Biology Grade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SH3053802</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Immersion Oil 1.516</td>
			<td style="width:71px;">GE Healthcare Life Sciences</td>
			<td align="right" style="width:119px;">29162940</td>
			<td>Manufactured by GE Healthcare Bio-Science</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-glutamine Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MCF-7 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Grade Formamide</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">PAH5051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus 60x/1.42 NA Objective</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Parafilm</td>
			<td style="width:71px;">Bemis Company, Inc.</td>
			<td style="width:119px;">52858-076</td>
			<td>Distributed by VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicilin-Streptomycin Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT3001Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ribonucleoside Vanadyl Complex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">101229-716</td>
			<td>Manufactured by New England Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase Away</td>
			<td style="width:71px;">Ambion</td>
			<td align="right" style="width:119px;">9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pyruvate, 100 mM</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">11-360-070</td>
			<td>Manufactured by Gibco</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermo Scientific Glass Coverslips, #1.5</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-545-81P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100, 100 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td>Manufactured by Fisher BioReagents</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA Solution 0.25%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Antifade Mounting Medium 10 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC9265087</td>
			<td>Manufactured by Vector Laboratories</td>
		</tr>
	</tbody>
</table>

single cell analysis of transcriptionally active alleles by single molecule fish

遺伝子転写は細胞生物学に不可欠なプロセスであり、細胞が内部および外部の手掛かりを解釈して応答することを可能にします。mRNA レベルを測定する従来のバルク集団法(ノーザンブロット、PCR、およびRNAseq)は、応答の細胞間変動に関する情報を提供する能力を欠いています。精密な単一細胞およびアレスリックな視覚化および定量は、単一分子RNA蛍光を使用して、その場合のハイブリダイゼーション(smFISH)で可能です。RNA-FISHは、標識されたオリゴヌクレオチドプローブを用いて標的RNAをハイブリダイズすることによって行われる。これらは中/高スループットのモダリティで画像化することができ、画像解析パイプラインを通じて、成熟したRNAと新生のRNAの両方に関する定量的なデータを単一のセルレベルで提供します。固定、パーメアビライゼーション、ハイブリダイゼーション、イメージングのステップは、ここに記載されているモデルシステムを使用して長年にわたって実験室で最適化されており、smFISH標識の単一細胞分析が成功し、堅牢になります。サンプルの準備と処理の主な目的は、信号対雑音比が高く、誤検出を低減し、より正確なデータを提供する高品質の画像を生成することです。ここでは、サンプルの準備からデータ分析までのパイプラインを、特定のサンプルに合わせて調整するための提案と最適化手順と組み合わせて説明するプロトコルを紹介します。

smFISHを介したホルモン応答を分析するために、例として、NIEHSスーパーファンド研究プログラム7への参加の文脈で環境災害における内分泌破壊化学物質の存在を決定するために、高スループットアッセイに使用されるエストロゲン受容体(ER)モデルを選びました。本実験では、付着性MCF-7乳癌細胞を、ERアゴニスト17β-エストラジオール(E2,10nM)または車両(DMSO)で24時間治療した?...

Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

この場合の単一分子RNA蛍光蛍光(smFISH)は、単一細胞レベルで特異的なRNAのレベルおよび局在を正確に定量する方法です。ここでは、特定のRNAの単一細胞定量化のためのウェットベンチ処理、イメージングおよび画像解析のための検証済みのラボプロトコルを報告します。

単一分子FISHによる転写活性アレスの単一細胞解析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

single-cell-analysis-transcriptionally-active-alleles-single-molecule

Research

JoVE Journal

Biochemistry

3.1K ビュー.  GCC Center for Advanced Microscopy and Image Informatics. この場合の単一分子RNA蛍光蛍光(smFISH)は、単一細胞レベルで特異的なRNAのレベルおよび局在を正確に定量する方法です。ここでは、特定のRNAの単一細胞定量化のためのウェットベンチ処理、イメージングおよび画像解析のための検証済みのラボプロトコルを報告します。

ビデオ: Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

磁気ピンセットで G quadruplexes の単一分子マニピュレーション

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

生化学

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

力の単一分子分光法のための蛋白質の共有結合固定

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

プロテオーム的分類の単一分子レベルでの均一性の定量化

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

ライブセル超解像顕微鏡と単一分子トラッキングのための従来のBODIPYコンジュゲート

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

キャップ依存翻訳キネティクス解析のための インビトロ 単分子イメージングアッセイ

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

制限エンドヌクレアーゼ媒介性DNA切断の1分子滞留時間解析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

リボソームタンパク質合成の単一分子蛍光エネルギー転移研究

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

DNA上に集合したEWS-FLI1凝縮体の1分子イメージング

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9精製超加工キネシン-3ファミリーモーターの1分子解析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

<meta charset="utf8"></head><body>1分子研究のためのin situヌクレオソーム再構成

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>著者スポットライト:単一分子技術のための効率的なヌクレオソーム再構成

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

単一分子相関力および蛍光顕微鏡法のためのIn Situヌクレオソームアセンブリ

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...