中国国立自然科学財団（No 21205078）と中国高等教育博士号プログラム（No.20123120110002）の支援を感謝します。この研究は、中国の国家主要研究開発計画（2016YFB1102303）、中国の国家基礎研究計画（​​973プログラム2015CB352001）、および中国国立自然科学財団（61378060）によって部分的に支持された。

1. SGEチップの基本設計<ol><li>ポリメタクリル酸メチル（PMMA）やポリカーボネートなどの透明プラスチックを使用してください。 注：SGEチップは図1Bに示されています。 SGEチップは、TBEバッファー用の円筒状の穴と、DNA分離用のチャンネルと、電極用の穴に沿って埋め込まれた2つのレーンとからなる。 </li><li>レーザー彫刻機を使用して、PMMAブロック内のSGEチャネルのアレイを製作します。 注：チップの幾何学的パラメータは、分離されるべきDNAのサイズに依存する。例えば、DNAフラグメントが1,000bpよりも小さい場合、SGEチップの最適なチャネルパラメータは、2.5mm×4.0mm×90mm（幅×奥行き×長さ）である。 </li><li> SGEデザインに基づいて、DNAサンプルをロードするためのチップ内にウェルを作成するために櫛を作製する。 </li></ol> 2.アガロースゲルの調製<ol><li> 10×TBE（1×TBE =89mMのトリス、89mMのホウ酸、および2mMのEDTA; pH 8.4）および蒸留水を1:19の比で含む。 </li><li> 100-bp DNAラダーを分離するために1.0％アガロースゲルを調製する。 注：0.5×TBE緩衝液中のアガロースの濃度は、分離すべきDNA断片のサイズに依存する。 </li><li>フラスコに0.1gのアガロースを入れ、0.5x TBE緩衝液10mLを加える。 備考：調製した溶液の容量は、フラスコの容量の1/3未満でなければならない。 </li><li>フラスコを保存用フィルムでシールし、次いで、アガロース/緩衝液混合物をマイクロ波（培地、1.0分）中で加熱する。フラスコをマイクロ波に入れる前に、爆発の場合には防腐膜に穴をあけてください。 </li><li>融解したアガロース溶液2.7 mLをSGEチップの4チャンネルに注ぎます。櫛をアガロース溶液に入れてウェルを作成します。 注：融解したアガロース溶液は、室温で3分後にゲル状態に冷却される。 </li><li> gから櫛を取り除くゲルを覆うため0.9mLの0.5×TBE緩衝液を添加する。 </li></ol> 3. SGEチップにおける電気泳動の実行<ol><li> LED光源を点灯させ、425〜505 nmのバンドパスフィルターを光源の上に置きます。 </li><li>ボルテックス装置を用いて14.4μLの100 bp DNAラダーと1.6μLのSYBR Greenを混合する。 </li><li> 4μLの混合液をSGEチップのアガロースゲルの各ウェルにロードします（ 図2 ）。 </li><li>電極をSGEチップの2つのレーンに入れます。 </li><li> SGEチップの上に550〜700 nmのバンドパスフィルタを配置します。 </li><li>電源を入れます。電圧は180V（20V / cm）に設定してください。スマートフォンをオンにしてDNA断片分離プロセスを記録します（ 図3 ）。 </li><li> 10分後に電気泳動が終了したら、電源とLEDライトをオフにします。 </li><li> SGEチップを清掃してください。 </li></ol>

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利害の衝突は宣言されていない。

アガロースゲル電気泳動は、DNA、RNAおよびタンパク質の分離に広く用いられている。この研究は、伝統的なゲル電気泳動プロトコルを置き換える新しい方法を提案している。結果は、50,100,1000bpのDNAラダーが、このように小型の組立装置で良好に分離できることを示している。この方法の大きな利点は、化学的消費の少ない核酸を分離できるだけでなく、分離プロセスも記録できることである...

<html>スラブゲル電気泳動（SGE）は、DNA断片分離1,2,3,4,5に最も有効な方法であり、生化学的および生物学的分析における多目的ツールであると考えられている6,7,8。しかし、多くの実験では、SGEが以下の4つの問題によって制限されていることが示されている。（1）分離に数時間および数日かかる。 （2）化学消費量が非常に高い。 （3）複雑な装置（ 例えば、 2D電気泳動セル、電気泳動電源、およびゲルイメージングシステム）を必要とする。 （4）ゲルイメージングシステムは、実験が終了したときにのみ、分離されたDNA断片を観察することができる。さらに、SGE9で一般的に使用されている臭化エチジウム（EtBr）は、突然変異原性であり、発癌性である11,12 。従って、EtBrを含むゲルを手渡すときは常に手袋を着用しなければならない。 キャピラリー電気泳動（CE）には、自動操作、短時間の分離、低消費など、SGEと比較して13,14,15,16,17の利点があります。しかし、CE機器は非常に高価です。従って、これらの制限を克服するために、DNAの分離のためのシステムが開発されている（ 図1 ）。このようなシステムは、化学物質の消費を大幅に削減し、SGE実験時間（8分未満）を節約するだけでなく、スマートフォンによるアガロースゲル中のDNA分離プロセスのリアルタイム追跡も行うことができる。このプロトコルに記載されている手順に従って、SGEチップの設計と製作、チップ内でのアガロースゲルの調製、スマートフォンによるシンプルなSGEシステムのセットアップ、アガロースゲルへのDNAマイグレーションプロセスの記録が可能です。

<table border="0" cellpadding="0" cellspacing="0" width="683">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10&#215;TBE</td>
			<td style="width:287px;">Beijing Solarbio Science &#38; Technology Co., Ltd.</td>
			<td style="width:181px;">T1051</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50bp DNA ladder</td>
			<td style="width:287px;">Takara Bio Inc.</td>
			<td style="width:181px;">3421A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100bp DNA ladder</td>
			<td style="width:287px;">Takara Bio Inc.</td>
			<td style="width:181px;">3422A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1kbp DNA ladder</td>
			<td style="width:287px;">Takara Bio Inc.</td>
			<td style="width:181px;">3426A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR GREEN</td>
			<td style="width:287px;">Takara Bio Inc.</td>
			<td style="width:181px;">5760A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:287px;">Sigma-Aldrich Corporate</td>
			<td style="width:181px;">V900510</td>
		</tr>
	</tbody>
</table>

real-time tracking of dna fragment separation by smartphone

スラブゲル電気泳動（SGE）は、DNAフラグメントの分離のための最も一般的な方法である;生物学などの分野に広く応用されている。しかし、従来のSGEプロトコルは非常に面倒であり、実験には長い時間がかかります。さらに、SGE実験における化学消費は非常に高い。この研究は、SGEチップに基づくDNA断片の分離のための簡単な方法を提案する。チップは彫刻機で作られています。 2つのプラスチックシートが、光信号の励起波長および発光波長に対して使用される。 DNAバンドの蛍光シグナルはスマートフォンで収集されます。この方法を検証するために、50,100および1,000bpのDNAラダーを分離した。この結果は、この方法を使用する場合、従来のSGE法の理想的な代替物であることを示しており、5,000bpより小さいDNAラダーが12分以内に高分解能で分離できることを示している。

<html> 図4 、 図5および図6は、 50,100および1,000bpのDNAラダーのゲル電気泳動後の典型的な結果を表す。実験後、DNA断片は十分に分離されていた。さらに、同じサンプルをSGEチップの4チャンネルで分離したところ、同じサイズのDNA断片が各実験において同じ距離を移動することが示された。 DNAラダーの分離?...

Watch this Scientific Journal Video about Real-time Tracking of DNA Fragment Separation by Smartphone at JoVE.com

Real-time Tracking of DNA Fragment Separation by Smartphone

スマートフォンによるDNA断片分離のリアルタイム追跡

伝統的なスラブゲル電気泳動（SGE）実験は、複雑な装置および高い化学消費を必要とする。この研究は、短い時間枠内でDNA断片を分離する低コストの方法を記載したプロトコールを提示する。

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

real-time-tracking-of-dna-fragment-separation-by-smartphone

Research

JoVE Journal

Biochemistry

14.4K ビュー.  University of Shanghai for Science and Technology. 伝統的なスラブゲル電気泳動（SGE）実験は、複雑な装置および高い化学消費を必要とする。この研究は、短い時間枠内でDNA断片を分離する低コストの方法を記載したプロトコールを提示する。 

ビデオ: Real-time Tracking of DNA Fragment Separation by Smartphone

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

アクリルアミドゲル電気泳動法で可視化した近赤外蛍光標識 DNA を用いた DNA ポリメラーゼ活性測定法

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

生化学

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

リアルタイム細胞解析搭載アレイの金電極の再生

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

Rad51 を介した DNA 鎖交換反応のリアルタイム観察

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

ハイスループットプリマゼプロファイリングを用いたDNAプリマゼによるDNA配列認識

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

初代ヒト気道上皮細胞の気道表面液 pH をリアルタイムで半自動で蛍光測定

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

GPCR-β-アレストリン1/2リアルタイムリビングシステムにおける相互作用をモニタリングし、創薬を加速

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

インタクトな機能的な膜タンパク質へのヌクレオチド結合をリアルタイムで測定

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

タンパク質トポグラフィ変化の測定のためのタンパク質の高速光化学酸化におけるリアルタイム補正の実現

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

ヘルムホルツ・ツェントルムベルリンにおける結晶学的フラグメントスクリーニングのワークフローとツール

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

DNAカーテン法によるヒストン集合の分子機構の解明

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...