Name: single-molecule manipulation of g-quadruplexes by magnetic tweezers
Uploaded: 2017-09-19T14:00:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

著者が原稿を校正孟パンをありがちましょう。この作品はサポートによってシンガポール省の教育学術研究基金第 3 層 (MOE2012-T3-1-001) J.Y.; にJ.Y.; ことシンガポール メカノバイオロジー研究所を通じて国立研究財団国立研究財団、その NRF Investigatorship プログラム (NRF Investigatorship 賞号の下で総理大臣のオフィス、シンガポール、J.Y.; に NRF-NRFI2016-03H. Y に中央大学 (2017KFYXJJ153) の基礎的研究基金。

1 です単一分子の伸張のための準備の G4 DNA <ol><li>準備 5 &#39;-チオール ラベル及び 5 &#39;-5 を使用してラムダ ファージ DNA テンプレートの DDNA ポリメラーゼを用いた PCR によるビオチン dsDNA ハンドル &#39;-チオールと 5 &#39;-ビオチン。プライマー 14 ( 図 1)。両方 dsDNA のハンドルがある高い GC の内容 (&#62; 60%) DNA を防ぐために溶融高力または overstretching DNA 中に DNA を開催移行 15</li>。
	<li>浄化の PCR の製品のメーカーによると BstXI の制限の酵素と商業浄化キットとダイジェストを使用して &#39; s プロトコル</li>。
	<li>縛る G4 成形 ssDNA、および製造元によると T4 DNA リガーゼを使ってフランク ssDNA と dsDNA ハンドル &#39; s プロトコル。メーカーによると商業精製キットを使用してゲルの抽出による結紮製品を浄化 &#39; s プロトコル</li>。
</ol> 2。流路の準備 <ol><li>Coverslip のクリーニングと表面機能化 <ol><li>場所下の染色 (各 jar coverslips の 7 個セット出来る瓶カバー ガラスのトップ coverslips (#1.5、20 × 20 mm) に coverslips (#1.5、22 × 32 mm)ボリュームが ~ 20 mL)。リンス、coverslips 蒸留水の瓶で 2-5 倍です</li>。
		<li>追加 ~ 5 ~ 40% に各 jar ファイル、および 30 分の超音波洗浄で洗剤溶液の 20 mL 用蒸留水ですすぎ &#62; 洗剤を削除する 10 倍</li>。
		<li>乾燥オーブンで瓶に coverslips (~ 150 ° C;注意、ホット)、または N 2 ガス。ドライ キャビネットの乾燥トップ coverslips を格納します</li>。
		<li>使用プラズマ (O 2 ガス) coverslips 10 分の瓶の中をきれいにします。10 分間の 1% (3-アミノプロピル) プタデカフルオロテトラヒドロデシルトリエトキシシラン (APTES) 液をメタノール 20 mL を準備 (注意 毒性・可燃性)。APTES をメタノールに溶解する化学発煙のフードを使用します 。
		注: は、APTES ソリューションを格納するときに湿気を避けるため。古い APTES しばしば coverslips の表面機能性の問題が発生します</li>。
		<li>プラズマ洗浄後すぐに瓶に 1 %aptes 溶液のすべてを追加し、1 h. 注ぐ 1 %aptes メタノールの特定廃棄物のボトルに廃棄物のためのインキュベートします。可燃性の化学薬品の発煙のフードでメタノールに関連の手順を実行します</li>。
		<li>メタノールと一度瓶を洗うと &#62; 10 倍蒸留水と (~ 150 ° C のオーブンで、ドライ注意、熱い)。2 週間使用中はともかく乾燥キャビネット APTES コーティング下 coverslips を格納します 。
		注: 2.1.4 を 2.1.1 から各サブ手順の後洗浄プロセスを一時停止できる次のステップにします</li>。
	</ol></li> <li>アセンブル流路 <ol><li>2 つの準備 &#34; スペーサー &#34;、すなわち、パラフィルムまたは両面テープ (〜 4 × 20 mm) 各チャネルの。長い端に沿って下部 coverslip のスペーサーの 2 つの作品を配置します。間にフローセルを形成、スペーサーのトップ coverslip の場所 (~ 10 mm × 20 mm 領域、 図 2 a B).</li>
		<li>は、パラフィルム、スペーサーとして使用する場合はヒーター (60-120 ° C; に流路を配置します。注意、ホット) 2 つ coverslips をパラフィルムで一緒に固執するトップ coverslip の両側を軽く押して中に 5-10 秒のため。結果の流路の高さがあり ~ 100 μ m、それによりチャネルのボリュームが ~ 20 μ L</li>。
		<li>漏れを避けるためにシリコーン接着剤でチャネルの長い端を密封します。シリコーン接着剤を使用して、エントリとソリューションの出口として機能する流路の開放端の小さなシンクのような構造を作る 。
		注: エントリと終了も可能 など、他の方法でワックスを使用して付着した小さなプラスチック製のリングで</li>。
		<li>4 週間乾燥キャビネットにチャンネルを保存します</li>。
	</ol></li> <li>流路の底面にテザー DNA <ol><li>アミノ酸コーティングのポリスチレン ビーズを希釈 (直径: 3 μ m) 蒸留水 1 X 200 X によるリン酸緩衝生理食塩水 (PBS) バッファー。渦ビーズ ソリューションとチャンネルの流れ。孵化のためのチャネルでビーズ ソリューション 〜 30 分 1 × PBS バッファーの 200 μ L で洗浄することにより任意のバラバラのビーズを削除します</li>。
		<li>調整 (増加/減少) 50 mm × 50 mm の面積あたり 1-5 ビーズの表面密度を達成するために培養時間。3 日間参照ビーズ堆積チャンネルを格納します</li>。
		<li>Dilute sulfosuccinimidyl 4-(N maleimidomethyl) シクロヘキサン-1-カルボン酸 (スルホ SMCC) パウダー 1 × PBS ソリューション (〜 0.5 mg/mL)。渦ソリューションとチャネルに、フローします 。
		注: 使用する前に新鮮なスルホ SMCC ソリューションを準備し、SMCC の加水分解を避けるためにチャンネルにすぐにそれを追加します</li>。
		<li>加温 30 分のためのチャネルの SMCC ソリューション大量洗浄 SMCC ・ ソリューションを取り外します (1 mL 〜 チャンネル ボリューム X 50) 1 × PBS 溶液 。
		注: 余分な SMCC を丁寧に洗います。この手順は、上の DNA の結合のために非常に重要です</li>。
		<li><ol><li>DNA テザリング の希釈、チオール-ビオチンは DNA の結果 DNA 濃度の 1 X PBS に 〜 0.3 nM。ソリューションをミックス、SMCC 被覆したチャネルに DNA の解決の流れ、室温で 30 分間インキュベートして優しくピペット (~ 23 ° C).</li>
		</ol></li> <li>1x PBS 10 Mg/ml とウシ血清アルブミン (BSA) と 0.01 %2-メルカプトエタノールを含むソリューションをブロックの 200 μ L で自由な DNA を優しく洗い流す</li>。
		<li>のためのソリューション (10 mg/mL 0.01 %2-メルカプトエタノール BSA) ブロック内のチャネルの孵化によってチャネル表面ブロック &#62; 2 h; これはステップ後チャネルは実験のため準備ができています。4 ° C で準備されたチャネルを保つことができる 〜 1 日 。
		注: ブロックのステップは DNA と、coverslips に磁気ビーズの非特異的結合を減らすために重要です</li>。
	</ol></li></ol> 3。磁気ピンセット セットアップおよび単一の識別 dsDNA テザー <ol><li>磁気ピンセット セットアップ <ol><li>スタート磁気ピンセット制御プログラム。ここで、磁気ピンセットは社内記述の LabVIEW プログラムによって制御されたします</li>。
		<li>は、チャネルをマウントする前に磁石の中心を合わせます。4 X 対物レンズを使用して顕微鏡の光軸に磁石の x 軸と y 軸を調整します</li>。
		<li>はコンピューター制御の電動マニピュレーターを使用して z 方向 ( 図 2 a) 磁石を移動し、d との距離を設定 = 0 (d: coverslip の磁石との間の距離) 磁石を付けるとき顕微鏡を観察</li>。
		<li>プログラム (つまり、定数 d) 一定の力を含む力の制御を達成するためにマニピュレーターを磁石の動きと時間変動力 F (t) (すなわち、時間変化 d(t)).</li>
		<li>は明るい目的をビーズの後方散乱照明用光源は発光ダイオード (LED) を使用します。電荷結合素子 (CCD) カメラで 100 Hz のサンプリング レートでビーズ画像を収集 </l

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H., Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+G-quadruplexes+in+gene+promoters:+a+novel+anticancer+strategy?.">Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?.</a> Nat Rev Drug Discov. 10 (4), 261-275 (2011).</li><li>Amato, J., 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=Toward+the+Development+of+Specific+G-Quadruplex+Binders:+Synthesis,+Biophysical,+and+Biological+Studies+of+New+Hydrazone+Derivatives.">Toward the Development of Specific G-Quadruplex Binders: Synthesis, Biophysical, and Biological Studies of New Hydrazone Derivatives.</a> J Med Chem. 59 (12), 5706-5720 (2016).</li><li>Wells, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-B+DNA+conformations,+mutagenesis+and+disease.">Non-B DNA conformations, mutagenesis and disease.</a> Trends Biochem Sci. 32 (6), 271-278 (2007).</li></ol>

著者が明らかに何もありません。

上記、G4 DNA の力学的安定性と G4 を使用して蛋白質の相互作用を研究するためのプラットフォームとして、単一分子磁気ピンセットが報告されます。プラットフォームを伴い、G4 DNA テザーと折り畳み展開ダイナミクス計測とナノメートル特別決議と G4 の構造安定性を見つけることの非常に効率的なプロトコルが開発されています。フォーカル プレーン ロックにより非常に安定したのドリフ?...

4 本鎖 DNA や RNA の G4 の構造は、多くの重要な生物学的プロセス1に重要な役割を果たします。多くの蛋白質は、G4 のバインディングとテロメア結合タンパク (テロメラーゼ、POT1、RPA、TEBPs、TRF2) などの規制に関与している1,2, 転写因子 (ヌクレオリン化における PARP1)3、RNA プロセシング蛋白質 (hnRNP A1hnRNP A2)4ヘリカーゼ (BLM、FANCJ、RHAU、警告、Dna2、Pif1)5、DNA 複製関連蛋白質 (Rif1、REV1, PrimPolymerase)6に。蛋白質の結合を安定化したり G4 構造を不安定にします。したがって後続の生体機能を調節します。G4 の安定性は、熱溶融性 uv (紫外線) や円偏光二色性 (CD) の方法7を使用して測定しました。ただし、そのような条件は、生理学的な関連、タンパク質7のバインドの効果の研究に適用することは困難。
1 分子操作技術の急速な発展は、折りたたみとリアルタイム8ナノメートルの分解能での単一分子レベルでのタンパク質や DNA など、生体分子のアンフォールディングの研究を可能にしました。原子間力顕微鏡 (AFM)、光ピンセットや磁気ピンセットは、最も一般的に使用される単一分子操作方法です。原子間力顕微鏡、光ピンセット9と比較して、磁気ピンセットはドリフト防止法10,11を使用して、日間折り畳み展開単分子動態の安定した測定を許可します。
ここでは、結合蛋白質による G4 安定性の規制を検討する磁気ピンセットを用いた単一分子操作プラットフォームは報告された12,13です。この作品では、サンプルと流れチャネルの準備、磁気ピンセットのセットアップ力校正など、基本的な方法について説明します。コンスタント ・ フォース (力クランプ) など、さまざまな力のコントロールの下で長い時間測定を可能にする力の制御とドリフト防止プロトコルに記載されている手順 3 として、読み込み定数 (力ランプ)、速度し力ジャンプ測定。手順 4 で説明した力校正プロトコルによりの力校正 &#60; 広い力短い綱を 1 μ m の範囲まで 100 pN は、10% 以内の相対誤差と。RNA ヘリカーゼの安定性の規則の例は、RNA G4 はこのプラットフォームの13のアプリケーションをデモンストレーションするために使用の解決に重要な役割を果たしている AU 豊富な要素 (RHAU) ヘリカーゼ (別名 DHX36、G4R1) に関連付けられています。

<table><tbody><tr><td>DNA PCR primers</td><td>IDT</td><td></td><td>DNA preparations</td></tr><tr><td>DNA PCR chemicals</td><td>NEB</td><td></td><td>DNA preparations</td></tr><tr><td>restriction enzyme BstXI</td><td>NEB</td><td>R0113S</td><td>DNA preparations</td></tr><tr><td>coverslips (#1.5, 22*32 mm, and 20*20 mm)</td><td>BMH.BIOMEDIA</td><td>72204</td><td>flow channel preparation</td></tr><tr><td>Decon90</td><td>Decon Laboratories Limited</td><td></td><td>flow channel preparation</td></tr><tr><td>APTES</td><td>Sigma</td><td>440140-500ML</td><td>flow channel preparation</td></tr><tr><td>Sulfo-SMCC</td><td>ThermoFisher Scientific</td><td>22322</td><td>flow channel preparation</td></tr><tr><td>M-280, paramganetic beads,streptavidin</td><td>ThermoFisher Scientific</td><td>11205D</td><td>flow channel preparation</td></tr><tr><td>Polybead Amino Microspheres 3.00 &amp;mu;m</td><td>Polysciences, Inc</td><td>17145-5</td><td>flow channel preparation</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M6250-250ML</td><td>flow channel preparation</td></tr><tr><td>Olympus Microscopes IX71</td><td>Olympus</td><td>IX71</td><td>Magnetic tweezers setup</td></tr><tr><td>Piezo-Z Stages P-721</td><td>Physik Instrumente</td><td>P-721</td><td>Magnetic tweezers setup</td></tr><tr><td>Olympus Objective lense MPLAPON-Oil 100X</td><td>Olympus</td><td>MPLAPON-Oil 100X</td><td>Magnetic tweezers setup</td></tr><tr><td>CCD/CMOS camera</td><td>AVT</td><td>Pike F-032B</td><td>Magnetic tweezers setup</td></tr><tr><td>Translation linear stage</td><td>Physik Instrumente</td><td>MoCo DC</td><td>Magnetic tweezers setup</td></tr><tr><td>LED</td><td>Thorlabs</td><td>MCWHL</td><td>Magnetic tweezers setup</td></tr><tr><td>Cubic Magnets</td><td>Supermagnete</td><td></td><td>Magnetic tweezers setup</td></tr><tr><td>Labview</td><td>National Instruments</td><td></td><td>Magnetic tweezers setup</td></tr><tr><td>OriginPro/Matlab</td><td>OriginLab/MathWorks</td><td></td><td>Data analysis</td></tr></tbody></table>

single-molecule manipulation of g-quadruplexes by magnetic tweezers

非正規の核酸の二次構造 G quadruplexes (G4) は、DNA 複製、転写、RNA プロセシング、テロメア伸長などの多様な細胞プロセスに関与しています。これらのプロセスの中に様々 な蛋白質はバインドし、職務の遂行に G4 構造を解決します。G4 の関数は、多くの場合その折り畳まれた構造の安定性に依存、G4 結合蛋白質が G4 の安定性を調節する方法を調査することが重要です。この作業は、リアルタイムで G4 分子に G4 結合タンパク質の制御の研究を可能にする磁気ピンセットを使用して単一の G4 分子を操作するためのメソッドを示します。一般的には、このメソッドは、タンパク質・ リガンド相互作用の様々 な DNA または RNA 二次構造に規制研究アプリケーションの広い範囲に適しています。

この図の拡大版を表示するのにはここ。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56328/56328fig5.jpg"> 
G4 の展開図 5: ジャンプ力測定。 力中にビード高さ変化 (A) 代表的な時間トレース 1 pN と 54 pN 10 DmRHAU、なしで測定のサイクルをジャンプ nM DmRHAU が、ATP、なしと両方が 10 nM DmRHAU と 1 mM ATP、図パネルに記載されています。(B...

Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

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

G quadruplexes を操作する単一分子磁気ピンセット プラットフォームによって報告されます、G4 の安定性と制御の研究によって、様々 なタンパク質。

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

single-molecule-manipulation-of-g-quadruplexes-by-magnetic-tweezers

Research

JoVE Journal

Biochemistry

7.9K ビュー.  Huazhong University of Science and Technology. G quadruplexes を操作する単一分子磁気ピンセット プラットフォームによって報告されます、G4 の安定性と制御の研究によって、様々 なタンパク質。

ビデオ: Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

酵素的に活性G4 Resolvase1の精製のためのG-四重鎖DNA親和性のアプローチ

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

生化学

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

生体分子構造決定のための単分子レベルでの高精度FRET

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

磁気ピンセットを用いた多重化単分子フォースタンパク質分解測定

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

生物工学

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

ツイストとトルクの計測のための磁気ピンセット

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

光ピンセットによる単一のRNA分子のナノマニピュレーション

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

化学マッピングを用いたDNAテンプレート中のG-四重鎖の同定

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

著者スポットライト:Bis-3-クロロピペリジンベースの化学マッピングによるDNA G-四重鎖の特性評価 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

イン・ビトロBis-3-クロロピペリジンによるG-四重鎖DNA構造のケミカルマッピング

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

力に敏感な素子のナノメカニカル測定のための高速磁気ピンセット

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

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

要約

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この動画の章

酵素的に活性G4 Resolvase1の精製のためのG-四重鎖DNA親和性のアプローチ

生体分子構造決定のための単分子レベルでの高精度FRET

磁気ピンセットを用いた多重化単分子フォースタンパク質分解測定

ツイストとトルクの計測のための磁気ピンセット

光ピンセットによる単一のRNA分子のナノマニピュレーション

Magnetic Tweezers in a Microplate Format

イン・ビトロBis-3-クロロピペリジンによるG-四重鎖DNA構造のケミカルマッピング

イン・ビトロBis-3-クロロピペリジンによるG-四重鎖DNA構造のケミカルマッピング

イン・ビトロBis-3-クロロピペリジンによるG-四重鎖DNA構造のケミカルマッピング

力に敏感な素子のナノメカニカル測定のための高速磁気ピンセット