The authors thank Kerstin Glensk for preparing the MTases M.BseCI and Set7/9 and gratefully acknowledge funding by the Excellence Initiative of the German Federal and State Governments and RWTH Aachen University. The authors are happy to provide 6BAz and SeAdoYn or other cofactor analogues for collaborative research.

1.一般说明<ol><li>商店氮丙啶辅因子6BAz（在DMSO中）和蛋白质转移酶SET7 / 9在-80℃，所有其他试剂包括双活化的辅助因子SeAdoYn和DNA转移酶M.BseCI（在50％甘油）在-20℃下。 </li><li>使用消光系数ε269nm（6BAz）=16000厘米-1 M -1和ε260nm处 （SeAdoYn）=15400厘米-1 M -1，在去离子水通过紫外/可见光谱确定6BAz和SeAdoYn的浓度。通过Bradford测定法在280nm确定MTases的浓度，或者，如果消光系数是可用的， 通过直接吸收。 </li><li>尽量避免产生气泡密集移液器或涡流，以防止酶失去活性。相反，轻轻吹打上下混合。 </li><li>当从储备溶液的DMSO加入氮丙啶辅因子确保测定中的终DMSO浓度为少超过5％。总是包含在试验缓冲液，以防止非特异性反应用DNA的10mM镁离子。 </li><li>当从酸性储备溶液添加双活化的辅助因子使用小体积（高度浓缩的储备溶液），以避免pH值的变化，并确保所述测定溶液的pH值不显著变化。避免硫醇， 例如 ，β巯基乙醇或二硫苏糖醇（DTT），在测定缓冲液中，因为它们可以用通过所需要的铜离子络合点击干扰反应。 </li></ol> DNA 通过氮丙啶辅因子2.一步法标签<ol><li>序列特异性甲基转移酶诱导标签ING（笑）质粒DNA与DNA M.BseCI和转移酶的辅助因子氮丙啶的6BAz。 <ol><li>解冻，在20℃的辅因子溶液中，制备反应混合物在冰上。 </li><li>除了测定执行“辅因子”控制，以可视化的任何非特定的修改，以及一个＆＃8220;酶“的控制，以确保在转移酶制剂是不含天然辅因子的AdoMet的。 </li><li>用于测定混合物2微升10×修饰缓冲液（含有100mM的Tris-HCl，100mM的MgCl 2的，20毫β巯基乙醇，pH为7.4），2微升的pBR322（0.5微克/微升），10当量的。 M.BseCI每对DNA识别序列（在pBR322中1识别序列）和氮丙啶辅因子6BAz为60微米的最终浓度为20微升的总体积内。添加辅助因子与DNA转移酶最后。 注：β巯基乙醇是有毒的，腐蚀性，破坏环境。 </li><li>对于“辅”控制加去离子水，而不是M.BseCI并为“酶”控件添加的，而不是6BAz去离子水。 </li><li>轻轻吹打上下混合溶液。 </li><li>孵育管在55℃下1小时。 </li><li>短暂离心以收集所有的液体在管的底部。 </li></ol></li><li>限制修饰测定以验证DNA修饰。 <ol><li>通过混合10微升10×R.TaqI缓冲液（含有100mM的Tris-HCl，50mM的氯化镁 ，1M氯化钠，1毫克/毫升牛血清白蛋白，pH 8.0）中，80微升去离子水和3.3微升的制备溶液限制性内切酶（REase）从水生栖热菌 （R.TaqI，10U /微升）。确保添加了REase中的最后一步。 </li><li>从2.1.7每管加入2微升的10×R。TaqI位缓冲液和28微升溶液从上方（2.2.1）。 </li><li>轻轻吹打上下混合溶液。 </li><li>孵育管在65℃下进行30分钟。 </li><li>短暂离心以收集所有的液体在管的底部。 </li></ol></li><li>有链霉亲和电动汽车变动分析（EMSA）来验证功能修改。 <ol><li>从各管（2.2.5）中取出25微升，加入2.4微升的链霉亲和解决方案（1毫米相对于链霉米onomer在含有100mM的Na 2 HPO 4的链霉缓冲液，100 mM氯化钠，pH值7.5; 4现金等价物共计生物素）。加入2.4微升链霉缓冲到剩余管中。 </li><li>孵育所有试管在37℃下1小时。 </li></ol></li><li> 通过琼脂糖凝胶电泳分析。 <ol><li>添加5微升的6×加样缓冲液（0.25％溴酚蓝，30％甘油）到每个管中。 </li><li>轻轻的混合解决方案。 </li><li>负载10微升各样品进琼脂糖凝胶（1％琼脂糖在0.5×TBE缓冲液含有1x GelRed从10000原液）的孔中。 </li><li>运行在0.5X TBE缓冲凝胶具有80 V约。 1小时。 </li><li>可视化上的UV表（312纳米）用CCD照相机装有一个过滤器（540±50毫微米）的DNA条带。 注：UV光损害眼睛和皮肤。 </li></ol></li></ol> 通过双激活辅因子3.两步蛋白标记<ol><li> Methyltransf擦除导演的活化基团（MTAG）与SET7 / 9和双激活辅SeAdoYn转移的组蛋白H3赖氨酸4标记（改性工序）。 <ol><li>解冻的组件，并准备将反应混合物在冰上。注意：始终保持SeAdoYn冷却，以免恶化。 </li><li>除了测定执行“辅因子”控制，以可视化的任何非特定的修改，以及一个“酶”的控制，以排除荧光探针的非特异性反应。 </li><li>制备含有改性缓冲液（50mM的Tris-HCl，5％甘油，pH值8.5），10μM组蛋白H3，10μMSET7 / 9和600μMSeAdoYn（在硒两个差向异构体的混合物）的测定溶液（20微升）。在过去的步骤添加辅酶，然后转移酶。 </li><li>对于“辅”控制准备检测解决方案的3.1.3，并添加60毫米的AdoMet的合成辅酶竞争。对于“酶”控件添加的，而不是SeAdoYn去离子水。</li><li>通过慢慢地上下吹打混匀的解决方案。加入1微升每个解决方案，以pH值带（ - 10 pH范围5）的上现场检查pH值。 </li><li>孵育在37℃下2小时。 </li><li>与此同时制备一个12％的SDS聚丙烯酰胺凝胶（凝胶运行：357毫的Bis-Tris pH值6.5-6.8，0.1％（重量/体积）的APS，0.04％（体积/体积）TEMED和12％丙烯酰胺/双丙烯酰胺37.5：1 ;装载凝胶：357毫的Bis-Tris pH值6.5-6.8，0.1％（重量/体积）的APS，0.04％（体积/体积）TEMED和5％丙烯酰胺/双丙烯酰胺37.5：1）。 注：丙烯酰胺/双丙烯酰胺是有毒的，有害健康。在此过程中，应戴上手套。 </li></ol></li><li> alkinylated赖氨酸4的通过铜催化的叠氮化物-炔环加成（CuAAC）（标记步骤）化学标记中组蛋白H3。 <ol><li>只是改性反应结束之前制备含有3mM的硫酸铜 ，3毫三（3-羟基丙基三唑基甲基）胺（THPTA），250毫抗坏血酸钠和6毫TAMRA叠氮化物用5倍点击混合的20微升总体积。 </li><li>加入5微升新鲜制备的5×点击混合到每个管中以启动CuAAC和骤冷的改性反应。 </li><li>吹打向上和向下轻轻混匀。 </li><li>保护用铝箔所有管从光，以避免光致漂白的荧光团。 </li><li>孵育在20℃进行1小时。 </li></ol></li><li>蛋白质沉淀，以除去过量的游离TAMRA荧光团。 <ol><li> 34 -为了避免荧光标记的组蛋白H3通过密集的凝胶荧光自由TAMRA荧光的风头甚至盖过，由蛋白质（3.3.4 3.3.2）的沉淀去除多余的荧光。 </li><li>添加75微升甲醇，18.8微升氯仿和50微升每次加入后短暂的去离子水，以每管并涡旋。离心在16000×g离心5分钟。除去上层相，而不会干扰所述界面层，其中包含的蛋白质。 </li><li>添加56.3微升甲醇剩余期n各自管中，涡旋并离心在16000×g离心5分钟以沉淀蛋白质。去除上清。重复此步骤，洗涤沉淀。 </li><li>覆盖开放管与无尘组织，让他们干15 - 30分钟。 </li></ol></li><li> 通过 SDS-PAGE分析。 <ol><li>溶解从3.3.4所述沉淀的蛋白质在20μlSDS上样缓冲液（50mM的Tris-HCl，2.5％（重量/体积）SDS，10％（体积/体积）甘油，320毫β巯基乙醇和0.05％（重量/ ⅴ）溴酚蓝，pH6.8）中。一定要通过冲洗管的壁用吸管完全溶解沉淀。 </li><li>孵育样品在95℃下进行10分钟，并让它们向下冷却到20℃。 </li><li>短暂离心以收集所有的液体在管的底部。 </li><li>加载每个样品的总量成SDS聚丙烯酰胺凝胶（3.1.7）的孔中。使用的50mM MOPS，50毫摩尔Tris-X（Tris碱），5毫摩尔EDTA，0.1％（重量/体积）SDS作为电泳缓冲液电泳。 </li><li>运行与120 V凝胶约。 90分钟。 </li><li>可视化在凝胶上荧光的UV表（312纳米）用CCD照相机装有一个过滤器（540毫微米±50纳米）。 注：UV光损害眼睛和皮肤。 </li></ol></li></ol>

The authors disclose the following competing financial interest: E.W. is inventor on related patents.

DNA与DNA MTases和氮丙啶辅助因子（笑DNA）中的一个步骤的标签是一个强大的方法，但计划实验时，有些方面应该考虑。 氮丙啶辅：用于DNA标记的6BAz浓度M.BseCI为60微米。当使用其它DNA MTases辅因子浓度应被优化， 例如 ，浓度低至20μM的已使用用该DNA转移酶M.TaqI 19。低6BAz浓度具有四倍过量的链霉（结合位点相对于生物素在测定的总量）的能够孵育REase后直接...

核酸1,2和蛋白质3,4特定的标签是功能性表征，医学诊断和（纳米）生物技术重大利益的。在这里，我们对这些生物聚合物是基于 S -adenosyl -1-甲硫氨酸（的AdoMet或SAM）依赖性甲基转移（MTases）提出了一种酶标记方法。这类酶（EC 2.1.1。）的目标的核酸和蛋白质的特定残基中的各个位置上的亲核（氮，氧，硫和碳原子）和自然传输的辅助因子的AdoMet（ 图1A）5的活化的甲基。此外，MTases可以利用合成的辅助因子的类似物与亲和标记，荧光团或其它标签（ 图1B）6特异性标记。已经开发了两个班的AdoMet类似物：氮丙啶辅助因子局长 equence 专用的M ethyltransferase- 我 </strong&gt; EP3受体激动剂诱导L阿贝尔荷兰国际集团 （笑）7和双启动的AdoMet类似物对M ethyltransferase定向T的一个 ctivated 摹 roups转让（BOT）（MTAG）8。 <img alt="图1" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig1highres.jpg" width="700" /> 图1：反应由甲基转移（MTases）从天然辅因子的AdoMet（SAM）的各种基材，包括DNA，RNA，蛋白质和小生物分子A.甲基转移的核酸和蛋白质B.标识/官能（NNNNN = 催化。碱基对的DNA，核苷酸的RNA和氨基酸的蛋白质; XXXXX =识别与绿色目标残渣），与​​合成辅酶类似物转移酶的序列。含记者组氮丙啶辅助因子（蓝色球）附着到腺嘌呤环是序列与靶残基（左）和双 - 活化的AdoMet类似物具体地说耦合导致传送携带化学记者Y（右），它可以通过在第二步骤中生物正交点击反应进行标记扩展烷基链。 <a href="https://www.jove.com/files/ftp_upload/52014/52014fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 氮丙啶辅助因子与DNA MTases效果最好。它们含有与氮原子9（或N -mustard 10,11）代替锍中心作为反应性基团的3元环。该氮原子的质子化激活的氮丙啶环用于由靶核苷酸从而导致共价全辅因子与DNA的偶联亲核攻击。通过将报告基团到腺嘌呤环的氮丙啶的辅助因子，可以组合使用与DNA MTases以在一个步骤中标记的DNA（ <stron<html>克&gt;图1B，左图）7,12。这表现了详细的DNA与6BAz 13的生物素- 15（氮丙啶辅助因子与生物素附着于腺嘌呤环的6位）和腺嘌呤特异性DNA转移酶来自嗜热脂肪芽孢杆菌 （M.BseCI）16（ 图2，见协议第2节： 通过氮丙啶辅助因子DNA的一步标签）。除了​​M.BseCI（5&#39;-ATCG T-3&#39;识别序列）的DNA MTases从水生栖热菌 （M.TaqI，5&#39;-TCG -3“），从嗜血heamolyticus（M.HhaI，5 &#39;-GÇGC-3&#39;）和从螺原体 （M.SssI，5&#39;- C G-3&#39;）已被成功地用于生物素化的DNA与6BAz 17。此外，氮丙啶辅因子可用于一步法荧光DNA标记18,19。 ontent“FO：保持together.within页=”总是“&gt; <img alt="图2" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig2highres.jpg" width="700" /> 图2：DNA与M.BseCI和6BAz序列特异性一步法生物素化的DNA转移酶M.BseCI识别双链DNA序列5&#39;-ATCG T-3&#39;和自然甲基化的第二腺嘌呤的氨基残渣（绿色）使用的AdoMet。随着氮丙啶辅6BAz的反应过程被改变，M.BseCI导致通过耦合整个辅助因子包括生物素（蓝色）与目标腺嘌呤测序特异的DNA生物素化。 <a href="https://www.jove.com/files/ftp_upload/52014/52014fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 双活化的AdoMet类似物包含扩展的不饱和侧链而不是在锍中心甲基（ 图1B </strong&gt;的吧）20。不饱和双键或三键的β位置到锍中心电子补偿由共轭稳定化中的过渡状态不利位阻效应。因为两者的锍中心和不饱和键激活侧链酶促转移，这些辅因子被命名为双活化的AdoMet类似物。通常情况下，它们被用来在第二步骤8,21传送侧链具有独特的化学基团（化学记者），象氨基，炔和叠氮基团，化学-选择性标记。在一般情况下，双启动的AdoMet类似物不仅可以用作辅助因子DNA MTases 8,20,21也为RNA MTases 22,23和蛋白质MTases 24 - 28，允许RNA和蛋白质的额外的标签。然而，在扩展侧链立体比甲基更苛刻的和扩大的转移酶的活性位点由蛋白质工程是经常连接需要获得有效的传输速率。另一个解决这个问题的方法是使用一个的AdoMet类似物具有小炔基（3个碳原子），其中所述末端炔提供两个功能：酶促转移期间1.稳定过渡态和2反应手柄以下由铜化学修饰催化叠氮炔环加成（CuAAC）点击化学。原来，产生的丙炔的AdoMet模拟29只在有限的使用中性或弱碱性条件下，相当不稳定。这个缺点可以固定通过用硒代替硫原子。所得的辅助因子的5&#39; - [（Se）的[（3 S）-3-氨基-3-羧基丙基〕丙-2- ynylselenonio〕-5&#39;-脱氧腺苷（SeAdoYn， 图3）由野生型DNA的接受，核糖核酸和蛋白质MTases 30 - 32，其废除在许多情况下，需要进行蛋白质工程。这是通过荧光亲例举<html>蛋白标记的组蛋白H3赖氨酸4（H3K4）转移酶SET7 / 9 33（ 图3，看到协议部3： 通过双活化的辅助因子的两步蛋白标记）。 <img alt="图3" fo:content-width="7in" src="/files/ftp_upload/52014/52014fig3highres.jpg" width="700" /> 图3：序列特异性的两步荧光组蛋白H3与SET7 / 9的标签，SeAdoYn和TAMRA叠氮化物的蛋白质转移酶SET7 / 9自然甲基化使用的AdoMet的氨基中的组蛋白H3的赖氨酸4的（H3K4，绿色）。与双活化的辅助因子SeAdoYn的转移酶转移一个小炔基（红色）与赖氨酸残基。所附终端三键，然后有选择地修改在生物正交点击反应（铜 - 催化的叠氮化物 - 炔环加成，CuAAC）与叠氮化物衍生化TAMRA（四甲基，蓝）的荧光团。加载/ 52014 / 52014fig3highres.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Reagent/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">6BAz</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Weinhold et al., Patent number US 8,129,106, published March 6, 2012.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">&#946;-Mercaptoethanol</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">28625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acetic acid</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10304980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">10688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">UltraPure Agarose</td>
			<td style="width:139px;">Invitrogen</td>
			<td align="right" style="width:119px;">16500100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ammonium persulfate (APS)</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">13375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bis-Tris</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Boric acid</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bromophenol blue Na salt</td>
			<td style="width:139px;">Serva</td>
			<td align="right">15375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Copper(II) sulfate</td>
			<td style="width:139px;">Aldrich</td>
			<td style="width:119px;">C1297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Chloroform</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right">10020090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Coomassie Brilliant Blue</td>
			<td style="width:139px;">Serva</td>
			<td align="right" style="width:119px;">17525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EDTA disodium salt</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">GelRed (10000x in water)</td>
			<td style="width:139px;">Biotium</td>
			<td align="right" style="width:119px;">41003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Glycerol (99.5%)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">FastRuler Low Range DNA Ladder</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SM1103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Histone H3</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Philipp Voigt and Prof. Danny Reinberg; expression and isolation according to T. J. Richmond et al., J. Mol. Biol. 1997, 272, 301-311.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M.BseCI</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Dr. Michael Kokkinidis; expression and isolation according to Kapetaniou et al., Acta Cryst. 2006, F63, 12-14.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Methanol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">10675112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesiumchloride&#160; hexahydrate</td>
			<td style="width:139px;">J.T. Baker</td>
			<td align="right" style="width:119px;">4003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MOPS</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium chloride</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pH strip (Neutralit)</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">1,095,330,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pBR322</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">SD0041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">R.TaqI (10u/&#181;l)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>ER0671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SeAdoYn</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Synthesized according to Willnow et al., ChemBioChem 2012, 13, 1167-1173.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Set7/9</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Expressionplasmid obtained from Prof. Danny Reinberg, expression and isolation according to D. Reinberg et al., Genes Dev.2002, 16, 479-489.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Streptavidin</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">3058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">(+)-Sodium L-ascorbate</td>
			<td style="width:139px;">Sigma Life Science</td>
			<td style="width:119px;">A7631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">SDS Granular</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">di-Sodiumhydrogenphosphat</td>
			<td style="width:139px;">Merck</td>
			<td align="right" style="width:119px;">106,586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TAMRA-azide</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TaqI buffer (10x)</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td style="width:119px;">B28</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:245px;">N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td style="width:139px;">Acros Organics</td>
			<td align="right" style="width:119px;">42058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-HCl</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tris-X (TRIS-base)</td>
			<td style="width:139px;">Gerbu</td>
			<td align="right" style="width:119px;">1018</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Tris(3-hydroxypropyltriazolyl-methyl)amine (THPTA)</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">762342</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues

S -Adenosyl -1-甲硫氨酸（的AdoMet或SAM）依赖性甲基转移（转移酶）催化激活甲基来自的AdoMet中的DNA，RNA，蛋白质和小生物分子转移到特定的位置。这种自然甲基化反应可扩展到各种各样的使用合成的辅助因子的类似物的烷基化反应。更换的AdoMet的反应性锍中心与氮丙啶环的导致辅因子可加上由各种DNA MTases的DNA。这些氮丙啶辅助因子可配记者团在腺嘌呤部分的不同位置，并用于ethyltransferase- EP3受体激动剂诱导DNA（笑DNA） 的 L-阿贝尔ING S equence 专用的M。作为一个典型的例子，我们给出一个协议的pBR322的质粒DNA的生物素化在5&#39;- ATCG T-3&#39;序列与该DNA转移酶M.BseCI和氮丙啶辅因子6BAz在一个步骤。延长活化甲基与不饱和烷基结果另一类的AdoMet类似物的是用以米 ethyltransferase定向T的一个ctivatedģroups（MTAG）转让（BOT）的。由于延长侧链由锍中心和不饱和键激活时，这些辅因子被称为双活化的AdoMet类似物。这些类似物不仅用作辅因子用于DNA MTases，如氮丙啶辅因子，而且还用于RNA，蛋白质和小分子MTases。它们通常用于转移酶底物与在第二化学步骤标记报告基团独特的官能团的酶促修饰。这被例示在一个协议组蛋白H3蛋白质的荧光标记。一个小炔基是从辅因子类似物SeAdoYn由组蛋白H3赖氨酸4（H3K4）转移酶SET7 / 9随后的单击标签转移到蛋白质炔基化组蛋白H3与TAMRA叠氮化物。与辅酶类似物转移酶介导的标签是一个有利的技术，许多令人兴奋的应用，包括识别和功能研究转移酶底物，以及DNA基因分型和甲基化检测。

DNA通过氮丙啶辅因子一步法标签 本实施例中进行反应用DNA转移酶M.BseCI，该修改的双链5&#39;- ATCG T-3&#39;序列中的第二个腺嘌呤残基和具有一个识别位点上的pBR322的质粒（ 图4A）。为了测试质粒标签，pBR322中是用限制性内切酶（REase）R.TaqI（5&#39;-TCGA-3&#39;）的挑战。 R.TaqI具有七个位点上的pBR322，其中之一包括在M.BseCI站点。如果出现标签，所述M.Bs...

Watch this Scientific Journal Video about Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues at JoVE.com

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

DNA和蛋白质序列特异性标记的亲和力，或使用DNA或蛋白质的甲基和合成的辅助因子的类似物的荧光报告基团。取决于酶，氮丙啶或双活化的辅助因子类似物的辅因子特异性的用于一个或两个步骤的标签。

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

sequence-specific-labeling-nucleic-acids-proteins-with

Research

JoVE Journal

Biology

13.9K 次观看.  RWTH Aachen University.  DNA和蛋白质序列特异性标记的亲和力，或使用DNA或蛋白质的甲基和合成的辅助因子的类似物的荧光报告基团。取决于酶，氮丙啶或双活化的辅助因子类似物的辅因子特异性的用于一个或两个步骤的标签。 

视频: Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

小学组织两个胸苷类似物的免疫荧光检测（CldU IDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

科研

生物学

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...