这项工作得到了美国国家科学基金会成就奖（0953583）和健康NCI / R21-CA116102的研究所，NCI / R21-CA125088，NIBIB / R01-EB012065，NCI / R01-CA157766支持。

在这个协议中，一个寡核苷酸（RBMB1）被标记在它的5&#39;末端与CF640R报告染料和具有序列：5&#39;-mCmUmUmC mGmUmC mCmAmC mAmAmA mCmAmC mAmAmC mUmCmC亩mGmAmAmG mGmAmC mGmGmC mAmGmC mGmUmG mCmAmG mCmUmC木木-3&#39;。自身互补结构域，其驱动所述发夹结构的形成中，用粗体表示。第二个寡核苷酸（RBMB2）被标记在3&#39;端有一个爱荷华黑色RQ-SP猝灭剂和具有序列：5&#39;-mGmAmG mCmUmG mCmAmC mGmCmU mGmCmC mGmUmC-3&#39;。 1，准备RBMBs的<ol><li>执行包含RBMB1和RBMB2寡管的快速旋转，以确保在干燥的寡核苷酸是在管的底部，并重新悬浮在DNA酶和RNA酶的水至100μM的最终浓度。例如，寡核苷酸的10纳摩尔样品应重新悬浮于100微升的水。 </li><li>测量尤尔RBMB1和RBMB2股票样本的紫外 - 可见光谱的准确浓度。 <ol><li>混合3微升117微升DPBS的RBMB样品（无钙和镁）在微量离心管中。 </li><li>空白的分光光度计用DPBS和从200-800纳米测量吸光度。注：峰值吸光度在260 nm和650 nm的应该是可见的。 </li><li>计算出的库存浓度基于260nm处的吸光度（或650纳米为RBMB1）的RBMB样品，使用下式： 原料浓度（M）= A 260×稀释因子/消光系数，其中A 260是在260nm处的吸光度和稀释因子为40。 注意：消光系数为RBMB可以由寡核苷酸生产商提供的说明书手册中找到。 CF640R的在650nm处的消光系数为103,000。 </li></ol></li><li>混合20微升100μM的RBMB1，30微升100μM的RBMB2，6微升的10倍磷酸盐缓冲液（10倍的磷酸盐缓冲液：480 mM的K 2 HPO 4，45 mM的KH 2 PO 4，140 mM的的NaH 2 PO 4，pH 7.2）中，并加入4μlDNA酶和RNA酶的水。在室温下孵育该混合物30分钟。注：这是典型的足够大约50个研究。 </li><li>准备用75制备级的Superdex删除任何未杂交RBMB2寡核苷酸液相层析柱。使用8毫升（0.7×20厘米），液相层析柱用〜4毫升床体积，净化小体积的混合探针。 </li><li>洗涤并用注射器泵以流量为0.6毫升/分钟的流速运行平衡了的Superdex以〜50毫升的1X磷酸盐缓冲液中。之前所有的流体进入床体积时，停止注射泵，取下，并让剩余的液体去通过该柱由重力。 </li><li>加载RBMB混合物（60微升）到层析柱中。 <ol><li>经过RBMB样品已经完全进入该床，慢慢加入另一个250微升1X磷酸盐缓冲液加入到床的顶部，以确保整个样品已完全进入柱。 </li><li>柱填充与1X磷酸盐缓冲液的边缘。合上，然后启动注射泵 - 注射器应该充满〜50毫升的1X的PBS中，并运行在0.6毫升/分钟的流速。 </li><li>一旦RBMB接近该柱的底部 - 的RBMB样本通常可以很容易地由于可视化，以掺入探针的染料的颜色 - 通过流在2滴每离心管（1.5ml）中收集的。停止收集样品，一旦离心管内的颜色变得清晰。 </li></ol></li><li>结合含彩色RBMB样品管 - 通常是5-6管 - 并装入离心过滤装置（10,000兆瓦截止）。离心样品以10,000 RCF离心20分钟，或直到所需的体积。注意：这些速度和时间将典型地屈服〜30微升的最终体积。 </li><li>测量通过UV-Vis光谱纯化RBMB样品的准确浓度。 <ol><li>取3μL浓RBMB样品，并在微量离心管117微升的DPBS混合。 </li><li>空白的分光光度计用DPBS和从200-800纳米测量吸光度。注：峰值吸光度在260 nm和650 nm的应该是可见的。 </li><li>计算出的库存浓度基于该吸光度在650nm处的杂交RBMB的，使用等式： 股票浓度（M）= 650 *稀释倍数/灭绝系数，其中A 650是吸收在650nm处，稀释倍数为40。 注：在650nm的消光系数CF640R是103,000和消光系数为衣阿华黑RQ是〜20,000。消光系数是相加的，并且不杂交敏感。因此，股票RBMB样品具有approximatel的组合消光系数Ÿ123000在650纳米。 </li></ol></li><li>同的名称和浓度和存储适当标记的管中于-20℃以备将来使用。 </li></ol> 2，制备聚-D-赖氨酸涂层8孔腔室盖玻片<ol><li>溶解在无菌的环境在25毫升无菌水5毫克聚-D-赖氨酸（冻干粉，G-辐照）的准备0.2毫克/毫升的溶液聚-D-赖氨酸。 </li><li>加入200微升的0.2毫克/毫升的聚-D-赖氨酸溶液中，以8孔腔玻璃罩的各孔中，在无菌环境中。 </li><li>在室温下孵育16-18小时，在细胞培养罩。 </li><li>吸出聚-D-赖氨酸和用无菌蒸馏水洗井3次。注：涂布室玻片可以储存于4℃。 </li></ol> 3，探针递送注意：使用应该包含表达的RNA与至少4顺序绑定一个集成的基因构建体的细胞系统荷兰国际集团的站点中的3&#39;-非翻译区（UTR）的RBMB。靶序列应该是互补的RBMB的循环。它是表示作为阴性对照，同样的细胞系被改造成表达同一基因构建，但没有串联重复。在这个协议中，人纤维肉瘤细胞系，HT1080，被改造成表达GFP的RNA与在3&#39;-UTR的RBMB靶序列的96个串联重复。对照细胞系被改造成表达野生型GFP的RNA。 <ol><li>板块细胞的前一天探针递送T25培养瓶中，在40〜50％汇合。培养的细胞用补充有1％青霉素/链霉素和10％胎牛血清（FBS）的DMEM培养基，并在37℃，5％CO 2中。 </li><li>第二天，打开microporator并设置微穿孔参数950 V，2个脉冲，25毫秒。注意：这些参数进行了优化，对于HT1080细胞，但它们是细胞类型依赖性的，可以是adjus泰德为其它细胞类型如制造商的说明中描述。 </li><li>填补了微穿孔管4毫升电解缓冲液，并将其放置在微穿孔站。 </li><li>取出纯化RBMB的股票样本及解冻。稀几微升至12μM用1×磷酸盐缓冲液中的最终浓度。需要为每个微穿孔1微升。 </li><li>吸管1毫升培养基与胎牛血清但不含抗生素到微量离心管中。注意：这将被用于微穿孔后，立即中止细胞，并能预留，该微穿孔装置附近，暂时。在培养基中加入抗生素将微穿孔之后降低细胞活力。 </li><li>从改造的HT1080细胞（60-80％汇合），除去细胞培养基，一旦用1ml 的 Ca 2 +和Mg 2 +的 -free DPBS洗涤细胞，并孵育1毫升胰蛋白酶为1-2分钟。 </li><li>停止trypsinization可添加补充有10％胎牛血清1毫升DMEM培养基，不含抗生素和酚红，并将细胞转移到2个1.5 ml离心管中。 </li><li>降速的细胞在微量离心管中，在200×g离心5分钟。去除上清，重悬，并结合细胞沉淀在1毫升DPBS的最终体积。 </li><li>取10微升的充分混合的细胞悬浮液并计数细胞。 </li><li>吸取细胞上下数次，以确保它们都很好地分散和转移300000细胞用DPBS到一个新的1.5 ml离心管和自旋向下在200 XG 5分钟。 </li><li>弃上清，小心不要打扰细胞沉淀。重悬在11微升的再悬浮缓冲液和吸液管上下数次沉淀，以确保细胞被很好地分散。确保不产生任何气泡。注：气泡将微穿孔时引起火花，导致穷人RBMB传递和细胞死亡。 </li><li>加入1μl的稀释RBMB（12μM），并通过上下吹打几次混匀。同样要注意不要产生任何气泡。 </li><li>吸液10微升RBMB细胞混合物，采用微穿孔吸管，然后将吸管进入微穿孔管。按下启动按钮，启动微穿孔。注意：不应该有明显的气泡一角。 </li><li>当microporator的屏幕显示完成，从车站取出吸管，驱逐10微升混合物倒入先前准备的，与FBS但没有抗生素1毫升培养基的离心管中。摇动该管一侧到另一侧几次轻轻混匀。 </li><li>旋转该细胞在200×g离心5分钟，并用胎牛血清但不含抗生素洗涤细胞两次，在1ml的无酚红培养基中，以除去那些没有递送到细胞中的任何RBMBs。悬浮与FBS，但没有400μL无酚红培养基抗生素。 </li><li>板的微穿孔细胞进入聚-D-赖氨酸包被的8孔腔玻璃罩以每孔或在所需的汇合200微升。 </li><li>可选：如果希望将图像的细胞核中，赫司特33342添加到细胞中，以0.01毫克/毫升的最终浓度。 </li><li>将腔玻璃罩到细胞培养孵化器。孵育的细胞1-2小时前成像，以使细胞有足够的时间来沉淀下来的玻璃罩表面。注：成像能够尽快进行30分钟后，微穿孔。 </li></ol> 4，图像采集<ol><li>转活细胞阶段顶部孵化系统和平衡它，直到它达到37℃，5％CO 2和75％的湿度。 </li><li>打开显微镜和荧光光源和打开的Metamorph软件。注：也可用于显微镜控制和图像采集中的类似软件包。 </li><li>适用Immersol油的目标。 </li><li>用微穿孔细胞转移腔玻璃罩到活细胞的阶段顶部孵化体系。孵化阶段顶部，直到系统温度和二氧化碳含量稳定。 </li><li>打开采集卡在软件的Metamorph。点击查看实时按钮来查找字段，调整在白光下的焦点，并单击停止现场按钮。 </li><li>根据采集选项卡，单击打开流采集弹出按钮，设置所需的影片采集参数。掌握使用的Cy5 / CF640R过滤器150帧的图像保存到硬盘驱动器。 </li></ol>

<ol>
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马克博士Behlke和凌黄博士是由IDT采用它提供的寡核苷酸出售类似的一些手稿中描述的化合物。 IDT的是，然而，并不是一家上市公司，他们个人不拥有任何股份/股权IDT。

使用传统的宽视场显微镜的能力，形象单一的工程RNA转录物在活细胞中，需要与每个RNA转录和低荧光背景与未结合的荧光探针发出的相关明亮，耐光荧光信号。在该方法中，明亮的荧光信号是由杂交多个（最多96个）基于寡核苷酸的荧光探针， 即 RBMBs，在每个RNA转录物来实现的。然而，仅仅四个绑定位点是足够16。集体信号产生，可以在实时的跟踪明亮的荧光斑点。多个探针的存在...

RNA转录物的表达调控是一个复杂的动态过程，主要是负责控制细胞的行为和命运。虽然RNA的口述细胞功能的重要性已经知道了一段时间，它往往是很难得出两个因为大多数RNA分析工具缺乏捕捉重要的调节事件，如转录爆破所需的空间和时间分辨率，RNA之间明确的连接贩卖人口和本地化RNA加工。这导致了一些技术，使个体的RNA转录物，以在实时1活细胞可视化的出现。也许，最突出的，这些技术利用了GFP-MS2融合蛋白靶向已被工程化以包含在3&#39;-UTR 2,3 MS2的结合位点的串联重复序列的RNA。通过将多个绿色荧光蛋白分子紧密接近，个别RNA转录出现明亮的荧光斑点通过荧光显微术。将GFP-MS2系统提供了前所未有的洞察RNA的行为，包括直接的可视化和转录的测量耐破4,5，检测独特的亚细胞定位和处理6-9，和RNA运输10的实时成像。然而，尽管GFP-MS2系统的巨大潜力，未结合的GFP-MS2融合蛋白可以创建限制了这种技术的通用性和动态范围的高背景荧光信号。几种方法已经被开发，以限制该背景信号，包括未结合的GFP-MS2 10的亚细胞区室化，蛋白片段互补11，并选择性RNA结合蛋白和目标12。然而，所有这些方法仍与RNA靶和GFP-MS2融合蛋白的相对和总表达敏感。 作为Alternative到GFP-MS2系统，分子信标也被用于检测工程化的RNA转录物的互补结合位点的3&#39;端非编码区13,14的串联重复序列。分子信标是被标记在一端与猝灭剂和在与荧光报道的另一端发夹形成的寡核苷酸探针。当未结合于靶R​​NA的荧光报道分子和猝灭剂保持在接近，从而导致低荧光状态。杂交时，该荧光报道分子和猝灭剂被强迫分开，荧光恢复。类似于GFP-MS2系统，多个分子信标结合到一个单一的RNA转录物的结果中，可以通过荧光显微镜来识别一个明亮的荧光斑点;然而，背景荧光预期要低的多，由于未结合的分子信标在骤冷结构。不幸的是，尽管巧妙活化机制并入米olecular信标设计方案，现在有越来越多的证据表明，未杂交的分子信标不留在发夹构象时引入活细胞。因此，它们产生假阳性信号，表明显著降低了信号 - 背景。为了克服这一缺点，我们最近开发了一种新的合成探针成像的RNA在活细胞中，比率双分子信标（RBMBs; 图1A）15,16。 RBMBs组成的构成的混合结构用从两个短发夹RNA（shRNA）和分子信标功能2 2&#39;-O-甲基寡核苷酸链。循环和荧光激活机制是类似的分子信标，而长的双链与3&#39;-UU突出域名的shRNA的多个特征。 shRNA的功能旨在推动核电出口，这是我们发现增加细胞内的生命周期，以&gt; ​​24小时，以最小的可观察到的退化，防止环路的非特异性开口。其结果RBMBs呈现显著更高的信号 - 背景比的分子信标。 但是应当注意的是，RBMBs不能使用的DNA寡核苷酸制备的，由于基于DNA的探针不具有相同的核输出能力，基于RNA的探针。在结构上，RBMB环通常被设计为15和21个碱基长之间，以创建经RNA杂交的特异性和选择性之间的平衡。短杆，其形成从两个自互补结构域通常被设计为4个碱基。如果较长的茎被选择，则RBMB环和靶RNA之间的杂交的速率显著减慢17,18。相反地​​，当选择了短茎序列的熔化温度通常太低，以维持茎 - 环结构，在37℃，从而导致高背景信号。该RBMB的特异性也红了uced作为干长度被缩短。由于RBMB茎-环也可以影响RBMB性能的序列，它必须仔细选择19。特别是，理想的循环序列应该具有最小的二级结构，杂交的RNA序列以最小的二级结构，避免蛋白质结合位点，并避免结合的脱靶。既RBMB和RNA二级结构的预测可以使用软件如MFOLD 20来获得。互补脱靶部位可使用核苷酸基本局部分配搜索工具（BLAST）确定。然而，因为在模型预测和确定蛋白质伺机网站难度不一致的，所有RBMBs的特异性，最终必须通过实验验证。 如果需要的话，一个非淬火参考染料是不敏感的杂交状态可以被添加到RBMB 15。又多了一个参考染料可公关ovide的标记探针递送和用于比例成像，如果细胞总荧光的更精确的测量是必需的。参考染料允许测量要调节为由于细胞 - 细胞间的变化在传递中背景的差异。然而，成像个体的RNA时誊基准染料是没有必要的。值得注意的是，一些参考染料可以与RBMBs的离核的出口干扰，导致在细胞核中稍高的背景信号。 当设计得当，RBMBs可用于图像个体的RNA转录物在单个活细胞中，如果靶RNA被工程化以包含至少四个RBMB结合位点（ 图1B）16。 RBMBs能够高效率地递送到大范围通过微穿孔的细胞类型的，很少或几乎没有作用，对细胞活力21，和基因表达的定量测量可在30分钟内获得的。此外，该方法是相当不敏感RBMB浓度和靶RNA水平，由于来自未结合RBMBs荧光被有效地淬灭。这里我们提供了用于制备和纯化RBMBs的方法的详细描述，以及用于递送RBMBs进入活细胞通过微穿孔的一般过程和单RNA转录物中的实时成像。

<table border="0" cellpadding="0" cellspacing="0" width="977">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nuclease-free Water</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">AM9932</td>
			<td style="width:244px;">no DEPC-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DPBS</td>
			<td style="width:207px;">Life Technologies</td>
			<td>14190-144</td>
			<td>No Ca2+ and Mg2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cary 100 UV-Vis Spectrophotometer</td>
			<td style="width:207px;">Agilent Technologies</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium Phosphate Dibasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">P290-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium Phosphate Monobasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">P284-500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sodium Phosphate Monobasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">S381-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microcentrifuge tubes</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:220px;">22364111</td>
			<td>1.5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Chromatography Column</td>
			<td style="width:207px;">Kimble Chase Life Science</td>
			<td style="width:220px;">420400-0720</td>
			<td>0.7x20cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Superdex 75 Prep Grade</td>
			<td style="width:207px;">GE healthcare</td>
			<td style="width:220px;">17-1044-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Syringe Pump</td>
			<td style="width:207px;">Braintree Scientific</td>
			<td style="width:220px;">BS-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Syringe</td>
			<td style="width:207px;">BD</td>
			<td style="width:220px;">301035</td>
			<td>60mL, Luer-lok tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Centrifugal Filter Units</td>
			<td style="width:207px;">Millipore</td>
			<td style="width:220px;">UFC501096</td>
			<td>Mw 10,000 cutoff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Centrifuge 5418</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td style="width:220px;">P7280-5MG</td>
			<td>lyophilized powder, g-irradiated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">8-well chambered coverglass</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">155409</td>
			<td>Working volume 0.2-0.5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HT1080</td>
			<td style="width:207px;">ATCC</td>
			<td style="width:220px;">CCL-121</td>
			<td>Human Fibrosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cell Culture Flask</td>
			<td style="width:207px;">Corning</td>
			<td style="width:220px;">430639</td>
			<td>25cm2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">11965084</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM without Phenol Red</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">21063029</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fetal Bovine Serum</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:220px;">F2442-500ML&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Penecillin/Streptomycin</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Trypsin</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">25300054</td>
			<td>0.05% Trypsin-EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Neon transfection system</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">MPK5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neon transfection system kit&#160;</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">MPK1096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Hemacytometer</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">02-671-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoescht 33342</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">H1399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">IX-81 Inverted fluorescence microscope&#160;</td>
			<td style="width:207px;">Olympus</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">SOLA light engine</td>
			<td style="width:207px;">Lumencor</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Metamorph</td>
			<td style="width:207px;">Molecular Devices</td>
			<td style="width:220px;"></td>
			<td>Software controlling microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersol oil 518F</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">12-624-66B&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of single engineered rna transcripts in living cells using ratiometric bimolecular beacons

人们越来越认识到这两个基因表达的时空调控可能对细胞功能的重要后果，导致了不同的技术的发展，以可视个体的RNA转录物在单个活细胞。最近已经描述的一个有前途的技术利用的寡核苷酸为基础的光学探头，比率双分子信标（RBMB），以检测该被工程化以包含在3&#39;-端非翻译区的RBMB靶序列的至少四个串联重复序列的RNA转录物。 RBMBs是专门设计成发射后，杂交到互补RNA明亮的荧光信号，但在其他方面仍骤冷。在这种方法中，使用合成探针允许耐光的，红移，并且将用于成像高度发光有机染料。多RBMBs的宽视场荧光显微镜下观察时，结合改造的RNA转录产生离散的荧光斑点。因此，个体的RNA转录物的运动可以容易地通过取一个时间序列的荧光图像的可视化中的实时性。在这里，我们描述RBMBs的制备和纯化，递送到细胞微穿孔和活细胞的单个RNA转录物成像。

不久下列HT1080细胞中RBMBs的存在下的微穿孔，被改造的那个人的RNA转录物，以含有多个RBMB结合位点在其3&#39;-UTR时通过宽视场荧光显微镜（ 图2）拍摄显示为明亮的荧光斑点。而个体的RNA转录物与少至4 RBMB结合位点可以在活细胞进行成像，越RBMB结合在3&#39;-UTR部位，越强的荧光信号。而且，越是RBMBs绑定到每个成绩单的时间越长的RNA可以可视化前的信号丢失，由于漂白。在这个协议中，R...

Watch this Scientific Journal Video about Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons at JoVE.com

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

比例式双分子信标（RBMBs）可用于将图像的单个设计RNA转录活细胞中。在这里，我们描述RBMBs，交货RBMBs到细胞通过微穿孔和的单个RNA转录物中的实时荧光成像的制备和纯化。

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

real-time-imaging-single-engineered-rna-transcripts-living-cells

Research

JoVE Journal

Biology

11.7K 次观看.  University of Pennsylvania. 比例式双分子信标（RBMBs）可用于将图像的单个设计RNA转录活细胞中。在这里，我们描述RBMBs，交货RBMBs到细胞通过微穿孔和的单个RNA转录物中的实时荧光成像的制备和纯化。 

视频: Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

科研

生物学

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

单分子荧光原位杂交实验中单个大肠埃希菌细胞的转录标记方法

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...