我们感谢托尼 Kulesa, 罗宾帕特里克和塞帕斯 Gatzogiannis 的协助与初步仪器设置和测试。我们进一步感谢托尼 Kulesa 的重要帮助编写和评估定制的单一粒子跟踪软件。这项工作得到了启动资金和职业奖的支持, 在科学接口 (PCB) 从宝来惠康基金会。

1. 生物素和 #955 的制备. dna 注意: 生物素-#955;-dna 分子通过结扎生物素标记的寡核苷酸, 5 和 #39;-AGGTCGCCGCCC (a) 20 -biotin-3 和 #39; 到和 #160;和 #955;-脱氧核糖核酸分子 20 . 
<ol> <li> 在 TE 缓冲区中准备 0.1 mM 生物素标记为寡聚. </li>
	<li> 热0.5 毫克/毫升和 #955;-65 和 #176 的 DNA 库存; C 六十年代, 并立刻陷入湿冰. 
	注: 快速淬火冷却降低和 #955;-DNA concatemerization. </li>
	<li> 吸管100和 #181; L #955;-DNA 溶液成离心管. </li>
	<li> 添加1和 #181; 0.1 mM 的 l 为9和 #181; l 缓冲. </li>
	<li> 添加2和 #181; 在含有 #955 的离心管的低聚体溶液中;完全混合. 
	注意: 寡聚存在于大约12倍的摩尔超过互补和 #955;-DNA 末端. </li>
	<li> 热 #955;-65 和 #176 的 DNA/寡糖混合物, 六十年代 C. </li>
	<li> 将混合物慢慢冷却到室温. 
	注意: 这一步允许寡聚退火到互补和 #955;-DNA 末端. </li>
	<li> 将混合物放在冰上。添加11和 #181; L T4 DNA 连接反应缓冲器 (最终缓冲浓度 1x)。轻轻地混合。然后添加2和 #181; L (800 单位) 的 T4 DNA 连接酶。轻轻地混合. </li>
	<li> 在16和 #176 中孵育2小时的混合物; c 或隔夜在4和 #176; c. </li>
	<li> 使用 100 kDa 的公称分子量限制的离心过滤管净化产品。具体地说, 将步骤1.9 混合物转换为离心式滤筒, 在 1.4万 x g 处离心5分钟, 用400和 #181 清洗; 两次缓冲, 收集纯化产品. 
	注: 作为纯化的生物素-#955; 在 TE 缓冲器中的 dna 在-20 和 #176 是稳定的; C 为一年. </li>
</ol> 2。片功能化 注意: 玻璃片通过与硅烷-PEG-生物素的反应功能。在我们的经验中, 这种 one-step 反应协议比标准的 two-step 反应协议 20 更简单、更可靠。片功能化与硅烷 PEG-生物素创建结合点的亲和, 并最大限度地减少非特异性 DNA 和蛋白质对片表面的吸附. 
<ol> <li> 几种五1片在95% 乙醇中的一个染色罐内为10分钟, 然后用超纯水冲洗三次. 
	注: 第五片提供备用以防破损. </li>
	<li> 用1米的 KOH 填充着色罐, 几种10分钟, 然后用超纯水冲洗三次. </li>
	<li> 重复 2.1-2.2 循环两次. </li>
	<li> 干燥片在干净的干燥 N 之下 2 气体流动. </li>
	<li> 通过在 900 mTorr 压力下使用空气等离子处理, 进一步清洁和干燥片5分钟. </li>
	<li> 孵育片与25毫克/毫升硅烷 peg-生物素溶于95% 乙醇在室温下为 2 h. 特别地, 三明治50和 #181; 在两个清洁干燥片之间, 并孵化片和 #34; 三明治和 #34;在一个封闭的吸管提示盒内, 底部有大约10毫升水 (不与片接触), 以减少溶剂蒸发. </li>
	<li> 清洗多余的 peg 分子与超纯水和干燥的 peg 涂层片下清洁干 N 2 气体流量. 
	注意: 功能化片可在环境条件下存储三天. </li>
</ol> 3。流单元结构 注意: 流单元是通过夹 double-adhesive 带预通道在 PEG 官能化片和含有入口和出口孔的聚能板之间建立的。每个流单元集成多个通道. 
<ol> <li> 设计 CAD 软件中的流通道, 并通过使用磁带切割器 (通常为每个流单元集成八通道) 在 double-adhesive 磁带上切割通道 (每个通道的宽度、深度和长度分别为1、0.13 和 12 mm)。移除磁带残余以形成流通道. </li>
	<li> 使其与5克的交联试剂完全混合45克的硅橡胶板 (足以使十二5毫米厚的 PMDS 板可以无限期地储存在环境条件下), 将混合物倒入两个 10 cm 的培养皿中, 然后离开在真空室里的盘子, 直到基本上所有的气泡都消失了 (当盘子从真空室中取出时, 如果有任何残留, 手动清除气泡)。然后把盘子转到 80 #176; C 烤箱, 直到硅氧烷固化 (约2小时). </li>
	<li> 切割出一个与预通道的 double-adhesive 磁带大小相匹配的硅橡胶板。剥去 double-adhesive 胶带上的一层保护膜, 并将薄膜粘附在一个平板表面上。使用23针的活检孔冲孔器, 在硅橡胶板上打出出口和入口孔. </li>
	<li> 将第二保护膜剥离在 double-adhesive 胶带上, 并将其粘贴到片的功能化表面 (确保片是平整的。在 图 1a 中查看完全组装的流单元的图片. </li>
</ol> 4。TIRF 成像 注意: 生物素-和 #955;-dna 被拴在流道表面, 层流被应用到流动伸展的脱氧核糖核酸分子 ( 图 1b )。#62; 80% 伸展的 DNA 分子用作观察荧光标记分子的结合和运输活动的空间扩展模板。通过延时 TIRF 成像 ( 图 1c 和 #38; e ) 跟踪单个分子的轨迹. 
<ol> <li> 在显微镜阶段固定流单元并加载 pre-degassed 空白缓冲区 (10 毫米磷酸钠, 2 毫米氯化钠, 50 和 #181; 米 EDTA, 20 毫米乙醇, 5% (v/五) 甘油, 0.01% Tween-20, 1% (v/v) 和 #946;-基, pH 7.4), 0.2 毫克/毫升亲和溶液, 1 毫克/牛血清白蛋白 (BSA) 溶液和 100 pM 生物素-#955;-DNA 溶液到四个储层中, 每个都有一个 Tygon 油管连接到一个螺线管值和另一个 Tygon 油管连接到 peek 管通过套管 Tygon 管在更小的 peek 管材 (防止试剂回流。通过隔夜接触真空或较短时间接触真空, 以搅拌 (直到气泡不再可见) 的小体积的缓冲. </li>
	<li> 将空白缓冲液库的 PEEK 管插入到流动单元的一个入口孔中。通过在空白缓冲器中流动, 将三其他 PEEK 管插入进进孔中, 将每个试剂的气压驱动流量由电磁阀控制, 并通过计算机脚本完全自动化。注意不要在通道内诱发气泡形成. </li>
	<li> 将 Tygon 油管与从出水孔到废料容器的短 PEEK 管连接起来。在出口的短 PEEK 油管有助于抑制空气气泡形成在输液过程中保持积极的压力在通道内. </li>
	<li> 将生物素和 #955;-DNA 分子到流道表面. 
	<ol> <li> 在0.2 毫克/毫升亲和溶液中流动, 孵育5分钟, 然后在空白缓冲区中流动, 以冲洗未绑定的亲和. </li>
		<li> 在1毫克/毫升 bsa 溶液中流动, 孵育1分钟, 然后在空白缓冲区中流动以冲洗未绑定的 bsa. 
		注意: BSA 进一步抑制 DNA 和样品对表面的吸附. </li>
		<li> 流在 100 pM 生物素-#955;-DNA 溶液, 孵育10分钟, 然后在空白缓冲液中流动, 洗涤未绑定的 dna. 
		注: 在 dna 与表面结合后, 避免快速流动, 以防止对栓 dna 分子的损伤. </li>
	</ol>
	</li><li> 检查功能化片的质量, 在要使用的检测条件下 (如下所示) 在 DNA 染色染料 (如 Sytox 橙) 中流动, 并在 532 nm 激光照射下启动荧光成像. 
	注意: 功能化片的质量通常很好, 流动的 DNA 分子的密度也会很高。重要的是要有足够高的流动密度的 dna 分子, 使 dna 结合和 sliding/1D 扩散事件可以观察到. </li>
	<li> 微调 TIRF 角度, 以获得最佳的信号噪声比图像的延伸 DNA 分子 ( 图 1d ). </li>
	<li> 记录单分子沿着 dna 移动的轨迹, 在一个新通道内重复步骤 4.2-4.4 (没有 dna 染色染料), 然后用注射器泵将 pre-degassed sub-nanomolar 荧光标记的分子注入足够高的流速收集帧速率为 100 Hz 的延时荧光图像. 
	注: 流速相当于應数 (Wi: 最长聚合物弛豫 time-approximately 0.2 s 的无量纲乘积, #955;-DNA-和剪切速率-从片表面的距离的流速变化) 的500内通道是建议的单分子成像的帧速率为 100 Hz 21 。
	<ol> <li> 在一个字段-视图 (视) 中收集1万帧, 以生成影片, 并在每个示例中从多个 (通常为十) FOVs 收集相似的影片. 
		注: 单粒子在一个视的密度不应该太 high-which 将复杂的数据分析, 使对象分配跨帧不明确, 也不太 low-which 可能导致低发生单分子事件的 DNA. </li>
		<li> 优化光照强度, 使与片表面绑定的分子可以快速地进行照片漂白, 以抑制背景, 而在 DNA 上扩散的分子不会太快地漂白, 以便能够检测到更长的轨迹. 
		注意: 我们通常在视中使用 200-800 W/厘米 2 的功率密度. </li>
	</ol> </li> <li> 在步骤4.6 的末尾, 注入 dna 染色染料以确认 dna 分子仍然可以流动. </li>
	<li> 同时运行正和负控制示例. 
	注: 良好的正向控制是 tetrapeptide, 甲基 (德涅斯特河沿岸摩尔多瓦共和国)-KRRR (德涅斯特河沿岸摩尔多瓦共和国耦合到 n-端胺), 其中有一个平均1D 扩散常数10.5 和 #177; 0.7 (标准错误) M (bp 2 /秒) 在中性 pH 值 11 。一个好的负控制是测量在没有 dna 栓的通道中检测缓冲的感兴趣的标记样本, 在那里没有 dna 扩散活动. </li>
</ol> 5。数据分析: 注意: 和 #160; 一个自定义的单粒子跟踪软件用于识别沿 DNA 扩散的单个分子的轨迹, 并估计传播常数。该软件首先确定了高精度的单粒子的质心位置, 确定了粘在片表面上的粒子, 然后将粒子链接到不同的帧, 形成了延时轨迹。从这些轨迹, 估计1D 扩散常数. 
<ol> <li> 若要启动数据分析, 请打开一个脚本软件 (请参阅材料表), 然后转到单个粒子跟踪软件的目录。打开名为 #34 的脚本; largedataprocess3 #34;。要定义的一个重要参数是单粒子检测的阈值. </li>
	<li> 若要确定最佳阈值, 请运行 #34;D etermine_threshold_value. m 和 #34; 脚本。此脚本将指示阈值如何影响单个粒子检测。在确定最佳阈值后, 运行 #34; largedataprocess3 #34; 脚本. </li>
	<li> 在完成单粒子跟踪分析后, 通过运行和 #34 来过滤原始轨迹; Trajectory_filtering #34; 脚本。内置图形用户界面 (GUI) 以可视化筛选步骤. </li>
	<li> 在 GUI 面板上, 将最小位移沿 dna, MinXDisp, 设置为2像素; 将最大位移横向设置为 dna、MaxYDisp、2像素; 设置最小的帧数, minFrames, 到 10; 设置最小的三连音, minTriplets 的状态数,10;设置最小扩散常数沿脱氧核糖核酸, D_par, 到 0;设置最大估计扩散常数横向到脱氧核糖核酸, D_trans, 对 10 M (bp 2 ) S -1 ;设置最小统计参数, Chi 2 _stat, 到-5;将沿 dna 的最小位移比率设置为横向到 dna, 2. 
	注意: 通过这些过滤参数的所有原始轨迹都列在表1中, 而那些不在表3中列出. </li>
	<li> 单击表1中的轨迹号, 轨迹将在图形1和 #38 中偏移; 2。点击 #34;P 放置和 #34; 按钮播放原始荧光图像。单击 #34; Add_to_Table2 和 #34; 将轨迹识别为单分子滑动轨迹。最后, 在关闭 GUI 时, 添加到 Table2 的所有轨迹都将被保存. </li>
	<li> 计算平均扩散常数, 运行脚本和 #34; Calculate_mean_diffusion_constant #34;。平均扩散常数是从已通过所有筛选步骤的轨迹集估计的, 并已添加到表2中. </li>
</ol>

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Expt. , e1689 (2010).</li><li>Parthasarathy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+accurate+particle+tracking+by+calculation+of+radial+symmetry+centers.">Rapid, accurate particle tracking by calculation of radial symmetry centers.</a> Nat Methods. 9, 724 (2012).</li><li>Vestergaard, C. L., Blainey, P. C., Flyvbjerg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+estimation+of+diffusion+coefficients+from+single-particle+trajectories.">Optimal estimation of diffusion coefficients from single-particle trajectories.</a> Physical review. E, Statistical, nonlinear, and soft matter physics. 89, 022726 (2014).</li><li>Schroeder, C. M., Blainey, P. C., Kim, S., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydrodynamic Flow-stretching Assay for Single-Molecule Studies of Nucleic Acid-Protein Interactions. , (2008).</li><li>Blainey, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Molecule Studies of Protein-DNA Interaction: Diffusive Search and Sequence-Dependent Motors. , (2007).</li><li>Gorman, J., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+one-dimensional+diffusion+of+proteins+along+DNA.">Visualizing one-dimensional diffusion of proteins along DNA.</a> Nat Struct Mol Biol. 15, 768-774 (2008).</li><li>Lin, Y. H., 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=Using+the+Bias+from+Flow+to+Elucidate+Single+DNA+Repair+Protein+Sliding+and+Interactions+with+DNA.">Using the Bias from Flow to Elucidate Single DNA Repair Protein Sliding and Interactions with DNA.</a> Biophysical journal. 96, 1911-1917 (2009).</li><li>Blainey, P. C., 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=Nonspecifically+bound+proteins+spin+while+diffusing+along+DNA.">Nonspecifically bound proteins spin while diffusing along DNA.</a> Nat Struct Mol Biol. 16, 1224-1234 (2009).</li><li>Cuculis, L., Abil, Z., Zhao, H., Schroeder, C. 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宽广的学院可能选择递交专利申请, 包括在这里介绍的工作的方面。

从传统的 two-step 片功能化反应协议切换到 one-step 反应协议时, 我们注意到片功能性的可靠性得到了显著的提高。由于片准备的总动手时间少于三十分钟, 我们建议每天准备新的片单分子成像计划。为了减少硅烷-PEG-生物素的变质, 试剂应在收到时在-20 ° c aliquoted 和储存在干 N2气体下。在这里, 我们不使用产生-首席运营官-或 CH3终端组的 PEG 分子在过去的20中使用。使用的负电荷-组和疏水 CH3组分别用于防止 DNA 分子和特定蛋白质样品在表面上的非特异性吸附。我们观察到在完全生物素终止的 peg 表面上的小肽样品对表面的吸附非常低, 而硅烷 peg-羧基和/或硅烷 peg-CH3可能对某些蛋白质样品的化验或化验条件 (如 pH 值和 #60) 有用。促进 DNA 表面的相互作用。
从玻璃滑动流细胞到硅橡胶流细胞的转变加速了流动细胞的构建, 并使多个通道易于集成。我们经常为每个流动单元设计八通道, 使每一个功能化的片有八独立的实验。为了进一步提高每片的吞吐量, 甚至可以使用更大数量的微制造通道。
建议在初始充填后, 在任何一点上防止气泡在通道内形成。通常情况下, 气泡不会导致 dna 的丢失 (虽然这是可能的), 但会扰乱流动, 并可能导致 dna 黏附在片。缓冲除气和小表面活性剂数量的增加通常是有效的防止气泡形成在油管和流道。在将气泡引入油管的情况下, 试着在缓慢的气流中冲洗气泡, 并确保它们不会流经 dna 被栓的区域。
由于生成的数据量巨大, 因此简化和高效的数据分析软件至关重要。我们通常收集70万高分辨率的图像, 从测量的一个流电池, 可以处理使用我们的定制单一粒子跟踪软件在一天内的台式计算机的四核处理器和 32 Gb 内存。通过软件 (步骤 5.5) 检测1D 扩散轨迹的假阳性率是样本依赖性的, 并且可以相当低, 因为: 1) 视中的单个粒子的密度足够低, 因此在帧之间连接粒子的模糊性很小 (蛋白质样本一般更容易对片比肽样品的非特异性吸附, 因此, 每个蛋白质样品的缓冲和功率密度需要优化);2、单粒子的信噪比高, 使粒子的误识别概率低。尽管如此, 软件还是会偶尔出错, 我们建议使用所提供的 GUI 对数据进行手动检查, 以评估软件的性能。在对假阳性轨迹检测特别敏感的应用程序中, 可以更严格地设置包括阈值、minFrames、minTriplets 和 Chi2_stat 的过程参数, 以降低对真实绑定的敏感度轨迹识别中的事件和潜在的更大的统计偏差。我们在这里分享和描述我们的单粒子跟踪软件, 希望能帮助标准化数据分析方法, 并激发关于最佳实践的讨论。
在我们的协议中, 需要在成像过程中连续流动以保持 dna 模板分子的拉伸状态, 流体流动可能会使某些蛋白质沿 dna 模板传播, 如果它们通过跳跃机构扩散, 或者如果水动力标记的荧光的半径为大22,23。虽然这种偏倚可以在大多数数据集中进行测量和修正, 但以小荧光标记的蛋白质的传输和滑动机构的扩散通常不会被2,4的流量所检测到. 24。值得注意的是, 流速对沿 dna 传递蛋白质的影响, 已被用来研究蛋白质沿 dna 扩散的机制23。一个紧密相关的替代方法, 使测量在缺乏流动利用 dna 模板与生物素在两个总站, 瞬时伸展在流动和拴在两端的片, 使 dna 分子保持在当流关闭时的扩展配置25,26,27,28。double-tether 方法具有允许无流传输实验的优点, 但需要对 DNA 模板分子的两端进行修改, 在束缚过程中进行细致的流量控制, 并对两个栓点之间的距离进行表征。此外, 必须评估非均匀张力和 dna 构象动力学在 dna 分子上对分子测定的影响。
总而言之, 除了研究 dna 中分子的1D 扩散外, 作为报告的单分子检测可以使许多在试管中在单分子水平上的生物化学和生物物理研究受益, 包括 dna 目标搜索过程蛋白质, 酶在 DNA 上的活动, 等等。
故障
问题 1: 流道泄漏。
解决方案: 首先, 确保设计的流量通道允许至少1.5 毫米的边缘, 以密封周围和渠道之间;然后, 在流动单元的装配过程中, 确保片、double-adhesive 胶带和板坯之间的接触面是平坦的、干燥的和无碎屑的, 用于形成密封的压力是均匀的, 密封操作是在室温下执行。
问题 2: 栓系 dna 的密度过低。
解决方法: 当 PEG 功能化效率较低或 DNA 分子不能与生物素结合时, 就会出现此问题。从我们的经验来看, 栓 DNA 的密度应该高 #62; 90% 的片是由新鲜的或适当储存的硅烷-PEG-生物素试剂制成的。当系栓 dna 密度较低时, 经证实的生物素-λ-dna, 尝试在 peg 功能化过程中提高硅烷-peg-生物素的浓度, 在 dna 的亲和和生物素-λ-dna 的浓度。如果这失败, 更换 PEG 和亲和试剂。
问题 3: dna 粘片d 不能被水流拉长。
解决方法: 当 PEG 的功能化效率较低时, 也会出现此问题, 并因低 pH 值而加剧。尝试流动更多的 BSA 或提高 pH 值 (如 8.0), 以帮助抑制 DNA 对表面的吸附 (步骤 4.3.2)。如果 DNA 吸附在特定的测定条件下是一个持久的问题, 尝试在 peg 功能化过程中包括多达90% 的硅烷 peg 羧基。
数据分析说明:
我们使用定制的单粒子跟踪软件来识别沿 DNA 扩散的单分子的轨迹, 它们的1D 扩散常数, D1 估计。为了改进数据分析, 我们实现了径向对称算法18用于定位粒子和 covariance-based 估计器19 , 用于估计1D 扩散常数, 这极大地加快了数据分析, 而不会损害精度.我们以前通过分析模拟数据11来验证自定义软件。主要步骤如下所述。
粒子标识:
这一步是检测和分割粒子-衍射限制点-从背景。首先, 空间带通滤波器的图像, 以提高信号的粒子在3-5 像素范围 (对应于我们的系统上的点传播功能的大小), 然后阈值的基础上的强度 (见脚本: spt2_SD_sandbox)。
质心分配
将图像的本地化分子分割成像素级的空间分辨率。要将分子定位到更高的精确度, 请使用径向对称超分辨率算法18对图像进行筛选。(此方法基于分析计算, 从而大大降低了计算负担, 与其他流行的高精度方法 (如2D 高斯拟合) 相比 (参见脚本: spt2_SD_sandbox)。
粒子跟踪:
要通过时间跟踪粒子的轨迹, 我们必须重新相同的粒子在帧之间移动。我们认为启动、闪烁和终止阶段是轨迹的组成部分。我们将当前帧中的粒子与前一帧中的颗粒之间的连接限制为每个帧所允许的最大位移。在可能存在多个联系的情况下, 使用琼克-Volgenant 算法来生成全局最优链接集 (参见脚本: traceTraj4_kx. 3. m)。
D1 估计:
为了从轨迹中估计 D1 值, 我们使用基于协方差的估计器 (CVE)19。在分析计算的基础上, 这种方法比其他常用的方法, 如平均平方位移回归 (参见脚本: runspt5_SD), 具有更高的运算效率和统计严谨性。
弹道滤波:
有些轨迹包含工件, 如与表面结合分子的连结, 必须移除。我们实现了几个特点, 如最小的位移平行于 dna, 最大位移横向到 dna, 最小的弹道长度, 最小的三重连续检测到的位置, 和最低概率得分 (见雄等人 al11为定义), 可以过滤, 以隔离一组最小的偏差的轨迹可能代表分子扩散的 DNA (见脚本: sptViewFig2_update3x. m)。

生物学中的一个长期存在的问题是, 在基因组中的特定部位的内源性蛋白质如何能够迅速地找到他们的 DNA 目标, 从而使生物体生存并对其环境做出有效的反应。过去四十年的研究提出并大体上支持一个假设, 即蛋白质的 DNA 靶向搜索的动力学可以通过促进扩散来加速, 其中蛋白质在体积和1D 扩散的3D 扩散之间交替 (包括滑动和跳跃过程) 沿 DNA1。现在已知许多涉及基因调控、核酸代谢和其他过程的蛋白质都能在 DNA 上滑动2,3,4,5,6,7 ,8,9。此外, 最近的研究报告说, 即使是小肽也能与携带货物的能力结合在一起并滑动到 DNA 上;例如, 蛋白质分子或 PCR 引物, 沿 DNA10,11,12,13,14。
在过去的15年中, 单分子流拉伸试验已被广泛用于研究分子的结合和扩散的 DNA2,15,16。在这种类型的检测中, 化双 dna 分子被固定在表面上, 层流被应用于 dna 的流动。#62; 80% 伸展的 dna 充当空间扩展的模板, 用于研究标记有荧光的分子的结合和运输活动, 其中单分子沿 DNA 的轨迹通过延时荧光成像来跟踪。在我们的实施, 优化的重现性和易用性, 该试验包括五主要步骤: 生物素-λ-DNA 的制备, 片功能化, 流细胞结构, 荧光成像和数据分析。在以前的协议17中, 玻璃片功能化的第一反应与 (3-氨基丙基) 烷 (APTES), 然后与胺反应聚乙二醇 (peg) 试剂 (如, NHS-peg-生物素), 形成一个 PEG 层, 抗拒对片表面的测定组分进行非特异性吸附。官能化片的质量主要取决于 PEG 试剂的质量和各步骤的反应条件。我们的协议描述了一个简化的功能化协议和多路流细胞结构, 不需要在当天的流动细胞上组装液体粘合剂或固化时间。我们还描述了一个简化和健壮的数据分析过程11 , 通过应用径向对称方法来消除计算密集型的回归步骤, 用于质心本地化18和 covariance-based 扩散常量估计器19。
在这里, 我们报告一个简单, 稳健和高吞吐量单分子流拉伸试验的实现, 并在片功能化, 流细胞结构和数据分析作出重大改进。特别是, 我们开发了 one-step 片功能化协议, 其中清洁干片直接反应与硅烷 PEG-生物素。该协议简化了片的制备, 提高了功能化表面质量与标准 two-step 反应协议的可靠性。我们描述了使用的片与多通道的多路硅橡胶流细胞, 使可靠的油管连接, 不粘胶。这些流动细胞进一步包括多个计算机控制的入口为每个流室, 使自动化的试剂流, 以减少动手时间在设置和增加化验吞吐量。

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:249px;">Biotin labeled oligo, 5&#8217;-AGGTCGCCGCCC(A)20-biotin-3&#8217;</td>
			<td style="width:249px;">IDT</td>
			<td style="width:159px;">Custom oligo synthesis</td>
			<td style="width:249px;">Standard desalting purification is sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">&#955;-DNA</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">N3011S</td>
			<td style="width:249px;">Make aliquots and minimize freeze-thaw cycle number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">T4 DNA ligase and ligation reaction buffer</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">M0202S</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Amicon centrifugal filter tubes with NMWL of 100k Dalton</td>
			<td style="width:249px;">EMD Millipore</td>
			<td style="width:159px;">UFC510008</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">No. 1 glass coverslips</td>
			<td style="width:249px;">VWR</td>
			<td style="width:159px;">48393-106</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Pure ethanol</td>
			<td style="width:249px;">VWR</td>
			<td style="width:159px;">89125-186</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Potassium hydroxide pellets</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:159px;">P250-500</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Plasma cleaner, model No. PDC-32G</td>
			<td style="width:249px;">Harrick Plasma</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:249px;">Silane-peg-biotin</td>
			<td style="width:249px;">Nanocs</td>
			<td style="width:159px;">PG2-BNSL-5k</td>
			<td style="width:249px;">Store at -20oC in dry nitrogen gas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">A CAD software</td>
			<td style="width:249px;">Autodesk</td>
			<td style="width:159px;">AutoCAD</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Double-adhesive tape</td>
			<td style="width:249px;">Adhesive Applications</td>
			<td style="width:159px;">8512-2-DP/DL</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Tape cutter, model No. CE5000-40-CRP</td>
			<td style="width:249px;">Graphtec America</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">PDMS and crosslink reagent kit</td>
			<td style="width:249px;">R.S. Hughes</td>
			<td style="width:159px;">RTV615 CLEAR 010</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Degassing chamber, model No. F42010-0000</td>
			<td style="width:249px;">BEL-ART Products</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Mixer, model No. ARV-310</td>
			<td style="width:249px;">Thinky</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:249px;">Hole puncher, Harris Uni-Cores, Size 0.5 mm</td>
			<td style="width:249px;">Harris Uni-Cores</td>
			<td style="width:159px;"></td>
			<td style="width:249px;">This can be substituted with a manual biopsy punch with a similar needle size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Tygon tubing</td>
			<td style="width:249px;">Cole Parmer</td>
			<td style="width:159px;">EW-06420-02</td>
			<td style="width:249px;">0.020&#34; ID, 0.060&#34;OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Peek tubing</td>
			<td style="width:249px;">Western Analytical</td>
			<td style="width:159px;">PK007-031-Y</td>
			<td style="width:249px;">0.007&#34; ID, 0.031&#34;OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Streptavidin</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">N7021S</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Bovine serum albumin (BSA)</td>
			<td style="width:249px;">New England Biolabs Inc.</td>
			<td style="width:159px;">B9000S</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Sytox orange nucleic acid stain</td>
			<td style="width:249px;">Invitrogen</td>
			<td style="width:159px;">S11368</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Ti-E Microscope</td>
			<td style="width:249px;">Nikon</td>
			<td style="width:159px;"></td>
			<td rowspan="5" style="width:249px;">Microscope, camera, objective, dichroic mirror and bandpass filter can be substituted for similar equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">100x Objective, NA/1.49, CFI Apo TIRF</td>
			<td style="width:249px;">Nikon</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">CMOS camera</td>
			<td style="width:249px;">Hammamatsu</td>
			<td style="width:159px;">Orca Flash 4.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Dichroic mirror</td>
			<td style="width:249px;">Semrock</td>
			<td style="width:159px;">FF560/659-Di01-25x36</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Bandpass filter</td>
			<td style="width:249px;">Semrock</td>
			<td style="width:159px;">FF560/659-Di01-25x36</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">532 nm laser</td>
			<td style="width:249px;">Rayscience Innovation Limited, Shanghai</td>
			<td style="width:159px;">OEMLL-FN-532nm-C</td>
			<td style="width:249px;">Select wavelength to correspond to stain of choice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Syringe pump</td>
			<td style="width:249px;">Chemxy</td>
			<td style="width:159px;">Fusion 200</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">TMR-KRRR, &#62;=85% purity</td>
			<td style="width:249px;">MIT Biopolymer lab</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">pVIc-Cy3B, HPLC purified</td>
			<td style="width:249px;">A gift from Dr. Walter Mangel at Brookhaven National lab</td>
			<td style="width:159px;"></td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Script programming software</td>
			<td style="width:249px;">MathWorks</td>
			<td style="width:159px;">Matlab</td>
			<td style="width:249px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:249px;">* see parts list for solenoid valves, computer interface, and assembly instructions at: https://sites.google.com/site/rafaelsmicrofluidicspage/valve-controllers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple, robust, and high throughput single molecule flow stretching assay implementation for studying transport of molecules along dna

我们描述了一个简单的, 健壮的和高通量的单分子流拉伸试验, 研究分子沿 DNA 的1D 扩散。在这种测定中, 玻璃片是功能化的 one-step 反应与硅烷 PEG-生物素。流动细胞是由夹在功能化的片和一个含有入口和出口孔的硅橡胶板之间的预通道上的粘胶带构造的。多个通道被集成到一个流动单元中, 并且每个通道的试剂流可以完全自动化, 这大大提高了化验的吞吐量, 并减少了每次化验的动手时间。在每个通道内, 生物素-λ-dna 在表面上是固定的, 层流被应用于 dna 的流动延伸。DNA 分子被拉伸至 #62; 80% 的轮廓长度, 并作为空间扩展模板, 研究荧光标记分子的结合和运输活动。通过延时全内反射荧光 (TIRF) 成像跟踪单个分子的轨迹。原始图像的分析使用简化的定制单粒子跟踪软件, 自动识别的轨迹, 单分子扩散沿 DNA 和估计其1D 扩散常数。

图 2a显示了 pVIc-Cy3B 的检测轨迹 (pVIc: GVQSLKRRRCF;Cy3B 是共轭的半胱氨酸残留物) 分子扩散在2毫米氯化钠缓冲在 pH 6.5。pVIc 的扩散是双向的, 并由环境热能量驱动。从这些轨迹中, 每个轨迹的1D 扩散常数 (D1 值) 估计 (参见图 2b中的直方图)。这个 pVIc 共轭的平均 D1 值被计算为22.2 ± 0.9 (标准误差) M (bp2/秒), 通过平均每个轨迹中所包含的三重连续位置调用的数量加权的每个 D1 值的轨迹。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55923/55923fig1.jpg"> 
图 1: 跟踪单分子沿 DNA 运动的装置. a) 一个八通道的硅橡胶微流控芯片附着在一个功能化的片上, DNA 可以被拴在一起 (与美国四分之一硬币的规模);b) 流动细胞变焦示意图, 显示流动延伸的λ-脱氧核糖核酸分子;c) TIRF 显微镜和用于拉伸 DNA 的流动系统示意图;d) TIRF 图像的流拉伸λ-DNA;e) TIRF 图像 pVIc-Cy3B (pVIc: GVQSLKRRRCF;Cy3B 是共轭的半胱氨酸残留物) 的分子绑定到流拉伸λ-DNA。此图已通过权限212进行了修改。<a href="//ecsource.jove.com/files/ftp_upload/55923/55923fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55923/55923fig2.jpg"> 
图 2: pVIc (GVQSLKRRRCF) 沿 DNA 的一维扩散. a). pVIc 沿流动延伸的λ-dna (137 弹道) 在2毫米氯化钠缓冲中的扩散在 pH 6.5。x (t) 和 y (t) 分别是沿和横向λ-DNA 的位移。虚线水平线表示染料闪烁导致的缺失点。b). 扩散常数的直方图, D1 pVIc 沿λ-dna 扩散。<a href="//ecsource.jove.com/files/ftp_upload/55923/55923fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA at JoVE.com

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

该协议展示了一个简单, 稳健和高吞吐量单分子流动拉伸试验研究 one-dimensional (1D) 分子沿 DNA 扩散。

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

a-simple-robust-high-throughput-single-molecule-flow-stretching-assay

Research

JoVE Journal

Biochemistry

8.0K 次观看.  Massachusetts Institute of Technology and Harvard Medical School. 该协议展示了一个简单, 稳健和高吞吐量单分子流动拉伸试验研究 one-dimensional (1D) 分子沿 DNA 扩散。

视频: A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

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.

单分子水平的高精度FRET用于生物分子结构测定

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

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

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

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

DNA序列识别利用高通量原酶分析的DNAPrimase

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

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

单分子鱼的转录活性等位基因单细胞分析

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

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...