由德意志研究联合会（DFG，佤族1386 / 17-1），金融支持的研究培训集团GRK 2039（DFG通过资助）和KIT表示感谢。

注意:使用前请咨询所有相关的材料安全数据表（MSDS）。几个在这些合成中使用的化学品是有毒和致癌性。请使用在有机化学实验室，如穿试验服，护目镜和手套通常所需的所有适当的安全措施。 1.染料的合成注意:两个染料可以通过相同类型的反应来合成图2示出了这些反应的概述。 <ol><li> 1-（3-叠氮基丙基）的合成-4-（2-（1-甲基-1H-吲哚-3-基）乙烯基）吡啶-1-鎓碘化物（染料1） <ol><li> 1-（3-碘丙基）-4-甲基吡啶-1-鎓碘化物的合成（图2A，步骤a） <ol><li>溶解466毫克4-甲基吡啶和5.91克1,3-二碘丙烷在10ml乙腈中的在20ml的顶部空间小瓶中，通过隔片盖紧密地密封。 </li><li>热至85℃16小时。 </li><li>允许冷却至室温，然后使用旋转蒸发器减压除去溶剂。 </li><li>加入20ml乙酸乙酯加入到残留的油和处理在120瓦超声浴将混合物3分钟。 </li><li>通过过滤收集形成的沉淀并用乙酸乙酯洗涤五次。在真空下干燥O / N固体产品。 </li></ol></li><li> 1-（3-叠氮基丙基）-4-甲基吡啶-1-鎓合成（图2A，步骤b） <ol><li>溶解900毫克1-（3-碘丙基）-4-甲基吡啶-1-鎓碘化物12毫升乙腈中20ml的顶部空间小瓶中，添加376毫克叠氮化钠，并且通过隔片盖紧密地密封。 </li><li>热至85℃16小时。 </li><li>允许冷却至室温，然后用旋转蒸发器在减压下除去溶剂。 </li><li>二氯甲烷（15mL）添加至残余物中。 </li><li>滤除并丢弃得到的沉淀。 </li><li>使用腐减压除去溶剂进制蒸发，得到产物，为棕色油状物。 </li></ol></li><li> 1-甲基-1H-吲哚-3-甲醛的合成（图2A，步骤c） <ol><li>在惰性气体（氩气），溶解1.45克吲哚-3-甲醛，1.52克碳酸钾2.70克的二碳酸在10ml绝对二甲基甲酰胺在50毫升圆底烧瓶装有回流冷凝器。 </li><li>搅拌在130℃的混合物19小时。 </li><li>允许冷却至室温，然后倒在冰上将混合物。 </li><li>萃取水层用150ml乙酸乙酯萃取三次。 </li><li>合并有机层，用水洗，晾干硫酸钠和用旋转蒸发器在减压下于50℃除去溶剂。 </li></ol></li><li> 耦合至1-（3-叠氮基丙基）-4-（2-（1-甲基-1H-吲哚-3-基）乙烯基）吡啶-1-鎓碘化物（染料1）（图2A，步骤d） <ol><li>在氩气下和在排除水分工作。 Dissol已经90毫克1-（3-叠氮基丙基）-4-甲基吡啶-1-鎓和48毫克1-甲基-1H-吲哚-3-甲醛在4ml乙醇中20ml的圆底烧瓶装有回流冷凝器。 </li><li>添加0.07毫升哌啶，并加热到80℃4小时。 </li><li>允许冷却至室温。 </li><li>通过过滤收集得到的沉淀，用乙醚洗涤三次。 </li><li>二乙醚添加到上清液并通过过滤收集得到的沉淀。用乙醚洗涤三次。 </li><li>结合沉淀。 </li></ol></li></ol></li><li> 1-（3-叠氮基丙基）的合成-4-（2-（1-甲基-2-苯基-1H-吲哚-3-基）乙烯基）喹啉-1-鎓碘化物（染料2） <ol><li> 1-（3-碘丙基）-4-甲基喹啉-1-鎓碘化物的合成（图2B，步骤a） <ol><li>溶解715毫克4-甲基喹啉和5.91克1,3-二碘丙烷在10ml乙腈中的在20ml的顶部空间小瓶中，通过隔片盖紧密地密封。 </li><li>热至85℃16小时。 </li><li>允许冷却至室温，然后用旋转蒸发器在减压下除去溶剂。 </li><li>加入20ml乙酸乙酯添加到剩余的油和处理在120瓦超声浴将混合物3分钟。 </li><li>通过过滤收集形成的沉淀并用乙酸乙酯洗涤五次。在真空下干燥O / N固体产品。 </li></ol></li><li> 1-（3-叠氮基丙基）-4-甲基喹啉-1-鎓合成（图2B，步骤b） <ol><li>溶解900毫克1-（3-碘丙基）-4-甲基喹啉-1-鎓碘化物12毫升乙腈中20ml的顶部空间小瓶中，添加333毫克叠氮化钠，并且通过隔片盖紧密地密封。 </li><li>热至85℃16小时。 </li><li>允许冷却至室温，然后用旋转蒸发器在减压下除去溶剂。 </li><li>二氯甲烷（15mL）添加至残余物中。 </li><li>滤除并丢弃得到的沉淀。 </li><li>除去溶剂ü升气管使用旋转蒸发，得到产物，为棕色油状物减压。 </li></ol></li><li> 1-甲基-2-苯基-1H-吲哚-3-甲醛的合成（图2B，步骤c） <ol><li>在惰性气体（氩气），溶解1.45克2-苯基-1H-吲哚-3-甲醛，0.996克碳酸钾和1.77克的二碳酸在10ml绝对二甲基甲酰胺在50毫升圆底烧瓶装有回流冷凝器。 </li><li>搅拌在130℃的混合物19小时。 </li><li>允许冷却至室温，然后倒在冰上将混合物。 </li><li>萃取水层用150ml乙酸乙酯萃取三次。 </li><li>合并有机层，用水洗，干燥它们经硫酸钠，并使用旋转蒸发器在减压下除去溶剂。 </li></ol></li><li> 耦合至1-（3-叠氮基丙基）-4-（2-（1-甲基-2-苯丙氨酸NYL -1H-吲哚-3-基）乙烯基）喹啉-1-鎓碘化物（图2B，步骤d） <ol><li> ARG下工作上和下排除水分。溶解90mg的1-（3-叠氮基丙基）-4-甲基喹啉-1-鎓和59.7毫克1-甲基-2-苯基-1H-吲哚-3-甲醛在4ml乙醇中20ml的圆底烧瓶中装有回流冷凝器。 </li><li>添加0.06毫升哌啶，并加热到80℃4小时。 </li><li>允许冷却至室温。 </li><li>通过过滤收集得到的沉淀，并用乙醚三次洗涤。 </li><li>二乙醚添加到上清液并通过过滤收集得到的沉淀。用乙醚洗涤三次。 </li><li>结合沉淀。 </li></ol></li></ol></li></ol> 2. DNA链的合成注意:DNA链的合成是通过使用亚磷酰胺方法在固相上，如在DNA合成由M. Caruthers 11所述。合成器的功能是在合成步骤之前进行测试，和试剂被更新，如果必要。 <ol><li> Prearrangements <ol><li>在1.2毫升亚酰胺稀释剂（额外干燥乙腈）的溶解市售2&#39;-O-炔丙基deoxyuridinephosphoramidite（CU）。移动溶液在小瓶给合成小瓶中，并拧入合成器。 </li><li>执行"泄漏测试"（根据制造商的说明），以确保没有氩气泄漏存在。如果"泄漏测试"失败，拧小瓶更紧密。 </li><li>素填充，连接到固相合成腔管中的CU溶液。 </li><li>清洗所有线路的乙腈。 </li></ol></li><li> DNA链的合成 <ol><li>使用连接计算机进入DNA序列和耦合方法，以下从制造商的协议的提示。相比40秒，7个脉冲为一常规的CU积木的偶联步骤需要168秒，7个脉冲（每个脉冲是16微升）亚磷作为构建块。 </li><li>装入含有与该第一基底的1微摩尔改性的CPG（可控孔度玻璃），为固相的柱到合成仪（DNA合成是从3&#39;至5&#39;执行）。 </li><li>开始对DNA合成11的合成。 </li></ol></li><li> 合成的DNA链的后处理 <ol><li>干燥，真空下O / N合成的DNA链的列。 </li><li>使用钳子打开柱并释放CPG入反应小瓶中。加入700μl浓缩的氨水溶液的去保护寡核苷酸，并从CPG释放的DNA链。 </li><li>关闭小瓶并应用安全盖到反应容器中，以防止其破裂，并加热至50℃18小时。 </li><li>通过在减压（100毫巴）离心，在30℃30分钟除去氨。 </li><li>在11000 XG FO离心机容器:从CPG开始过滤R 3分钟。取上清液，并将其与0.45微米的孔尺寸转移到一个离心设备。离心在1000 xg离心4分钟。 </li><li>同时在11000 xg离心加300微升双蒸水至CPG，涡旋20秒并离心3分钟。将上清转移到离心装置和离心机在1000×g离心4分钟。重复此洗涤步骤两次。 </li><li>通过在0.1毫巴和25℃CO / N离心除去从合并的水溶液的水。 </li></ol></li></ol> 3."单击"程序<ol><li>加入50μl双蒸馏水中，抗坏血酸钠溶液的25微升（在水中的0.4M），34微升三 - [（1-苄基-1H-1,2,3-三唑-4-基）甲基]胺（0.1 M在DMSO /叔丁醇3:1），114微升叠氮化物（0.01M在DMSO /叔丁醇3:1）和17微升四（乙腈）铜（I）​​六氟磷酸盐溶液（0.1M在DMSO /叔丁醇3: 1）以冻干炔修饰的DNA样品。 </l我&gt; <li>孵育在60℃下的样品1.5小时。 </li><li>冷却到室温。 </li><li>添加150微升的 Na 2 EDTA（0.05M水溶液）和450微升乙酸钠（在水中的0.3M）的DNA样品，并转移到10毫升管。 </li><li>加入10毫升乙醇中（100％），并保持在-32℃下16小时。 </li><li>离心容器在1000 XG 15分钟和上清。 </li><li>洗的DNA沉淀用2ml冷的乙醇（80％）。 </li><li>减压干燥沉淀。 </li></ol> 4. HPLC纯化注:之前分离DNA链，确保了HPLC工作正常，足够的溶剂可和列是干净的。用缓冲液/乙腈的起始浓度冲洗柱。 <ol><li>溶解在250微升双蒸馏水和转让的粗的DNA沉淀至HPLC小瓶中。 </li><li>用0％乙腈梯度开始的45分钟HPLC运行乙腈的15％为染料1和gradi<html> 0％乙腈耳鼻喉科乙腈染料2的17％通过检查260纳米（DNA吸收）和459纳米（染料1）或542纳米（染料2）监控运行。收集那些表现在两个波长信道的吸收分数。 </li><li>通过采用由3-羟基吡啶甲酸（3HPA）和diammoniumhydrogencitrate基质基质辅助激光解吸电离（MALDI）质谱法检查正确质量的收集的级分。 <ol><li>乙腈和水用100μl在水中的diammoniumhydrogencitrate溶液（100克/升）的1-混合物:通过在1混合900微升的饱和3-HPA溶液的制备基质。 </li><li>吸取目标1-2微升DNA探针，让它干燥的空气。 </li><li>添加0.2微升3-HPA / diammoniumhydrogencitrate矩阵于样品并混合至其结晶。 </li><li>执行MALDI质谱法。 </li><li>结合表现出正确的质量那些HPLC级分。 </li></ol></li></ol>_title"&gt; 5。浓度的测定注意:浓度是通过使用紫外/可见分光光度计，基于所述DNA碱基和染料的消光系数（ε260）测定260nm处的吸收来确定。 <ol><li>溶解在100 DNA的 - 200微升双蒸馏水。 </li><li>应用1μl的在紫外/可见分光光度计的DNA溶液。 </li><li>测量在260nm的吸收。重复三次。 </li><li>取平均值来计算该溶液的浓度。为消光系数的计算中，使用四种天然碱基12的ε260nm处和购买的积木Cu的ε260nm处 （被认为是自然尿苷）12，以获得整个DNA链的ε260nm处 。对于染料1，用ε= 260nm的 10200 L *摩尔-1 * -1和染料2，用ε= 260nm的 13100 L *摩尔&lt;SUP&gt; -1 * -1。计算以下基于所述消光系数和测定吸光度朗伯-比尔斯定律的溶液的浓度。13 </li></ol> 6.样品制备和光谱<ol><li> 单链样品的制备 <ol><li>制备分别将1ml各200mM的氯化钠，50mM的NAP i缓冲改性DNA1和DNA2的2.5微米的解决方案，pH值= 7。 </li></ol></li><li> 双链样品的制备 <ol><li>制备1ml含有DNA1 2.5μM和2.5μMDNA2的在200毫摩尔NaCl，50毫NAP i缓冲，pH值= 7在反应容器中的溶液。 </li><li>固定盖子与安全盖，并加热到90℃10分钟。然后关闭加热，让冷却到室温O / N。 </li></ol></li><li> 吸收光谱 注意:要确定规范的荧光激发波长troscopy吸收光谱被记录。 <ol><li> 单链测量 <ol><li>记录只含有200 mM氯化钠，50mM的NAP i缓冲，pH值= 7的空白测量。 </li><li>转移DNA1的制备的溶液到1厘米的石英玻璃比色杯，并记录吸收。纠正对空白数据的测定。确定染料的吸收最大值。 </li><li>重复DNA2。 </li></ol></li></ol></li><li> 荧光光谱 <ol><li> 单链测量 <ol><li>记录只包含200毫米氯化钠，50毫米NAP i缓冲空白测量，pH值= 7;用染料1的激发波长。 </li><li>转移DNA1溶液到1厘米的石英玻璃比色杯。 </li><li>记录荧光光谱。 </li></ol></li><li> 双链测量 <ol><li>转移双链溶液到石英玻璃比色杯。 </l我&gt; <li>记录用染料1的激发波长的荧光光谱。 </li></ol></li></ol></li><li> 可视化实验 注意:适当的保护眼睛应佩戴避免紫外线对眼睛的伤害！ <ol><li>为了更好地了解什么样的记录的光谱显示，用照射紫外线手持灯（ 图5）的试管。观察荧光的颜色由单一的双链DNA的变化。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Color+Readout+of+DNA+Hybridization+with+Thiazole+Orange+as+an+Artificial+DNA+Base.">Fluorescent Color Readout of DNA Hybridization with Thiazole Orange as an Artificial DNA Base.</a> Angew. Chem. Int. Ed. 48 (13), 2418-2421 (2009).</li><li>Bohl&#228;nder, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+Photostable+Energy-Transfer+Pair+for+&amp;#34;DNA+Traffic+Lights&amp;#34.">Synthesis of a Photostable Energy-Transfer Pair for &amp;#34;DNA Traffic Lights&amp;#34.</a> Eur. J. Org. Chem. 34, 7547-7551 (2014).</li><li>Walter, H. K., Bohl&#228;nder, P. R., Wagenknecht, H. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+deoxyoligonucleotides+on+a+polymer+support.">Synthesis of deoxyoligonucleotides on a polymer support.</a> J. Am. Chem. Soc. 103 (11), 3185-3191 (1981).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids. , 589 (1975).</li><li>Puglisi, J. D., Tinoco, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbance+melting+curves+of+RNA.">Absorbance melting curves of RNA.</a> Meth. Enzymol. 180, 304-325 (1989).</li><li>Johansson, M. K., Fidder, H., Dick, D., Cook, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecular+Dimers:+A+New+Strategy+to+Fluorescence+Quenching+in+Dual-Labeled+Oligonucleotide+Probes.">Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes.</a> J. Am. Chem. Soc. 124, 6950-6956 (2002).</li><li>Barrois, S., W&#246;rner, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Duplex+Stability+for+Wavelength-Shifting+Fluorescent+DNA+Probes:+Energy+Transfer+vs+Excition+Interactions+in+DNA+&amp;#34;Traffic+Lights&amp;#34;,+Photochem.">The Role of Duplex Stability for Wavelength-Shifting Fluorescent DNA Probes: Energy Transfer vs Excition Interactions in DNA &amp;#34;Traffic Lights&amp;#34;, Photochem.</a> Photobiol. Sci. 13, 1126-1129 (2014).</li></ol>

The authors have nothing to disclose.

这个协议显示了完整的过程来标记DNA后合成通过CuAAC由叠氮化物修饰的荧光染料。这包括染料和炔修饰的DNA的合成，以及作为标记过程。 染料的合成如下四个步骤。所有的产品可以通过一种相当简单的沉淀由于它们的正电荷而获得，不需要耗时柱色谱法。引入中央偶联步骤之前的叠氮化物官能的缩短了染料的合成四个而不是5个连续的反应步骤。避免了1,3-二碘丙烷代替3-碘丙?...

分子成像代表活细胞内理解生物过程的基本技术。1-3荧光基于核酸的探针，例如化学-生物应用的发展，已成为一个扩大的研究领域。这些荧光探针需要满足一些要求，成为细胞成像适当的工具。首先，施加染料应该具有高量子产率的荧光，大斯托克斯位移，而最重要的是，高光稳定性，以允许在体内成像长期。其次，他们应该表现出可靠的荧光读数。常规发色团-猝灭剂的系统是基于在荧光强度简单的变化的单一荧光颜色的读出。4这种方法承担的假阳性或假阴性结果的风险，由于细胞内成分或低信号对噪声比的自发荧光由于其他COM意外淬火ponents 4 我们最近报道的"DNA的交通灯"，通过使用两个不同的发色团显示出双重荧光颜色读数的概念。5-6的概念是基于从供体染料的能量传递（ET），以受体染料而变化的荧光颜色（参见图1）。这允许更可靠的读出，从而提供了用于荧光成像探针的有力工具。用荧光染料的寡核苷酸标签可通过两种不同的方法来实现。染料可以在化学DNA合成过程中，通过使用相应的改进的Phosphoramidite积木掺入在固相上。7该方法仅限于那些标准亚磷酰胺和脱保护条件下是稳定的染料。作为替代方案，后合成修饰方法建立在寡核苷酸化学。在这里，我们展示我们的新照片之一的合成表能量转移对8,9和DNA的用叠氮化物和炔烃（CuAAC）之间的铜催化的1,3-环加成的合成后标记。10

<table><tbody><tr><td colspan="4">&lt;strong&gt;synthesis&lt;/strong&gt;</td></tr><tr><td>4-Picoline</td><td>Sigma Aldrich</td><td>239615</td><td></td></tr><tr><td>1,3-Diiodopropane</td><td>Sigma Aldrich</td><td>238414</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>10660131</td><td>HPLC grade</td></tr><tr><td>Ethyl acetate</td><td>Fisher Scientific</td><td>10456870</td><td>technical grade</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td>p.a. grade</td></tr><tr><td>Dichloromethane</td><td>Fisher Scientific</td><td>10626642</td><td>technical grade</td></tr><tr><td>Indole-3-carboxaldehyde; 98%</td><td>ABCR</td><td>AB112969</td><td></td></tr><tr><td>Potassium carbonate, 99+%</td><td>Acros</td><td>424081000</td><td></td></tr><tr><td>dimethylcarbonate</td><td>Sigma Aldrich</td><td>517127</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide, 99.8%, Extra Dry over Molecular Sieve</td><td>Acros</td><td>348435000</td><td></td></tr><tr><td>Sodium sulfate</td><td>Bernd Kraft</td><td>12623.46</td><td></td></tr><tr><td>Ethanol, 99.5%</td><td>Acros</td><td>397690010</td><td></td></tr><tr><td>Piperidine, 99%</td><td>Acros</td><td>147181000</td><td></td></tr><tr><td>Diethylether</td><td>Fisher Scientific</td><td>10407830</td><td>technical grade</td></tr><tr><td>2-Phenylindole-3-carboxaldehyde; 97%</td><td>ABCR</td><td>AB125050</td><td></td></tr><tr><td>4-Methylquinoline</td><td>ABCR</td><td>AB117222</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;DNA synthesis&lt;/strong&gt;</td></tr><tr><td>Expedite 8909 Nucleic Acid Synthesizer</td><td>Applied Biosystems</td><td>&amp;nbsp;-</td><td></td></tr><tr><td>DMT-dA(bz) Phosphoramidite</td><td>Sigma Aldrich</td><td>A111081</td><td></td></tr><tr><td>DMT-dT Phosphoramidite</td><td>Sigma Aldrich</td><td>T111081</td><td></td></tr><tr><td>DMT-dG(dmf) Phosphoramidite</td><td>Sigma Aldrich</td><td>G11508</td><td></td></tr><tr><td>DMT-dC(bz) Phosphoramidite</td><td>Sigma Aldrich</td><td>C11108</td><td></td></tr><tr><td>Amidite Diluent for DNA synthesis</td><td>Sigma Aldrich</td><td>L010010</td><td></td></tr><tr><td>Ultrapure Acetonitrile for DNA synthesis</td><td>Sigma Aldrich</td><td>L010400</td><td></td></tr><tr><td>Cap A</td><td>Sigma Aldrich</td><td>L840000</td><td></td></tr><tr><td>Cap B</td><td>Sigma Aldrich</td><td>L850000</td><td></td></tr><tr><td>CPG dT Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>T461010</td><td></td></tr><tr><td>CPG dA(bz) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>A461010</td><td></td></tr><tr><td>CPG dG(ib) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>G461010</td><td></td></tr><tr><td>CPG dC(bz) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>C461010</td><td></td></tr><tr><td>ammonia (aqueous solution)&amp;nbsp;</td><td>Fluka Analytical</td><td>318612</td><td></td></tr><tr><td>centrifugal devices nanosep 0.45&amp;nbsp;&amp;micro;m</td><td>Pall</td><td>ODGHPC34</td><td></td></tr><tr><td>5-(Benzylthio)-1H-tetrazole (Activator)</td><td>Sigma Aldrich</td><td>75666</td><td></td></tr><tr><td>2&#039;-O-propargyl deoxyuridinephosphoramidite</td><td>Chem Genes</td><td>ANP-7754</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;workup&lt;/strong&gt;</td></tr><tr><td>vacuum concentrator</td><td>Christ</td><td></td><td></td></tr><tr><td>&lt;strong&gt;clicking procedure&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tetrakis(acetonitrile)copper(I) hexafluorophosphate</td><td>Sigma Aldrich</td><td>346276</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma Aldrich</td><td>S2889</td><td></td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Sigma Aldrich</td><td>A7631</td><td></td></tr><tr><td>EDTA disodium salt</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>TBTA-ligand</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>synthesized according to a literature procedure&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;HPLC&lt;/strong&gt;</td></tr><tr><td>HPLC-system</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>MALDI-Biflex-IV spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>LC-318 C18 column</td><td>Supelcosil via Sigma Aldrich</td><td>58368</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;determination of concentration&lt;/strong&gt;</td></tr><tr><td>ND 1000 Spectrophotometer</td><td>nanodrop</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;sample preparation and spectroscopy&lt;/strong&gt;</td></tr><tr><td>Cary 100 Bio</td><td>Varian</td><td></td><td></td></tr><tr><td>Fluoromax-3 fluorimeter</td><td>Jobin-Yvon</td><td></td><td></td></tr><tr><td colspan="4">&lt;sup&gt;1&lt;/sup&gt; R. Chan Timothy, R. Hilgraf, K. B. Sharpless, V. Fokin Valery, &lt;em&gt;Org Lett&lt;/em&gt; 2004, &lt;strong&gt;6&lt;/strong&gt;, 2853-2855.</td></tr></tbody></table>

synthesis of wavelength-shifting dna hybridization probes by using photostable cyanine dyes

在这个协议中，我们证明了2&#39;-炔合成的方法，用标准的亚磷酰胺化学改性通过自动固相合成脱氧核糖核酸（DNA）链。寡核苷酸是使用铜催化点击化学两个新的耐光菁染料后的综合标记。供体和受体染料的合成中描述并在连续的三个步骤进行。用该DNA作为周边结构，这两种染料经历能量转移时，他们通过杂交带入靠近。因此，两个单链DNA链的退火通过荧光颜色的变化可视化。这种颜色的变化的特征是荧光光谱，但也可以通过使用手持式紫外线（UV）灯被直接观察。双色荧光读出的概念使得这些寡核苷酸探针的分子成像尤其是当所描述的河粉优秀的工具tostable染料被使用。从而，成像探针的光漂白防止和生物过程可实时地观察了更长的时间。

的单链和双链DNA的吸收和荧光光谱被记录为如图4所示。 所记录的吸收光谱（ 图4右）显示吸收峰λ 最大为465纳米的单链DNA1（染料1）和单链DNA2（染料2）546纳米。退火的DNA1_2（染料1染料2）示出了在两个469纳米和567纳米的最大值。两个最大吸收显示出红移，对于染料1 4纳米和21纳米的染料。这些...

Watch this Scientific Journal Video about Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes at JoVE.com

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

synthesis-wavelength-shifting-dna-hybridization-probes-using

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Alkynes

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11.0K 次观看.  Karlsruhe Institute of Technology. Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

视频: Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

科研

生物化学

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全基因组阵列比较基因组杂交技术示范

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

生物学

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

用噻唑橙代替溴代或替代染料进行 DNA 电泳

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

生物工程

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

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 ...

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA &#39;roadblocks&#39;&#160;&#8212; is critical for ensuring faithful&#160;genome duplication.&#160;G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase &#948;, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.

This protocol outlines a fluorescence microscopy-based single-molecule DNA replication assay, enabling real-time visualization of interactions between DNA polymerases and obstacles such as G-quadruplex structures.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

摘要

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此视频中的章节

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

全基因组阵列比较基因组杂交技术示范

用噻唑橙代替溴代或替代染料进行 DNA 电泳

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

结合荧光染色体复制时序

强大的3D直接标记的探针DNA鱼

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

G-四链体 DNA 聚合酶动力学的单分子荧光可视化

通过使用光稳定菁染料波长移的DNA杂交探针的合成

摘要

探索更多视频

此视频中的章节

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

全基因组阵列比较基因组杂交技术示范

用噻唑橙代替溴代或替代染料进行 DNA 电泳

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

结合荧光染色体复制时序

强大的3D直接标记的探针DNA鱼

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

G-四链体 DNA 聚合酶动力学的单分子荧光可视化

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装