Configuring robust DNA strand displacement reactions for in situ molecular analyses.
Configuring robust DNA strand displacement reactions for in situ molecular analyses.
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DOI：
10.1093/nar/gkr1209
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发表时间：
2012-04
影响因子：
14.9
通讯作者：
Diehl MR
中科院分区：
文献类型：
--
作者：
Duose DY;Schweller RM;Zimak J;Rogers AR;Hittelman WN;Diehl MR
通过荧光显微镜可以在单个细胞和组织切片内可视化的不同生物分子的数量受到永久耦合到其目标的荧光染料分子的光谱重叠的限制。这个问题阻碍了细胞中不同分子途径组分之间重要功能关系的表征。然而，最近改进的理解DNA链置换反应现在提供了机会，创造可编程的标记和检测方法，通过不同的动态DNA复合物之间的受控瞬时相互作用。我们研究了是否可以创建可擦除的分子成像探针，利用这种机制来耦合，然后从固定的细胞样品内的DNA标记的蛋白质标记物中去除带有荧光团的寡核苷酸。我们表明，通过链置换的标记擦除的效率可以通过非立足点介导的立场交换过程，降低携带荧光团的链扩散出细胞的速率受到限制。描述了两种探针结构，其避免了这个问题，并允许从其目标有效地去除荧光团。通过这些修改，我们表明，通过重复原位标记和擦除细胞上的标记物，可以在相同细胞上可视化的蛋白质数量至少增加一倍。
The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells.
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影响因子：
14.9
作者：
Zhang DY;Winfree E
通讯作者：
Winfree E
影响因子：
15
作者：
Zhang, David Yu
通讯作者：
Zhang, David Yu
DOI：
10.1073/pnas.0407024101
发表时间：
2004-10-26
影响因子：
11.1
作者：
Dirks, RM;Pierce, NA
通讯作者：
Pierce, NA
影响因子：
4.7
作者：
Duose, Dzifa Y.;Schweller, Ryan M.;Hittelman, Walter N.;Diehl, Michael R.
通讯作者：
Diehl, Michael R.
影响因子：
46.9
作者：
通讯作者：
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