Department of Bioengineering, Rice University, 6100 Main Street, Houston, TX 77005, USA.
Nucleic Acids Res. 2012 Apr;40(7):3289-98. doi: 10.1093/nar/gkr1209. Epub 2011 Dec 11.
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.
通过荧光显微镜在单个细胞和组织切片中可视化的独特生物分子的数量受到与其靶标永久偶联的荧光染料分子的光谱重叠的限制。这个问题妨碍了细胞内不同分子途径成分之间重要功能关系的特征描述。然而，最近对 DNA 链置换反应的深入了解为创建可编程标记和检测方法提供了机会，这些方法通过不同动态 DNA 复合物之间的受控瞬态相互作用来运行。我们研究了是否可以创建可擦除的分子成像探针，利用这种机制将带有荧光团的寡核苷酸与固定细胞样品中标记 DNA 的蛋白质标记物偶联，然后将其去除。我们表明，通过链置换进行标记物擦除的效率可能会受到非引发链介导的链交换过程的限制，这些过程降低了带有荧光团的链从细胞中扩散出来的速率。描述了两种避免这个问题并允许荧光团从其靶标有效去除的探针结构。通过这些修改，我们表明，人们至少可以通过在细胞上进行重复的原位标记和擦除标记物，将可以在同一细胞上可视化的蛋白质数量增加一倍。
