Shah Shalin, Dubey Abhishek K, Reif John
Department of Electrical & Computer Engineering , Duke University , Durham , North Carolina 27701 , United States.
Department of Computer Science , Duke University , Durham , North Carolina 27701 , United States.
ACS Synth Biol. 2019 May 17;8(5):1100-1111. doi: 10.1021/acssynbio.9b00010. Epub 2019 Apr 16.
Many biochemical events of importance are complex and dynamic. Fluorescence microscopy offers a versatile solution to study the dynamics of biology at the mesoscale. An important challenge in the field is the simultaneous study of several objects of interest, referred to as optical multiplexing. For improved multiplexing, some prior techniques used repeated reporter washing or the geometry of nanostructures; however, these techniques require complex nanostructure assembly, multiple reporters, or advanced multistep drift correction. Here we propose a time-based approach, for improved optical multiplexing, that uses readily available inexpensive reporters and requires minimal preparation efforts. We program short DNA strands, referred hereby as DNA devices, such that they undergo unique conformation changes in the presence of the dye-labeled reporters. The universal fluorescent reporter transiently binds with the devices to report their activity. Since each device is programmed to exhibit different hybridization kinetics, their fluorescent time trace, referred to as the temporal barcode, will be unique. We model our devices using continuous-time Markov chains and use stochastic simulation algorithm to generate their temporal patterns. We first ran simulation experiments with a small number of DNA devices, demonstrating several distinct temporal barcodes, all of which use a single dye color. Later, using nanostructure-based devices, we designed a much larger pool of temporal barcodes and used machine learning for classification of these barcodes. Our simulation experiments and design principles can aid in the experimental demonstration of the DNA devices.
许多重要的生化事件都是复杂且动态的。荧光显微镜为在中尺度上研究生物学动态提供了一种通用的解决方案。该领域的一个重要挑战是同时研究多个感兴趣的对象，即光学复用。为了改进复用，一些先前的技术使用重复的报告分子洗涤或纳米结构的几何形状；然而，这些技术需要复杂的纳米结构组装、多个报告分子或先进的多步漂移校正。在此，我们提出一种基于时间的方法来改进光学复用，该方法使用现成的廉价报告分子，且所需的制备工作最少。我们对短DNA链进行编程，在此将其称为DNA装置，使其在存在染料标记的报告分子时经历独特的构象变化。通用荧光报告分子与这些装置瞬时结合以报告它们的活性。由于每个装置被编程为表现出不同的杂交动力学，它们的荧光时间轨迹，即时间条形码，将是独特的。我们使用连续时间马尔可夫链对我们的装置进行建模，并使用随机模拟算法生成它们的时间模式。我们首先用少量DNA装置进行模拟实验，展示了几个不同的时间条形码，所有这些都使用单一染料颜色。后来，使用基于纳米结构的装置，我们设计了一个大得多的时间条形码库，并使用机器学习对这些条形码进行分类。我们的模拟实验和设计原则有助于DNA装置的实验演示。
