Computational Sciences and Engineering Division, Health Data Sciences Institute , Oak Ridge National Lab , Oak Ridge , Tennessee 37831 , United States.
Nano Lett. 2019 Apr 10;19(4):2668-2673. doi: 10.1021/acs.nanolett.9b00590. Epub 2019 Mar 26.
Fluorescence microscopy enables simultaneous observation of the dynamics of single molecules in a large region of interest. Most traditional techniques employ either the geometry or the color of single molecules to uniquely identify (or barcode) different species of interest. However, these techniques require complex sample preparation and multicolor hardware setup. In this work, we introduce a time-based amplification-free single-molecule barcoding technique using easy-to-design nucleic acid strands. A dye-labeled complementary reporter strand transiently binds to the programmed nucleic acid strands to emit temporal intensity signals. We program the DNA strands to emit uniquely identifiable temporal signals for molecular-scale fingerprinting. Since the reporters bind transiently to DNA devices, our method offers relative immunity to photobleaching. We use a single universal reporter strand for all DNA devices making our design extremely cost-effective. We show DNA strands can be programmed for generating a multitude of uniquely identifiable molecular barcodes. Our technique can be easily incorporated with the existing orthogonal methods that use wavelength or geometry to generate a large pool of distinguishable molecular barcodes thereby enhancing the overall multiplexing capabilities of single-molecule imaging.
荧光显微镜能够同时观察大感兴趣区域中单分子的动态。大多数传统技术要么利用单分子的几何形状，要么利用其颜色来唯一识别（或编码）不同种类的感兴趣分子。然而，这些技术需要复杂的样本制备和多色硬件设置。在这项工作中，我们引入了一种基于时间的、无需扩增的单分子编码技术，使用易于设计的核酸链。标记染料的互补报告分子链暂时与编程核酸链结合，发出时间强度信号。我们将 DNA 链编程为发出可识别的独特时间信号，用于分子级别的指纹识别。由于报告分子与 DNA 器件短暂结合，我们的方法提供了相对抗光漂白的能力。我们使用单个通用报告分子链来制作所有 DNA 器件，使我们的设计极具成本效益。我们展示了 DNA 链可以被编程生成大量可识别的独特分子条码。我们的技术可以很容易地与现有的正交方法结合使用，这些方法利用波长或几何形状生成大量可区分的分子条码，从而增强单分子成像的整体多路复用能力。
