Stoichiometry and Dispersity of DNA Nanostructures Using Photobleaching Pair-Correlation Analysis

A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.