Publications

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFluS). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

JTD Keywords: dna-paint, hemagglutinin, influenza, neuraminidase, paint, recombinant proteins, single-molecule localization microscopy, single-particle analysis, virus-like particles, Dna-paint, Hemagglutinin, Influenza, Neuraminidase, Paint, Recombinant proteins, Single particle analysis, Single-molecule localization microscopy, Single-particle analysis, Super-resolution microscopy, Superresolution microscopy, Virus-like particles

Surface functionalization with targeting ligands confers to nanomaterials the ability of selectively recognize a biological target. Therefore, a quantitative characterization of surface functional molecules is critical for the rational development of nanomaterials-based applications, especially in nanomedicine research. Single-molecule localization microscopy can provide visualization of surface molecules at the level of individual particles, preserving the integrity of the material and overcoming the limitations of analytical methods based on ensemble averaging. Here we provide single-molecule localization data obtained on streptavidin-coated polystyrene particles, which can be exploited as a model system for surface-functionalized materials. After loading of the active sites of streptavidin molecules with a biotin-conjugated probe, they were imaged with a DNA-PAINT imaging approach, which can provide single-molecule imaging at subdiffraction resolution and molecule counting. Both raw records and analysed data, consisting in a list of space-time single-molecule coordinates, are shared. Additionally, Matlab functions are provided that analyse the single-molecule coordinates in order to quantify features of individual particles. These data might constitute a valuable reference for applications of similar quantitative imaging methodologies to other types of functionalized nanomaterials.

JTD Keywords: DNA-PAINT, Functional materials, Nanoparticles, Single-molecule localization microscopy, Super-resolution microscopy