(295d) DNA-Caged Polymer Micelles for Cell and Tissue Labeling

Purpose: This research leverages DNA nanotechnology (i.e., DNA tiles) to create ‘erasable’ labeling technologies for pathology imaging. Multiplexed images allow for the labeling of multiple targets simultaneously with high image resolution, which means that complicated pathways in samples such as tumors can be studied. However, the gold standard for pathology is single biomarker analysis in sequential slides, which precludes simultaneous evaluation of multiple signaling markers in the same cell. In conventional immunocyto- and immunohisto- chemistry processes, cells or tissue are stained with antibodies conjugated to fluorophores or colored dye reporter molecules designed for single use. However, one path forward is the use of erasable labels that would enable the same sample to be interrogated multiple times. The methods used in erasing fluorescent labels often involve harsh chemicals or photobleaching with UV light. Both of these processes have the potential to damage delicate structures found in thin tissue slices. Another type of erasable labeling uses single-stranded (ss)DNA. The ssDNA, usually labeled with a fluorophore, is attached to an antibody. Depending upon the attachment scheme, the fluorophore can then be photobleached or the entire strand can be removed by a strand displacement. This method is problematic in that the dyes employed are not protected from the environment and if only a single fluorophore or dye molecule is used, the signal may be weak. Here, we show the use of DNA-caged micelles for erasable pathology labeling. This system is based on gentle DNA dehybridization interactions that minimize tissue damage. Additionally, the dyes used in cell and tissue labeling are protected by the micelle coating.

Methods: DNA tiles are used as a surface coating for polymer nanocomposites based on electrostatic interactions, as proposed by Kurokawa et al.¹. The DNA tiles were modified to include fewer interlocking strands in favor of unique targeting strands that bind the labeling antibody. The targeting strands allow for reversible attachment of DNA caged particles to free ssDNA or to ssDNA-modified antibodies. Solution erase was demonstrated by the addition ssDNA tagged with fluorophores that were removed via strand displacement reactions.

Results and Implications: The first step is proving existence and formation of DNA cages. TEM images of micelles, DNA tiles, and DNA caged micelles were taken. Each image shows a different structure and particle size, established the formation of DNA cages. Next, to ensure that optimal structure formation and surface binding, FRET and saturation studies were conducted. FRET between dye on the micelle surface and quencher on the DNA shows that DNA does bind to the surface of the micelles rather than forming separate populations in solution. Additionally, DNA tile structures were designed to show fluorescence quenching when the repeating units interlock, showing cage formation rather than random surface absorption. Saturation studies show that there is a maximum amount of DNA that can bind to the micelle surface. We have also observed that DNA cages are stable in common buffers used such as 5% Triton X100 or Tween-20. When the DNA cages are used in solution erasing as described above, up to 85% of the signal can be repeatability erased in 8 to 15 minutes. Since the action of erasing uses a solution of ssDNA in PBS it is designed to be gentle on tissue. We then proved this hypothesis correct with the use of DNA cages and erasing solution in a variety of tissue samples with no evidence of damage to the samples. Finally, when DNA cages are used as a fluorescent reporter, up to 80% of the signal is erased after 20 minutes of exposure to ssDNA.

Our results indicated DNA cage formation on the nanocomposite surface and proof of concept labeling in cells and tissues. Proper diagnosis and treatment, especially of cancer, is highly dependent on cell and tissue labeling. DNA caged particles allow erasable labeling without harsh treatments. These approaches could be applied to label the same target in multiple cycles (e.g., super-resolution imaging) or enable different targets to be labeled sequentially. When coupled with advanced image analysis, this approach could be used to create 3D images. Therefore, DNA cages have the potential to significantly change cell and tissue labeling.

Reference

1 Kurokawa, C. et al. DNA cytoskeleton for stabilizing artificial cells. Proc Natl Acad Sci U S A 114, 7228-7233, doi:10.1073/pnas.1702208114 (2017).