Fluorescent DNA-Polymer Enables Signal Amplification and Precise Protein Localization in Immunofluorescence Imaging.

The DNA-barcoding method has revolutionized immunofluorescence imaging, offering a transformative approach to protein visualization and analysis. However, achieving effective signal amplification while maintaining precise target localization remains a critical challenge. In this study, we developed fluorescent DNA-polymer conjugates through enzyme-catalysis-mediated reversible addition-fragmentation chain transfer polymerization, enabling the attachment of multiple fluorescent molecules to a single DNA strand. Utilizing a straightforward one-step DNA complementation process, DNA-polymers facilitate in situ signal amplification without the need for complex DNA nanostructures. Unlike DNA self-assembly-based amplification techniques, this approach minimizes positional deviations caused by bulky DNA structures, thereby significantly enhancing the protein localization accuracy. We validated the precision of DNA-polymers in protein localization using stochastic optical reconstruction microscopy and demonstrated their signal amplification and multiplexed imaging capabilities in expansion microscopy and peripheral blood leukocyte analysis. This versatile platform enables a broad range of fluorescence imaging applications, including signal amplification, multiplexed protein imaging, and super-resolution imaging, positioning DNA-polymers as a powerful tool with immense potential to advance diverse areas of biological research.