Oligo DNA-based quantum dot (QD) single-particle tracking for multicolor single-molecule imaging

Sakuragi, S.; Kato, N.; Uchida, T.; Katagiri, T.; Enomoto, M.; Kato, R.; Yoshimura, H.; Oyama, C.; Katayama, I.; Chikuma, A.; Teramura, Y.; Bannai, H.

doi:10.1101/2025.10.06.680600

bioRxiv · 10.1101/2025.10.06.680600

Oligo DNA-based quantum dot (QD) single-particle tracking for multicolor single-molecule imaging

Sakuragi, S.·Kato, N.·Uchida, T.·Katagiri, T.·Enomoto, M.·Kato, R.·Yoshimura, H.·Oyama, C.·Katayama, I.·Chikuma, A.·Teramura, Y.·Bannai, H.

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Abstract

Quantum dot single-particle tracking (QD-SPT), a powerful tool for analyzing membrane domains that are critical to various cellular processes, is widely used in membrane molecular dynamics research. QDs, which possess both a broad excitation range and a narrow emission bandwidth, are inherently suited for multicolor imaging at various wavelengths. However, applying multicolor QD-SPT with multiple biomolecular targets has been challenging due to the limited methods for specifically conjugating QDs to biomolecules. Here, we propose a DNA hybridization-based QD labeling method that generates several specific interactions based on sequence. QD fused with 20-mer oligo DNA specifically labeled membrane lipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) covalently bound to complementary oligo DNA, forming a stable label that is suitable for SPT. The combination of polyA-polyT sequence as the linker oligo caused more QDs to fuse to DPPE compared with a random sequence linker. Oligo DNA-based QD-SPT accurately reflected the diffusion dynamics of DPPE measured using the single-fluorescence tracking technique and was compatible with conventional QD-SPT utilizing secondary antibody Fab. Using different pairs of oligo DNA sequences, we successfully achieved multicolor QD-SPT that distinguishes the lateral diffusion of DPPE and a membrane protein, GABAA receptor (GABAAR), within the same cell. The oligo DNA-based QD labeling method developed in this study is anticipated to substantially advance simultaneous multicolor QD-SPT of different living cell membrane molecules specific to DNA sequences.

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Sakuragi, S., Kato, N., Uchida, T., Katagiri, T., Enomoto, M., Kato, R., Yoshimura, H., Oyama, C., Katayama, I., Chikuma, A., Teramura, Y., Bannai, H.. 2025-10-06. Oligo DNA-based quantum dot (QD) single-particle tracking for multicolor single-molecule imaging. https://doi.org/10.1101/2025.10.06.680600

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