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bioRxiv · 10.64898/2026.01.13.699169

Recombinant Photo-tagging Enables Fluorescent Labelling of Biomolecules and Visualization of Liquid-Liquid Phase Separation

Agrawal, K.·Sivaprasad, P. S.·Singh, R.·Kumar, Y.·Pushpavanam, K.
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Abstract

Liquid-liquid phase separation (LLPS) of biomolecules has emerged as a fundamental principle governing biological systems. A key aspect to understand LLPS is the ability to visualize these coacervates, which requires labelling strategies. Fluorescent labelling of biomolecules remains one of the widely adopted techniques involving either small-molecule chemical conjugation or recombinant expression alongside a fluorescent protein. These small-molecule fluorophores are typically employed in excess, which further necessitates additional downstream processing steps, including separation of the conjugated molecules from the unreacted fluorophores. On the other hand, recombinantly appended fluorescent protein affects the functioning of the labelled biomolecule owing to its large size. There is still a need to develop a labelling technique with small molecular weight fluorophores that is also genetically encodable. We previously discovered that the C-terminal peptide fragment (CTPF) released during photo-exposure of a green-to-red photoconvertible fluorescent protein (PhoCl1) displayed red fluorescence. Notably, this chromophore displays broad excitation and emission wavelength profiles, rendering it robust under multiple excitation wavelengths. We leverage this phenomenon to achieve post-expression fluorescent labelling by genetically fusing PhoCl1 to the target peptide or protein to be labelled (TPTL). We successfully demonstrated this approach of conferring fluorescence to silica binding peptide, riboflavin kinase enzyme, TEV protease, Lanthanide Binding Peptide and maltose binding protein, all of which exhibited red fluorescence upon photocleavage. Finally, we demonstrate the applicability of this probe to visualize LLPS of CTPF tagged elastin-like polypeptide in the presence of polymeric crowder. We anticipate that this strategy for inducing red fluorescence in non-fluorescent biomolecules via a photocleavable protein will open new avenues for minimally perturbative fluorescent labelling.

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Agrawal, K., Sivaprasad, P. S., Singh, R., Kumar, Y., Pushpavanam, K.. 2026-01-13. Recombinant Photo-tagging Enables Fluorescent Labelling of Biomolecules and Visualization of Liquid-Liquid Phase Separation. https://doi.org/10.64898/2026.01.13.699169

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