Multiplexed bioluminescence microscopy via phasor analysis

Yao, Zi; Brennan, Caroline K.; Scipioni, Lorenzo; Chen, Hongtao; Ng, Kevin K.; Tedeschi, Giulia; Parag-Sharma, Kshitij; Amelio, Antonio L.; Gratton, Enrico; Digman, Michelle A.; Prescher, Jennifer A.

doi:10.1038/s41592-022-01529-9

Cited by 24 publications

(17 citation statements)

References 51 publications

(27 reference statements)

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“…BRET receivers should enable tracking of complex cellular interactions, including more than two populations of interest in heterogeneous environments. While the emission spectra are quite broad and overlapping, multiplexed imaging is possible using spectral unmixing or bioluminescent phasor analysis. , …”

Section: Resultsmentioning

confidence: 99%

“…We intend to examine these parameters in future studies using the wide range of SmBiT affinities reported. Future work will also apply LOTIIS to multiplexed and orthogonal imaging using the split-BRET reporters featured in this study and related variants. − Collectively, such probes will be able to capture both transient and long-lasting interactions across varying distances, improving our understanding of complex multicellular networks.…”

Section: Discussionmentioning

confidence: 97%

“…While the emission spectra are quite broad and overlapping, multiplexed imaging is possible using spectral unmixing or bioluminescent phasor analysis. 28,31 ■…”

Section: Lotiis Labeling Is Time-and Distance-dependentmentioning

confidence: 99%

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Generalized Bioluminescent Platform To Observe and Track Cellular Interactions

Prescher

2022

Bioconjugate Chem.

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Cell-to-cell communications are critical to biological processes ranging from embryonic development to cancer progression. Several imaging strategies have been developed to capture such interactions, but many are challenging to deploy in thick tissues and other complex environments. Here, we report a platform termed Luminescence to Observe and Track Intercellular Interactions (LOTIIS). The approach features split fragments of a luciferase enzyme that reassemble when target cells come into proximity. One fragment is secreted by “sender” cells, and the complementary piece is secreted by “receiver” cells. Split reporter assembly is facilitated by a single chain variable fragment (scFv)–peptide interaction on the receiver cell, resulting in localized light production. We demonstrate that LOTIIS can rapidly label cells in close proximity in a time- and distance-dependent fashion. The platform is also compatible with bioluminescence resonance energy transfer probes for multiplexed imaging. Collectively, these data suggest that LOTIIS will enable a variety of cellular interactions to be tracked in biological settings.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

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Generalized Bioluminescent Platform To Observe and Track Cellular Interactions

Prescher

2022

Bioconjugate Chem.

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“…Extending the approach used here to create similarly specific luciferases for synthetic luciferin substrates beyond DTZ and h-CTZ would considerably extend the multiplexing opportunities illustrated in Fig. 4 (particularly with the recent advances in microscopy 41 ), and enable a new generation of multiplexed luminescent toolkits. More generally, our family-wide hallucination method opens up an almost unlimited number of scaffold possibilities for substrate binding and catalytic residue placement, which is particularly important when the reaction mechanism and how to promote it are not completely understood: many alternative structural and catalytic hypotheses can be readily enumerated with shape and chemically complementary binding pockets but different catalytic residue placements.…”

Section: Discussionmentioning

confidence: 99%

De novo design of luciferases using deep learning

Yeh

Norn

Kipnis

et al. 2023

Nature

136

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De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence–structure relationships. Here we describe a deep-learning-based ‘family-wide hallucination’ approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M−1 s−1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.

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“…NLuc-E2 was visualized using a specially developed bioluminescence microscope [ 42 ]. Samples were placed in a temperature-, humidity-, and CO 2 -controlled box (Tokai) within an Olympus IX83 TIRF microscope, which was used in widefield mode.…”

Section: Methodsmentioning

confidence: 99%