Near-infrared fluorescent proteins engineered from bacterial phytochromes

Shcherbakova, Daria M.; Baloban, Mikhail; Verkhusha, Vladislav V.

doi:10.1016/j.cbpa.2015.06.005

Cited by 116 publications

(136 citation statements)

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“…The first one is an effective brightness of NIR FP in mammalian cells, which depends on its molecular brightness, intracellular stability, efficiency of BV incorporation and cell expression level. In contrast to GFP-like FPs, the effective brightness of BphP-based NIR FPs does not always correlate with their molecular brightness1. Decreased cellular fluorescence of some NIR FPs results from a low specificity of BV binding and a competition between BV and other haem-derived compounds, including protoporphyrin IX, for binding to NIR FP apoproteins1314.…”

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confidence: 99%

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

et al. 2016

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Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2–5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein–protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes.

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Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

et al. 2016

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“…As BV has the largest electron-conjugated system, it absorbs the most NIR-shifted light among all chromophores found in phytochromes. This makes BphPs the favorable templates to develop fluorescent proteins for applications in mammals 18,19 . BphPs exist in two interconvertible states, traditionally called the red-absorbing (Pr; absorbs at 660–700 nm) and the far-red-absorbing (Pfr; absorbs at 740–780 nm).…”

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Near-infrared optogenetic pair for protein regulation and spectral multiplexing

et al. 2017

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Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

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“…The superior probe for in vivo application therefore should not only merge the benefits of increased sensitivity of bioluminescence with the higher spatial and temporal resolution of fluorescence, but also have a near-infrared-shifted emission spectrum. The near-infrared (NIR) spectral range is preferable for deep-tissue and whole-body imaging due to both reduced light scattering and combined absorbance of hemoglobin, melanin and water9.…”

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Near-infrared bioluminescent proteins for two-color multimodal imaging

Rumyantsev

Turoverov

Verkhusha

2016

Sci Rep

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Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 104 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification.

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Near-infrared fluorescent proteins engineered from bacterial phytochromes

Cited by 116 publications

References 50 publications

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

Near-infrared bioluminescent proteins for two-color multimodal imaging

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