( e) Mean fluorescence intensity of cells transfected with Nb GFP, containing miRFP670nano3 inserted at G44/K45, S65/V66 or P90/E91 sites via Gly 4Ser linkers before and after 4 h of incubation with 10 μM bortezomib. Fluorescence intensity of cells transfected with the pNIR-Fb GFP and pEGFP-N1 plasmids with the ratio of 1:1 was assumed to be 100% for each FP. ( d) Fluorescence intensity of cells transfected with the same amount of the pNIR-Fb GFP plasmid and indicated ratios of the pEGFP-N1 plasmid to the pNIR-Fb GFP plasmid. ( c) Quantification of the data presented in (b). ( b) The fluorescence intensity distribution of cells transfected with Nb GFP, containing miRFP670nano3 inserted at G44/K45, S65/V66, or P90/E91 positions and co-expressed with or without EGFP. ( a) Structure of Nb against GFP (Nb GFP) (PDB ID: 3OGO) with indicated positions for insertion of miRFP670nano3. Representative images of three experiments are shown. ( a-b) GFP and miRFP670nano3 fluorescence images were acquired simultaneously using a 920 nm excitation light and emission filters 525/70 nm for EGFP and 645/75 nm for miRFP670nano3. Bottom, three example images from the xy fluorescence image stack at the indicated depths (z) from the pial surface. ( b) Top, xz sub-projection from a xy fluorescence image stack showing neurons (red) and microglia (green) in the lumbar spinal cord of a 12.5-weeks-old Cx3cr1 GFP/+ mouse five weeks after stereotactic AAV9-hSYN-miRFP670nano3 vector delivery into superficial dorsal horn laminae. Right, four example images from the xy fluorescence image stack at the indicated depths (z) from the pial surface. ( a) Left, xz sub-projection from a xy fluorescence image stack showing neurons (red) and microglia (green) in the somatosensory cortex of a 14-weeks-old Cx3cr1 GFP/+ mouse five weeks after stereotactic AAV9-hSYN-miRFP670nano3 vector delivery into deep cortical layers. Gating was performed as shown in Supplementary Fig. Fluorescence intensity in (e, g, h, i) was measured by flow cytometry using a 640 nm excitation laser and a 670 nm LP emission filter for miRFP670nano and miRFP670nano3, and a 488 nm excitation laser and a 510/15 nm emission filter for EGFP. ( i) Fluorescence intensity of live HeLa cells transiently transfected with miRFP670nano3, miRFP670nano, or EGFP 48 h and 120 h after transfection normalized to that at 48 h. ( h) Fluorescence intensity of live HeLa cells transiently transfected with miRFP670nano3, miRFP670nano, or EGFP before and after 4 h of incubation with 10 μM bortezomib. ( g) Fluorescence intensity of live HeLa cells transiently transfected with miRFP670nano3, miRFP670nano, or EGFP before and after 4 h of incubation with 20 μg/ml cycloheximide. ( f) Photobleaching kinetics of miRFP670nano3 in comparison with parental miRFP670nano in live HeLa cells. The effective brightness of miRFP670nano was assumed to be 100% for each cell type. Fluorescence intensity was analyzed 72 h after transfection. ( e) Effective (cellular) brightness of miRFP670nano3 and miRFP670nano in transiently transfected HeLa, N2A, U-2 OS, HEK293T, and NIH3T3 live cells. ( d) pH dependence of miRFP670nano3 fluorescence in comparison with parental miRFP670nano. ( c) Size exclusion chromatography of miRFP670nano3 and used molecular weight protein standards. ( b) Fluorescence excitation spectrum recorded at 730 nm emission and emission spectrum recorded at 600 nm excitation. The Author(s), under exclusive licence to Springer Nature America, Inc. Altogether, NIR-Fbs enable the detection and manipulation of a variety of cellular processes based on the intracellular protein profile. Applying NIR-Fbs as destabilizing fusion partners, we developed molecular tools for directed degradation of targeted proteins, controllable protein expression and modulation of enzymatic activities. NIR-Fbs allowed background-free visualization of endogenous proteins, detection of viral antigens, labeling of cells expressing target molecules and identification of double-positive cell populations with bispecific NIR-Fbs against two antigens. By exploring miRFP670nano3 as an internal tag, we engineered 32 kDa NIR fluorescent nanobodies, termed NIR-Fbs, whose stability and fluorescence strongly depend on the presence of specific intracellular antigens. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications.
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