mRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis

Abstract Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.

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Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

Communications Chemistry ◽

10.1038/s42004-020-00437-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Yusaku Hontani ◽

Mikhail Baloban ◽

Francisco Velazquez Escobar ◽

Swetta A. Jansen ◽

Daria M. Shcherbakova ◽

...

Keyword(s):

Real Time ◽

Rational Design ◽

Near Infrared ◽

Fluorescent Proteins ◽

Covalent Bond ◽

Binding Pocket ◽

Tissue Imaging ◽

Covalent Attachment ◽

Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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Imaging of red-shifted photons from bioluminescent tumours using fluorescence by unbound excitation from luminescence

10.1101/428771 ◽

2018 ◽

Cited By ~ 1

Author(s):

Fabiane Sônego ◽

Sophie Bouccara ◽

Thomas Pons ◽

Nicolas Lequeux ◽

Anne Danckaert ◽

...

Keyword(s):

Cell Line ◽

Near Infrared ◽

Tumour Size ◽

Visible Spectrum ◽

Tissue Imaging ◽

Tumour Detection ◽

Signal Sensitivity ◽

Bioluminescence Signal ◽

Deep Tissue Imaging

AbstractEarly detection of tumours is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. Bioluminescence imaging has been widely used in the field of oncology and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, photons that are emitted by the majority of commonly used luciferases are usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal and calling for optimised optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumour detection in two different subcutaneous tumour models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with the plasmid Luc2. Tumour size and bioluminescence were measured over time and tumour vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs)in the tumour site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumour detection.

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Fluorescent proteins for in vivo imaging, where's the biliverdin?

Biochemical Society Transactions ◽

10.1042/bst20200444 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2657-2667

Author(s):

Felipe Montecinos-Franjola ◽

John Y. Lin ◽

Erik A. Rodriguez

Keyword(s):

Hydrogen Peroxide ◽

In Vivo Imaging ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Imaging ◽

Infrared Light ◽

Red Fluorescent Protein ◽

Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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Quantitative assessment of near-infrared fluorescent proteins

10.21203/rs.3.rs-797586/v1 ◽

2021 ◽

Author(s):

Kiryl Piatkevich ◽

Hanbin Zhang ◽

Stavrini Papadaki ◽

Xiaoting Sun ◽

Luxia Yao ◽

...

Keyword(s):

Mammalian Cells ◽

Quantitative Assessment ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Oligomeric State ◽

C Elegans ◽

The Right ◽

Infrared Fluorescent Protein

Abstract Recent progress in fluorescent protein development has generated a large diversity of near-infrared fluorescent proteins, which are rapidly becoming popular probes for a variety of imaging applications. To assist end-users with a selection of the right near-infrared fluorescent protein for a given application, we will conduct a quantitative assessment of intracellular brightness, photostability, and oligomeric state of 19 near-infrared fluorescent proteins in cultured mammalian cells. The top-performing proteins will be further validated for in vivo imaging of neurons in C. elegans, zebrafish, and mice. We will also assess the applicability of the selected NIR FPs for expansion microscopy and two-photon imaging.

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Design of AIEgens for Near-Infrared IIb Imaging Through Structural Modulation at Molecular and Morphological Levels

10.26434/chemrxiv.10728086 ◽

2019 ◽

Author(s):

yuanyuan li ◽

Zhaochong Cai ◽

shunjie liu ◽

Haoke Zhang ◽

sherman Wong ◽

...

Keyword(s):

Blood Vessels ◽

Fluorescence Imaging ◽

Near Infrared ◽

Molecular Design ◽

Organic Dyes ◽

Tissue Imaging ◽

Deep Penetration ◽

Structural Modulation ◽

Organic Fluorophore

<p>Fluorescence imaging in near-infrared IIb (NIR-IIb, 1500-1700 nm) spectrum holds a considerable promise for tissue imaging with deep penetration and high spatial resolution owing to the minimized autofluorescence and suppressed photon scattering. While few inorganic NIR-IIb fluorescent probes have been reported, their organic counterparts are still underdeveloped, possibly due to the lack of efficient materials with long emission wavelength. Herein, we propose a new molecular design philosophy to develop organic NIR-IIb fluorophores with high quantum yield (QY) by manipulation of the effects of twisted intramolecular charge transfer and aggregation-induced emission at the molecular and morphological levels. A pure organic fluorescent dye emitting up to 1600 nm with a QY of 14.2% in the NIR-II region (1000-1600 nm) is developed. For the first time, NIR-IIb fluorescence imaging of blood vessels and deeply-located intestinal tract of live mice based on organic dyes is achieved. The results show that organic fluorophore performs superb imaging ability in both superficial blood vessels and internal organs with high resolution and enhanced signal-to-background ratio in NIR-IIb region. We hope this groundbreakingly study will inspire further research on the evolution of pure organic NIR-IIb probes for in vivo imaging.</p>

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Pretargeted Imaging with Gallium-68—Improving the Binding Capability by Increasing the Number of Tetrazine Motifs

Pharmaceuticals ◽

10.3390/ph11040102 ◽

2018 ◽

Vol 11 (4) ◽

pp. 102 ◽

Cited By ~ 4

Author(s):

Dominik Summer ◽

Sonja Mayr ◽

Milos Petrik ◽

Christine Rangger ◽

Katia Schoeler ◽

...

Keyword(s):

Pet Imaging ◽

Binding Capacity ◽

Alder Reaction ◽

Tissue Imaging ◽

In Vivo Studies ◽

Primary Amines ◽

Imaging Studies ◽

Gallium 68

The inverse electron-demand Diels-Alder reaction between 1,2,4,5-tetrazine (Tz) and trans-cyclooct-2-ene (TCO) has gained increasing attraction among extensive studies on click chemistry due to its exceptionally fast reaction kinetics and high selectivity for in vivo pretargeting applications including PET imaging. The facile two-step approach utilizing TCO-modified antibodies as targeting structures has not made it into clinics yet. An increase in the blood volume of humans in comparison to mice seems to be the major limitation. This study aims to show if the design of multimeric Tz-ligands by chelator scaffolding can improve the binding capacity and may lead to enhanced PET imaging with gallium-68. We utilized for this purpose the macrocyclic siderophore Fusarinine C (FSC) which allows conjugation of up to three Tz-residues due to three primary amines available for site specific modification. The resulting mono- di- and trimeric conjugates were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, stability, binding towards TCO modified rituximab (RTX)) and in vivo (biodistribution- and imaging studies in normal BALB/c mice using a simplified RTX-TCO tumour surrogate). The 68Ga-labelled FSC-based Tz-ligands showed suitable hydrophilicity, high stability and high targeting specificity. The binding capacity to RTX-TCO was increased according to the grade of multimerization. Corresponding in vivo studies showed a multimerization typical profile but generally suitable pharmacokinetics with low accumulation in non-targeted tissue. Imaging studies in RTX-TCO tumour surrogate bearing BALB/c mice confirmed this trend and revealed improved targeting by multimerization as increased accumulation in RTX-TCO positive tissue was observed.

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Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging

Molecular Imaging and Biology ◽

10.1007/s11307-020-01523-7 ◽

2020 ◽

Vol 22 (6) ◽

pp. 1523-1531

Author(s):

Giorgia Zambito ◽

Natasa Gaspar ◽

Yanto Ridwan ◽

Mary P. Hall ◽

Ce Shi ◽

...

Keyword(s):

Near Infrared ◽

Bioluminescence Imaging ◽

Spectral Characteristics ◽

Photon Emission ◽

Ccd Camera ◽

Spectral Unmixing ◽

Tissue Imaging ◽

Click Beetle

Abstract Purpose Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different d-luciferin (d-LH2) analogues in vivo. Procedures Transduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software. Results Our in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was d-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with d-LH2. Conclusion We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.

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Fluorescence Phenomena in Amyloid and Amyloidogenic Bionanostructures

Crystals ◽

10.3390/cryst10080668 ◽

2020 ◽

Vol 10 (8) ◽

pp. 668 ◽

Cited By ~ 1

Author(s):

B. Apter ◽

N. Lapshina ◽

H. Barhom ◽

B. Fainberg ◽

A. Handelman ◽

...

Keyword(s):

Amyloid Fibrils ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Protein Structures ◽

Visible Spectrum ◽

Biological Tissues ◽

New Generation ◽

Β Sheet

Nanoscale optical labeling is an advanced bioimaging tool. It is mostly based on fluorescence (FL) phenomena and enables the visualization of single biocells, bacteria, viruses, and biological tissues, providing monitoring of functional biosystems in vitro and in vivo, and the imaging-guided transportation of drug molecules. There is a variety of FL biolabels such as organic molecular dyes, genetically encoded fluorescent proteins (green fluorescent protein and homologs), semiconductor quantum dots, carbon dots, plasmonic metal gold-based nanostructures and more. In this review, a new generation of FL biolabels based on the recently found biophotonic effects of visible FL are described. This intrinsic FL phenomenon is observed in any peptide/protein materials folded into β-sheet secondary structures, irrespective of their composition, complexity, and origin. The FL effect has been observed both in natural amyloid fibrils, associated with neurodegenerative diseases (Alzheimer’s, Parkinson’s, and more), and diverse synthetic peptide/protein structures subjected to thermally induced biological refolding helix-like→β-sheet. This approach allowed us to develop a new generation of FL peptide/protein bionanodots radiating multicolor, tunable, visible FL, covering the entire visible spectrum in the range of 400–700 nm. Newly developed biocompatible nanoscale biomarkers are considered as a promising tool for emerging precise biomedicine and advanced medical nanotechnologies (high-resolution bioimaging, light diagnostics, therapy, optogenetics, and health monitoring).

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