scholarly journals Tracking RNA with light: selection, structure, and design of fluorescence turn-on RNA aptamers

2019 ◽  
Vol 52 ◽  
Author(s):  
Robert J. Trachman ◽  
Adrian R. Ferré-D'Amaré
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Structural Diversity ◽  
Rna Aptamers ◽  
Live Cells ◽  
Rna Molecules ◽  
Turn On ◽  
Green Fluorescent ◽  
Fluorescence Turn On

AbstractFluorescence turn-on aptamers,in vitroevolved RNA molecules that bind conditional fluorophores and activate their fluorescence, have emerged as RNA counterparts of the fluorescent proteins. Turn-on aptamers have been selected to bind diverse fluorophores, and they achieve varying degrees of specificity and affinity. These RNA–fluorophore complexes, many of which exceed the brightness of green fluorescent protein and their variants, can be used as tags for visualizing RNA localization and transport in live cells. Structure determination of several fluorescent RNAs revealed that they have diverse, unrelated overall architectures. As most of these RNAs activate the fluorescence of their ligands by restraining their photoexcited states into a planar conformation, their fluorophore binding sites have in common a planar arrangement of several nucleobases, most commonly a G-quartet. Nonetheless, each turn-on aptamer has developed idiosyncratic structural solutions to achieve specificity and efficient fluorescence turn-on. The combined structural diversity of fluorophores and turn-on RNA aptamers has already produced combinations that cover the visual spectrum. Further molecular evolution and structure-guided engineering is likely to produce fluorescent tags custom-tailored to specific applications.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

scholarly journals Genetically-encoded fluorescent biosensor for rapid detection of protein expression

2020 ◽  
Author(s):  
Matthew G Eason ◽  
Antonia T Pandelieva ◽  
Marc M Mayer ◽  
Safwat T Khan ◽  
Hernan G Garcia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

Studies on the Dynamic Localization of GFP-Myosin During Cytokinesis in Live Cells

1997 ◽  
Vol 3 (S2) ◽  
pp. 129-130
Author(s):  
James Sabry ◽  
Sheri Moores ◽  
Shannon Ryan ◽  
Ji-Hong Zang ◽  
James A. Spudich
Keyword(s):  
Fluorescent Protein ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Thick Filament ◽  
Live Cells ◽  
In Vitro Motility ◽  
Multimeric Complex ◽  
Ring Constriction ◽  
Green Fluorescent

Cell division is thought to be powered by the constriction of an actomyosin containing contractile ring found transiently in the cleavage furrow. Conventional myosin II plays a fundamental role in this process of cytokinesis where, in the form of a multimeric complex known as the bipolar thick filament, it is thought to be the molecular motor that generates the force necessary to cause ring constriction.In order to study the dynamics of this protein in the dividing cell, we have made a fusion protein of the green fluorescent protein (GFP) and the amino terminus of the Dictyostelium myosin heavy chain (GFP-myosin), and imaged the location of this protein in dividing Dictyostelium cells were it is the only myosin II present in the cell. The addition of GFP does not compromise the functioning of the myosin motor as evidenced by the fact that purified GFP-myosin has solution ATPase and in vitro motility kinetics similar to that of non-labelled myosin. In addition, GFP-myosin fully complements the myosin null mutation for both development and cytokinesis in suspension suggesting that GFP-myosin acts as a regulated motor when expressed in cells.

scholarly journals A bright and high-performance genetically encoded Ca2+ indicator based on mNeonGreen fluorescent protein

2020 ◽  
Author(s):  
Landon Zarowny ◽  
Abhi Aggarwal ◽  
Virginia M.S. Rutten ◽  
Ilya Kolb ◽  
Ronak Patel ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Model Organisms ◽  
Green Fluorescent Proteins ◽  
Comparable Performance ◽  
Green Fluorescent

AbstractGenetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost two decades of steady improvements in the Aequorea victoria GFP (avGFP)-based GCaMP series of GECIs, the performance of the most recent generation (i.e., GCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression towards ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

20 GENERATION AND CHARACTERIZATION OF TRANSGENIC-CLONED PIGS EXPRESSING THE FAR-RED FLUORESCENT PROTEIN MONOMERIC PLUM

2014 ◽  
Vol 26 (1) ◽  
pp. 124
Author(s):  
M. Kobayashi ◽  
M. Watanabe ◽  
H. Matsunari ◽  
K. Nakano ◽  
T. Kanai ◽  
...  
Keyword(s):  
Mixed Culture ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Cell Stage ◽  
Fetal Fibroblasts ◽  
Morula Stage ◽  
Clear Identification ◽  
Green Fluorescent

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.

Visualization ofToxoplasma gondiistage conversion by expression of stage-specific dual fluorescent proteins

Parasitology ◽  
2009 ◽  
Vol 136 (6) ◽  
pp. 579-588 ◽  
Author(s):  
A. UNNO ◽  
K. SUZUKI ◽  
T. BATANOVA ◽  
S.-Y. CHA ◽  
H.-K. JANG ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Host Cells ◽  
Live Cells ◽  
Green Fluorescence ◽  
Red Fluorescence ◽  
Genes Encoding ◽  
Transgenic Parasite ◽  
P38 Mapk Inhibitor ◽  
Green Fluorescent

SUMMARYTo recognize the stage conversion ofToxoplasma gondiibetween tachyzoite and bradyzoite in live host cells, a transgenicT. gondiiline, which expressed stage-specific red and green fluorescence, was constructed.T. gondiiPLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

scholarly journals Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1

1999 ◽  
Vol 337 (3) ◽  
pp. 575-583 ◽  
Author(s):  
Richard A. CURRIE ◽  
Kay S. WALKER ◽  
Alex GRAY ◽  
Maria DEAK ◽  
Antonio CASAMAYOR ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Lipid Vesicles ◽  
Live Cells ◽  
Ph Domain ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Green Fluorescent

3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of PtdIns(3,4,5)P3 (KD 1.6 nM) and PtdIns(3,4)P2 (KD 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P2. The binding of PtdIns(3,4,5)P3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Bα (PKBα) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P3, but did not affect the rate at which PDK1 activated a PKBα mutant lacking the PH domain in the absence of PtdIns(3,4,5)P3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBα to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein–PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P3 plays several roles in the PDK1-induced activation of PKBα. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P3 facilitates the rate at which it can activate PKB.

scholarly journals A genetically-encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

2019 ◽  
Author(s):  
David L. Prole ◽  
Colin W. Taylor
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cell Signalling ◽  
Intracellular Signalling ◽  
Live Cells ◽  
Green Fluorescent Proteins ◽  
Membrane Contact Sites ◽  
Green Fluorescent

AbstractBackgroundIntrabodies enable targeting of proteins in live cells, but it remains a huge task to generate specific intrabodies against the thousands of proteins in a proteome. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags.ResultsWe generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerizing or heterodimerizing modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria, and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane.ConclusionsThis toolkit greatly expands the utility of intrabodies for studying cell signalling in live cells.

scholarly journals Labeling RNAs in live cells using malachite green aptamer scaffolds as fluorescent probes

2017 ◽  
Author(s):  
V. Siddartha Yerramilli ◽  
Kyung Hyuk Kim
Keyword(s):  
Malachite Green ◽  
Tandem Repeats ◽  
Fluorescent Proteins ◽  
Rna Aptamers ◽  
Live Cells ◽  
High Background ◽  
Rna Molecules ◽  
High Background Noise ◽  
Fluorescent Tags ◽  
Malachite Green Aptamer

AbstractRNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamerfluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ~20 fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically-encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

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