Imaging of RNAs in live cells with spectrally diverse small molecule fluorophores.
Ontology highlight
ABSTRACT:
SUBMITTER: Carrocci TJ
PROVIDER: S-EPMC3873762 | biostudies-literature | 2014 Jan
REPOSITORIES: biostudies-literature
Publications
Imaging of RNAs in live cells with spectrally diverse small molecule fluorophores.
Carrocci Tucker J TJ Hoskins Aaron A AA
The Analyst 20131101 1
We have developed new protein-based tags for use in imaging of RNAs in living cells. These tags are based on the MS2 phage coat protein fused to either E. coli dihydrofolate reductase or the SNAP tag. Labeled RNAs can be visualized with fluorophores spanning the visible spectra range. ...[more]
Similar Datasets
Project description:Bright, long-lasting organic fluorophores enable a broad range of imaging applications. "Self-healing" fluorophores, in which intra-molecularly linked protective agents quench photo-induced reactive species, exhibit both enhanced photostability and biological compatibility. However, the self-healing strategy has yet to achieve its predicted potential, particularly in the presence of ambient oxygen where live-cell imaging studies must often be performed. To identify key bottlenecks in this technology that can be used to guide further engineering developments, we synthesized a series of Cy5 derivatives linked to the protective agent cyclooctatetraene (COT) and examined the photophysical mechanisms curtailing their performance. The data obtained reveal that the photostability of self-healing fluorophores is limited by reactivity of the COT protective agent. The addition of electron withdrawing substituents to COT reduced its susceptibility to reactions with molecular oxygen and the fluorophore to which it is attached and increased its capacity to participate in triplet energy transfer. Exploiting these insights, we designed and synthesized a suite of modified COT-fluorophores spanning the visible spectrum that exhibited markedly increased intra-molecular photostabilization. Under ambient oxygen conditions, the photostability of Cy3 and Cy5 fluorophore derivatives increased by 3- and 9-fold in vitro and by 2- and 6-fold in living cells, respectively. We further show that this approach can improve a silicon rhodamine fluorophore. These findings offer a clear strategy for achieving the full potential of the self-healing strategy and its application to the gamut of fluorophore species commonly used for biomedical imaging.
| S-EPMC5299821 | biostudies-literature
Project description:Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.
| S-EPMC6273459 | biostudies-literature
Project description:To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.
| S-EPMC4297622 | biostudies-literature
Project description:Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
| S-EPMC4344395 | biostudies-literature
Project description:Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.
| S-EPMC4142238 | biostudies-literature
Project description:Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.
| S-EPMC5167155 | biostudies-literature
Project description:CD38 is a type II transmembrane glycoprotein with multiple functions. It acts as an ecto-enzyme as well as a receptor. The enzymatic activity catalyzes the formation of two potent Ca(2+) releasing agents: cyclic adenosine diphosphate ribose (cADPR) from nicotinamide adenine dinucleotide (NAD) and nicotinic acid adenine dinucleotide phosphate (NAADP) from NAD phosphate (NADP). The receptor function of CD38 leads to the phosphorylation of intracellular signaling proteins and the up-regulation of cytokine production in immune cells. These two functions of CD38 underlie its involvement in various biological processes, such as hormone secretion, immune cell differentiation, and immune responses. Clinically, CD38 is used as a negative prognosis marker for chronic lymphatic leukemia (CLL). However, a clear molecular understanding of CD38's role in physiology and pathology is still lacking. To facilitate the study of CD38 at cellular and molecular levels, here we report a mechanism-based method for fluorescently labeling CD38 on live cells. This labeling method does not interfere with the receptor function of CD38 and the downstream signaling. The labeling method is thus a useful tool to study the receptor function of CD38 in live cells. In addition, since the mechanism-based labeling also inhibits the enzymatic activity of CD38, it should be useful for dissecting the receptor function of CD38 without interference from its enzyme function in complicated biological processes.
| S-EPMC2644825 | biostudies-literature
Project description:Though high brightness and biocompatible small NIR-II dyes are highly desirable in clinical or translational cancer research, their fluorescent cores are relatively limited and their synthetic processes are somewhat complicated. Herein, we have explored the design and synthesis of novel NIR-II fluorescent materials (H1) without tedious chromatographic isolation with improved fluorescence performance (QY ≈ 2%) by introducing 2-amino 9,9-dialkyl-substituted fluorene as a donor into the backbone. Several types of water-soluble and biocompatible NIR-II probes: SXH, SDH, and H1 NPs were constructed via different chemical strategies based on H1, and then their potential to be used in in vivo tumor imaging and image-guided surgery in the NIR-II region was explored. High levels of uptake were obtained for both passive and active tumor targeting probes SXH and SDH. Furthermore, high resolution imaging of blood vessels on tumors and the whole body of living mice using H1 NPs for the first time has demonstrated precise NIR-II image-guided sentinel lymph node (SLN) surgery.
| S-EPMC5418643 | biostudies-literature
Project description:A general approach for the sequential labeling of fusion proteins of O(6)-alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.
| S-EPMC454197 | biostudies-literature
Project description:We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.
| S-EPMC2688641 | biostudies-literature