High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670...     

Tracking the Dynamic Folding and Unfolding of RNA G‐Quadruplexes in Live Cells

Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1 , for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes.     

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.     

Fluorescence imaging of potassium ions in living cells using a fluorescent probe based on a thrombin binding aptamer-peptide conjugate

When a biotinylated FRET probe based on a peptide-thrombin binding aptamer conjugate was introduced together with streptavidin and biotinylated nuclear export signal peptide into HeLa cells, the resulting ternary complex enabled visualization of K(+) concentration changes in the cell.     

Multiplexed imaging detection of live cell intracellular changes in early apoptosis with aggregation-induced emission fluorogens

Apoptosis is an important process for maintaining tissue homeostasis and eliminating abnormal cells in multicellular organisms. Abnormality in apoptosis often leads to severe diseases such as cancers. Better understanding of its mechanisms and processes is therefore important. Accompanying molecular biology events of apoptosis is a series of cellular morphology changes: nucleus condensation, cell shrinkage and rounding, cell surface blebbing, dynamic blebbing, apoptotic membrane protrusions and nucleus fragmentations and finally, the formation and release of apoptotic bodies. It is difficult to detect cellular changes in the early phase of apoptosis due to the subtle changes at this phase. In the current study, we induced apoptosis in HeLa cells with H₂O₂ and used nuclear dye Hoechst 33258, mitochondria, lysosome and cytoplasmic protein specific aggregation-induced emission fluorogens (AIEgens), TPE-Ph-In, 2M-DABS and BSPOTPE to successfully perform live cell multiplexed imaging to investigate early apoptosis cellular events. We showed the gradual dissipation of mitochondria membrane potential until it is nondetectable by TPE-Ph-In. Increased mitophagy detected by TPE-Ph-In and 2M-DABS, condensed nucleus detected by Hoechst 33258, increased permeability and/or reduced integrity of nuclear membrane, and increased intracellular vesicles detected by 2M-DABS are some of the early events of apoptosis.     

Viral RNA export

Viruses can express intron-containing and intronless mRNAs, which are exported by alternative pathways. The study of the nuclear export of these unconventional mRNAs can provide key insights into the normal process of nuclear export and the alternative pathways provide an attractive target for the development of specific antiviral therapies.     

A Multifunctional Peptide‐Conjugated AIEgen for Efficient and Sequential Targeted Gene Delivery into the Nucleus

Gene therapy has immense potential as a therapeutic approach to serious diseases. However, efficient delivery and real‐time tracking of gene therapeutic agents have not been solved well for successful gene‐based therapeutics. Herein we present a versatile gene‐delivery strategy for efficient and visualized delivery of therapeutic genes into the targeted nucleus. We developed an integrin‐targeted, cell‐permeable, and nucleocytoplasmic trafficking peptide‐conjugated AIEgen named T_DNCP for the efficient and sequential targeted delivery of an antisense single‐stranded DNA oligonucleotide (ASO) and tracking of the delivery process into the nucleus. As compared with T_DNCP/siRNA‐NPs (siRNA functions mainly in the cytoplasm), T_DNCP/ASO‐NPs (ASO functions mainly in the nucleus) exhibited a better interference effect, which further indicates that T_DNCP is a nucleus‐targeting vector. Moreover, T_DNCP/ASO‐NPs showed a favorable tumor‐suppressive effect in vivo.     

RNA Hairpin Folding in the Crowded Cell

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.     

Subcellular Localization of Sulfonated Tetraphenyl Porphines in Colon Carcinoma Cells by Spectrally Resolved Imaging

Subcellular localization of the dye, 5,10,15,20-tetra(4-sul-fonatophenyl)porphine (TPPS₄) and the more hydrophobic dye, 5,10,15,20-tetra(1-sulfonatophenyl)porphine (TPPS₁), in murine colon carcinoma cells was studied by spectrally resolved imaging (SRI) combined with image processing techniques. Spectrally resolved imaging enabled the acquisition of multipixel fluorescence spectra (>10⁴) from a single cell. Demarcation of specific localization sites and segregation of the irrelevant fluorescence were based on the pixel spectra and by operating the functions of spectral similarity mapping (SSM), principal component analysis (PCA) and spectral classification. The SRI revealed the fine details of the photochemical process that clarify some aspects of subcellular damage. The SRI depicted the differences between TPPS₄ and TPPS, with respect to their initial localization and their fate at the end of the photochemical effect. The dye TPPS₄ was localized initially in lysosomal vesicles, and upon irradiation fluorescence was seen in the nucleus as well as in vesicles. Some of the vesicles were closely related to the nucleus, as resolved by SSM, PCA and spectral classification. Additional light exposure stimulated relocalization of TPPS₄ into the nucleus as well as into the nucleolus, which was clearly depicted by SSM and PCA. Spectral classification showed a third, weak residual cytoplasmic array around the nucleus. The dye TPPS, concentrated in a Golgi-like complex and was resolved in the nuclear envelope and in small vesicles: it was not redistributed into other compartments upon photosensitization. Serum supplementation to the incubation media of colon carcinoma cells treated with TPPS₄ or TPPS, did not change the localization patterns. Pixel spectra of the two dyes in the cells showed spectral shifts and expanded shoulders due to microenvironmental effects. Thus, the chemical nature of the sulfonated phenyl porphines, and not their interaction with serum proteins, was the main determinant of their binding to the lysosomes, nucleus, nucleolus, nuclear envelope or Golgi.     

Direct detection of RNAs in living cells using peptide-inserted Renilla luciferase

In this study, non-engineered RNAs were detected in living cells using bioluminescence. Two types of probe were utilized: a peptide inserted RLuc (PI-RLuc) probe and a split-RNA probe. Incorporation of the PI-RLuc and split-RNA probes enabled the direct detection of RNA introduced into living cells.     

Helical cyclic pentapeptides constrain HIV-1 Rev peptide for enhanced RNA binding

HIV-1 Rev is a 116 residue transporter protein that enters the host cell nucleus and uses its 17 amino acid segment (Rev_34–50) to bind and capture a specific piece of RNA, the Rev Response Element (RRE), for transport to the cytoplasm. This is critical for HIV replication. In isolation, Rev_34–50 shows negligible structure in water, but is alpha helical in a mixture of water and 2,2,2-trifluoroethanol (TFE) or when bound to RRE. Here we report that helix-constrained cyclic pentapeptides, either appended to the N-terminus or incorporated within Rev_34–50, are efficient helix nucleators in water. They induce up to 90% alpha helicity for isolated Rev peptides in water and confer high RNA-binding affinity.     

Tumor-Triggered Disassembly of a Multiple-Agent-Therapy Probe for Efficient Cellular Internalization

Integration of multiple agent therapy (MAT) into one probe is promising for improving therapeutic efficiency for cancer treatment. However, MAT probe, if entering the cell as a whole, may not be optimal for each therapeutic agent (with different physicochemical properties), to achieve their best performance, hindering strategy optimization. A peptide-conjugated-AIEgen (FC-PyTPA) is presented: upon loading with siRNA, it self-assembles into FC_siRNA-PyTPA. When approaching the region near tumor cells, FC_siRNA-PyTPA responds to extracellular MMP-2 and is cleaved into FC_siRNA and PyTPA. The former enters cells mainly by macropinocytosis and the latter is internalized into cells mainly through caveolae-mediated endocytosis. This two-part strategy greatly improves the internalization efficiency of each individual therapeutic agent. Inside the cell, self-assembly of nanofiber precursor F, gene interference of C_siRNA, and ROS production of PyTPA are activated to inhibit tumor growth.     

Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability

The delivery of free molecules into the cytoplasm and nucleus by using arginine‐rich cell‐penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine‐rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full‐length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP–GFP conjugates were obtained by using azido‐functionalized CPPs and an alkyne‐functionalized GFP. Our findings reveal that the cyclic‐CPP–GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full‐length proteins into live cells.     

Self-Assembled Peptidyl Aggregates for the Fluorogenic Recognition of Mitochondrial DNA G-Quadruplexes

The selective monitoring of G-quadruplex (G4) structures in living cells is important to elucidate their functions and reveal their value as diagnostic or therapeutic targets. Here we report a fluorogenic probe ( CV2 ) able to selectively light-up parallel G4 DNA over antiparallel topologies. CV2 was constructed by conjugating the excimer-forming CV dye with a peptide sequence (l -Arg-l -Gly-glutaric acid) that specifically recognizes G4s. CV2 forms self-assembled, red excimer-emitting nanoaggregates in aqueous media, but specific binding to G4s triggers its disassembly into rigidified monomeric dyes, leading to a dramatic fluorescence enhancement. Moreover, selective permeation of CV2 stains G4s in mitochondria over the nucleus. CV2 was employed for tracking the folding and unfolding of G4s in living cells, and for monitoring mitochondrial DNA (mtDNA) damage. These properties make CV2 appealing to investigate the possible roles of mtDNA G4s in diseases that involve mitochondrial dysfunction.     

Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell‐Nucleus Targeting

Functionalization of monodisperse superparamagnetic magnetite (Fe₃O₄) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus‐targeting Fe₃O₄ nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS‐coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non‐aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications.     

Brightness through Local Constraint—LNA‐Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking

Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild‐type organisms. We developed nuclease‐resistant DNA forced intercalation (FIT) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic acid (LNA) unit serves to introduce a local constraint. This closes fluorescence decay channels and thereby increases the brightness of the probe–target duplexes. As few as two probes were sufficient to enable the tracking of oskar mRNPs in wild‐type living Drosophila melanogaster oocytes.     

Intracellular localization of PNA in human cells upon its introduction by electroporation

Peptide nucleic acid (PNA) is one of the most useful DNA analogs in a wide variety of gene analysis in human cells. In order to exhibit its maximal functions, PNA must be localized to a desired place (e.g., nucleus, cytoplasm and other organelles). Here, we introduced PNAs into HeLa cells by electroporation and examined their localization at various time points. The PNA which binds to the mitochondrial COII gene was initially accumulated in the nucleus, and thereafter mostly transferred to cytoplasm. This time-dependent intracellular localization of PNA is ascribed to the breakdown of the nuclear envelope in the cell division. On the other hand, another PNA that binds to telomere repeat sequence mostly remained in the nucleus, even after the cell division occurred. The retention of this PNA in the nucleus was further enhanced when it was conjugated with Cy3.