Real-time monitoring of intracellular mRNA hybridization inside single living cells

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells.     

Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells

Monitoring gene expression is at the center of research for a wide variety of medical, biological, and biotechnological applications. Currently no method exists for true multiple gene expression monitoring inside of a single living cell that allows for the gene expression profile of the cell to be directly compared with another single living cell. By microinjecting multiple molecular beacons with different fluorophores inside of single breast carcinoma cells and monitoring with advanced fluorescent microscopy, the expression of multiple genes can be simultaneously monitored inside single living cells. Using ratiometric analysis as a basis for the measurements allows the different gene expression levels to be compared from cell to cell. Not only does this allow differentiation of individual mRNA expression levels between multiple single cells but it also allows for mRNA expression trend analysis at the single cell level.     

Rapid,High‐Throughput,and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire‐Incorporated and Pneumatic Pressure‐Driven Microdevice

Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real‐time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire‐incorporated and pneumatic pressure‐driven microdevice for rapid, high‐throughput, and direct MB delivery to human breast cancer MCF‐7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump‐associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire‐patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB‐coated nanowire array can be manipulated. The combination of the nanowire‐mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment.     

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.     

Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons

New techniques for single‐cell analysis enable new discoveries in gene expression and systems biology. Time‐dependent measurements on individual cells are necessary, yet the common single‐cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP‐E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP‐E is used to transfect a DNA‐based beacon that detects glyceraldehyde 3‐phosphate dehydrogenase and an RNA‐based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time‐dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.     

Molecular oxygen-sensitive fluorescent lipobeads for intracellular oxygen measurements in murine macrophages

Intracellular oxygen concentration is of primary importance in determining numerous physiological and pathological processes in biological systems. This paper describes the development and application of micrometer-sized oxygen-sensitive fluorescence lipobeads for intracellular measurements of molecular oxygen in J774 murine macrophages. A ruthenium diimine complex [Ru(bpy-pyr)(bpy)2]C12 (bpy = 2,2'-bipyridine, bpy-pyr = 4-(1"-pyrenyl)-2,2'-bipyridine) is used as the oxygen indicator. The indicator exhibits high chemical and photostability and high sensitivity to oxygen. The indicator molecules are immobilized in a phospholipid membrane that coats polystyrene microparticles. The fluorescence of the lipobeads is effectively quenched by molecular oxygen. The fluorescence intensity of the oxygen-sensitive lipobeads is 3 times higher in a nitrogenated solution than in an oxygenated solution. The lipobeads are internalized by murine macrophages through phagocytosis. They maintain their spectral properties for 24 h in living cells when the cells are stored in phosphate-buffered saline at pH 7.4. The photostability, reversibility, and effect of hypoxia, hyperoxia, and oxidative stress on the intracellular level of oxygen in J774 murine macrophages are described.     

Supercontinuum dynamically visualizes a dividing single cell

During cell division, various organelles behave dynamically. Visualization of these dynamic behaviors of organelles is a promising one step forward for understanding life at the molecular level. One- or two-photon excited fluorescence microscopy has so far been used for visualizing these cell dynamics. The fluorescent probe introduced into a living cell can visualize the spatial distribution of a target molecule in real time, enabling the tracing of cell dynamics at the molecular level. Introducing a fluorescent probe into a cell, however, may alter the physical and chemical conditions of the cell. Here we show a new method for direct (no need for staining cells) visualization of living cell processes with coherent anti-Stokes Raman scattering (CARS) spectroscopy. A new light source, supercontinuum generated from a photonic crystal fiber, has facilitated ultrabroadband (>3500 cm(-1)) multiplex CARS spectroscopy and imaging with high molecular specificity. Using this multiplex CARS technique, we have been successful in tracing the whole cell division process, the splitting of a mother cell into two daughter cells, appearance and disappearance of septum, and dynamic distribution changes of organelles consisting of lipid membrane. The supercontinuum has also facilitated simultaneous measurement of the CARS and two-photon excited fluorescence (TPEF) spectra, enabling what we call multiple nonlinear spectral imaging. Three-dimensional image reconstruction of a living cell with high speed is now possible to elucidate more detailed molecular-level dynamics inside a dividing living cell.     

Probing single molecules in single living cells

Single-molecule detection in single living cells has been achieved by using confocal fluorescence microscopy and externally tagged probe molecules. The intracellular background fluorescence is substantially higher than that in aqueous buffer, but this background is continuous and stable and does not significantly interfere with the measurement of single-molecule photon bursts. As a result, single-molecule data have been obtained on three types of fluorescent probes at spatially resolved locations (e.g., cytoplasm and nucleus) inside human HeLa cells. First, the iron transport protein transferrin labeled with tetramethylrhodamine undergoes rapid receptor-mediated endocytosis, and single transferrin molecules are detected inside living cells. Second, the cationic dye rhodamine 6G (R6G) enters cultured cells by a potential-driven process, and single R6G molecules are observed as intense photon bursts when they move in and out of the intracellular laser beam. Third, we report results on synthetic oligonucleotides that are tagged with a fluorescent dye and are taken up by living cells via a passive, nonendocytic pathway.     

Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells

Fluorescent particles are routinely used to probe biological processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biological environments. Here, we demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centres inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centres with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The experiments reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biological systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.     

Combination of differential interference contrast with prism-type total internal fluorescence microscope for direct observation of polyamidoamine dendrimer nanoparticle as a gene delivery in living human cells

A combined system of differential interference contrast (DIC) and total internal reflection fluorescence microscope (TIRFM) with a transmitted all-side polished dove prism was used for the direct monitoring of polyamidoamine (PAMAM) dendrimer nanoparticles as a gene delivery in living human embryonic kidney 293 (HEK 293) cells. The PAMAM dendrimer conjugated with fluorescein isothiocyanate (FITC) was used to form a fluorescent nanoparticle with the plasmid DNA (complexes) in order to directly monitor the entry of the complexes inside living cells. The DIC image provided precise information of the living HEK 293 cellular structures. Without moving the cell, the TIRFM images of the PAMAM nanoparticle-DNA complexes on the all-side polished dove prism provided precise information on the distance between the cell membrane and the complexes (< 200 nm) as well as the real-time localization of the individual complexes in the cells. The complexes were observed in cytosol within 4 h after incubating the cells with the complexes in Dulbecco's modified eagle's medium. The localization data of the complexes inside the cell obtained by TIRFM were reconfirmed using 3D confocal microscopy images of the complexes at the subcellular localization. These results suggest that the combined system of DIC and all-side polished dove prism-type TIRFM is a powerful tool for the direct real-time monitoring of the internalization and subcellular localization of nanoparticles carrying genes through a nonviral approach for gene therapy.     

Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase

We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.     

Label‐free and dynamic monitoring of cytotoxicity to the blood–brain barrier cells treated with nanometre copper oxide

A cytotoxicity study was conducted with a primary culture of the nervous system cells, including brain microvascular endothelial cells (BMECs) and astrocytes, which are important components of the blood–brain barrier. The real‐time cell analysis (RTCA) was used to determine the cytotoxicity of copper‐oxide nanoparticles (CuO NPs). The IC₅₀ values of CuO NPs in astrocytes and BMECs were determined by the RTCA at different exposure times and were used as base values for further research. DNA damage after exposure to CuO NPs for 3 and 24 h was assessed using comet assay at the IC₅₀ obtained from RTCA. The onset time of cytotoxicity induced by CuO NPs was 2 and 2–4 h post‐exposure in BMECs and astrocytes, respectively. Furthermore, the degree of cytotoxicity induced by exposure to CuO NPs for 24–48 h in the BMECs and astrocytes was similar. Treatment with CuO NPs at 1/2*IC₅₀ and 1/5*IC₅₀ for 3 h induced genotoxicity in both cells as assessed by a measurement of DNA damage, although no cytotoxicity was observed. However, significant DNA damage was observed at all concentrations of CuO NPs used in this study, when the treatment time was 24 h.Inspec keywords: biochemistry, blood, brain, cellular biophysics, copper compounds, DNA, molecular biophysics, nanoparticles, toxicologyOther keywords: label‐free cytotoxicity monitoring, dynamic cytotoxicity monitoring, blood‐brain barrier cells, nervous system cells, brain microvascular endothelial cells, astrocytes, real‐time cell analysis, copper‐oxide nanoparticles, comet assay, genotoxicity, DNA damage measurement, time 24 h to 48 h, time 2 h to 4 h, CuO     

Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton.     

An efficient and biocompatible fluorescence resonance energy transfer system based on lanthanide-doped nanoparticles

This work demonstrates an efficient and bio-friendly fluorescence resonance energy transfer (FRET) system based on lanthanide-doped inorganic nanoparticles. A facile aqueous route was used to synthesize the CePO(4):Tb nanorods with homogeneous colloidal dispersion, which emits a bright green light with a high quantum yield (～0.36) and a long fluorescence lifetime (～3.50 ms) upon UV excitation. Upon treatment of CePO(4):Tb with aqueous Rhodamine B (RhB), an efficient FRET occurs from the Tb(3+) to the RhB molecules, giving rise to well resolved and ratiometric emissions of donors and acceptors, respectively, with an energy transfer efficiency of up to 0.85. When incubated with HeLa cells at 37?°C, the CePO(4):Tb treated with RhB shows bright intracellular luminescence, indicating that it can be successfully internalized inside the cells and the FRET remains in the living cells. Moreover, the cytotoxic measurements demonstrate good biocompatibility and low cytotoxicity of our present FRET system. The advantages presented above including high quantum yield of donors, high energy transfer efficiency, ratiometric fluorescent emission and good biocompatibility, indicate the high potential of the CePO(4):Tb/RhB FRET system for monitoring biological events.     

Unimolecular catalytic DNA biosensor for amplified detection of L-histidine via an enzymatic recycling cleavage strategy

Fluorescence catalytic beacons have emerged as a general platform for sensing applications. However, almost all such sensing systems need covalent modification of the DNAzymes with fluorophore-quencher pairs, which may require elaborate design of the synthetic routes and many heavy and complicated synthetic steps and result in increased cost and lower synthesis yield. Here we report the construction of fluorescent cascadic catalytic beacons. With separation of the molecular recognition module from the signal reporter, this new design both avoids DNAzyme modifications and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification. This allows detection of L-histidine with high sensitivity (LOD = 200 nM) and excellent specificity. The proposed sensing system has also been used for detection of L-histidine in cellular homogenate with satisfactory results.     

Synthesis of water soluble quantum dots for monitoring carrier-DNA nanoparticles in plant cells

Fluorescent quantum dots (QDs) have shown great promise for use as biolabels in cell and animal biology and more recently in plant sciences. An important use of QDs is for monitoring the dynamics, intracellular trafficking, and fate of carrier-DNA nanocomplexes in cell transfection and potentially in plant transformation. In this study, a low cost aqueous procedure has been developed to efficiently prepare biocompatible QDs for monitoring nanoparticle-mediated gene transfer in conjunction with molecular breeding of Jatropha curcas. Water-soluble CdSe nanoparticles were synthesized by self-assembly using L-Cysteine as stabilizer and optimal synthesis scheme established by fluorescence spectroscopy. The QDs were used to label chitosan-DNA nanoparticles via electrostatic interaction and the resultant QD-labeled chitosan-DNA complexes were shown to have superior fluorescence properties with red shift of emission and absorption spectra relative to the CdSe QDs alone. This system is being explored as a superior alternative to Agrobacterium-mediated genetic transformation of Jatropha curcas cells. PCR amplification of the full length of the carried reporter gene (GFP) suggests that the DNA was not digested in Jatropha curcas cells transfected with CdSe/CS-DNA complexes. Furthermore, GFP gene expression in the transfected callus cells, as evidenced by fluorescence detection, suggests that the target DNA was integrated into the plant genome.     

Gold Nanoparticle‐Based Sensors Activated by External Radio Frequency Fields

A novel molecular beacon (a nanomachine) is constructed that can be actuated by a radio frequency (RF) field. The nanomachine consists of the following elements arranged in molecular beacon configuration: a gold nanoparticle that acts both as quencher for fluorescence and a localized heat source; one reporter fluorochrome, and; a piece of DNA as a hinge and recognition sequence. When the nanomachines are irradiated with a 3 GHz RF field the fluorescence signal increases due to melting of the stem of the molecular beacon. A control experiment, performed using molecular beacons synthesized by substituting the gold nanoparticle by an organic quencher, shows no increase in fluorescence signal when exposed to the RF field. It may therefore be concluded that the increased fluorescence for the gold nanoparticle‐conjugated nanomachines is not due to bulk heating of the solution, but is caused by the presence of the gold nanoparticles and their interaction with the RF field; however, existing models for heating of gold nanoparticles in a RF field are unable to explain the experimental results. Due to the biocompatibility of the construct and RF treatment, the nanomachines may possibly be used inside living cells. In a separate experiment a substantial increase in the dielectric losses can be detected in a RF waveguide setup coupled to a microfluidic channel when gold nanoparticles are added to a low RF loss liquid. This work sheds some light on RF heating of gold nanoparticles, which is a subject of significant controversy in the literature.     

Nuclease resistance of telomere-like oligonucleotides monitored in live cells by fluorescence anisotropy imaging

Telomeres carry important biological functions such as the protection of chromosomes. In this paper, we have developed a fluorescence anisotropy imaging system for monitoring DNA digestion inside live cells. The nuclease-resistant capability of telomere-like ssDNAs in nuclei of human breast cancer cells is studied. We found that those oligonucleotides were clearly more stable than regular DNA sequences during the time course of the experiments. We conclude that the G-quadruplex structure of the telomere-like ssDNA makes it inherently more stable in intracellular environments than non-G-quadruplex structures. This will help us understand why the G-quadruplex forming telomere sequences were adopted by almost all eukaryotic cells to protect the ends of chromosomes. This is the first time such a phenomenon was observed in live cells. Our fluorescence anisotropy imaging provides an efficient way to directly monitor DNA digestion in any region of live cells in real time, providing insights into many important and related intracellular processes.