Deirdre R. Meldrum

Stability of DNA Origami Nanoarrays in Cell Lysate

Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami-lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms.

Application of synthetic biology in cyanobacteria and algae

Cyanobacteria and algae are becoming increasingly attractive cell factories for producing renewable biofuels and chemicals due to their ability to capture solar energy and CO2 and their relatively simple genetic background for genetic manipulation. Increasing research efforts from the synthetic biology approach have been made in recent years to modify cyanobacteria and algae for various biotechnological applications. In this article, we critically review recent progresses in developing genetic tools for characterizing or manipulating cyanobacteria and algae, the applications of genetically modified strains for synthesizing renewable products such as biofuels and chemicals. In addition, the emergent challenges in the development and application of synthetic biology for cyanobacteria and algae are also discussed.

An FRET-based ratiometric chemosensor for in vitro cellular fluorescence analyses of pH

Ratiometric fluorescence sensing is an important technique for precise and quantitative analysis of biological events occurring under complex conditions by simultaneously recording fluorescence intensities at two wavelengths and calculating their ratios. Herein, we design a ratiometric chemosensor for pH that is based on photo-induced electron transfer (PET) and binding-induced modulation of fluorescence resonance energy transfer (FRET) mechanisms. This ratiometric chemosensor was constructed by introduction of a pH-insensitive coumarin fluorophore as an FRET donor into a pH-sensitive amino-naphthalimide derivative as the FRET acceptor. The sensor exhibited clear dual-mission signal changes in blue and green spectral windows upon pH changes. The pH sensor was applied for not only measuring cellular pH, but also for visualizing stimulus-responsive changes of intracellular pH values.

Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2.

(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO2 photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO2. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO2.

A New Highly Selective Fluorescent K+ Sensor

We describe the synthesis, properties, and application of a new fluorescent potassium chemosensor, KS2, for K(+) sensing and imaging in live cells. By virtue of a strong electron-withdrawing group, 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF), with a triazacryptand ligand, the new sensor can respond to K(+) up to 1.6 M. This is the first highly selective intracellular sensor suitable for sensing K(+) over a broad and high concentration range. Confocal fluorescence microscopy has established the utility of KS2 for live-cell K(+) detection. The application of KS2 combined with other sensors will be of great benefit for investigating cellular metabolism, detecting and diagnosing diseases including cancer, and monitoring responses to therapy.

A series of naphthalimide derivatives as intra and extracellular pH sensors

A series of new naphthalimide derivatives were synthesized and studied. Three of the materials (SM1, SM2, and SM3) possess methacrylate(s) moieties as pH sensor monomers, enabling these compounds to be polymerized with other monomers for thin film preparation for extracellular pH sensing. Herein, poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (PHEMA-co-PAM) was chosen as the polymer matrix. Structure influences on pH responses and pK(a) values were studied. The film P3 composed of the sensing moiety SM3 has a pK(a) close to the usual biological environmental pH of approximately 7. It was used as an extracellular pH sensor to monitor pH change during the metabolism of prokaryotic Escherichia coli (E. coil). On the other hand, the three sensor monomers are new intracellular biomarkers to sense lysosomes of eukaryotic cells since (1) their pK(a) values are in a range of 5.9-6.8; (2) their emission intensities at acidic conditions (such as at pH 5) are much stronger than those at a neutral condition of pH 7; (3) lysosomes range in size from 0.1 to 1.2 mum in diameter with pH ranging from 4.5 to 5.0, which is much more acidic than the pH value of the cytoplasm (usually with a pH value of approximately 7.2); and (4) the acidity of lysosomes enables a protonation of the amino groups of the pH probes making the sensors emit brightly in acidic organelles by inhibiting the photo-induced electron transfer from the amino groups to the fluorophores. Lysosome sensing was demonstrated using live human brain glioblastoma U87MG cell line, human cervical cancer HeLa cell line, and human esophagus premalignant CP-A and CP-D cell lines by observations of small acidic spherical organelles (lysosomes) and significant colocalizations (82-95%) of the sensors with a commercially available lysosome-selective staining probe LysoTracker Red under confocal fluorescence microscopy.

Automated Selection and Placement of Single Cells Using Vision-Based Feedback Control

We present a robotic manipulation system for automated selection and transfer of individual living cells to analysis locations. We begin with a commonly used cell transfer technique using glass capillary micropipettes to aspirate and release living cells suspended in liquid growth media. Using vision-based feedback and closed-loop process control, two individual three-axis robotic stages position the micropipette tip in proximity to the cell of interest. The cell is aspirated and the tip is moved to a target location where the cell is dispensed. Computer vision is used to monitor and inspect the success of the dispensing process. In our initial application, the target cell destination is a microwell etched in a fused silica substrate. The system offers a robust and flexible technology for cell selection and manipulation. Applications for this technology include embryonic stem cells transfer, blastomere biopsy, cell patterning, and cell surgery.

Using fluorine-containing amphiphilic random copolymers to manipulate the quantum yields of aggregation-induced emission fluorophores in aqueous solutions and the use of these polymers for fluorescent bioimaging

Two new series of aggregation-induced emission (AIE) fluorophore-containing amphiphilic copolymers possessing the segments of a monomeric AIE fluorophore, N-(2-hydroxypropyl)methacrylamide (HPMA), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (MATMA), and/or 2,2,2-trifluoroethyl methacrylate (TFEMA) were synthesized. Photophysical properties were investigated using UV-Vis absorbance and fluorescence spectrofluorometry. The increases of molar fractions of the hydrophobic AIE fluorophores and/or the trifluoroethyl moieties result in the higher quantum yields of the AIE fluorophores in the polymers. Using 1-mol% of AIE fluorophores with the tuning of molar fractions of TFEMA, 40% quantum yield was achieved, whereas only less than 10% quantum yield was obtained for the polymers without the TFEMA segments. The quantum yield difference indicates the importance of the fluorine segments for getting high quantum yields of the AIE fluorophores. These polymers were explored for fluorescent bioimaging using human brain glioblastoma U87MG and human esophagus premalignant CP-A cell lines. All the polymers are cell permeable and located in the cellular cytoplasma area. Cellular uptake was demonstrated to be through endocytosis, which is time and energy dependent. The polymers are non-cytotoxic to the two cell lines. Because the polymers contain (19)F segments, we studied the spin-lattice relaxation time (T1) and spin-spin relaxation time (T2) of these polymers. T1 and T2 are the two important parameters for the evaluations of the capacity of these polymers for further applications in (19)F magnetic resonance imaging ((19)F MRI). Structure influence on T1 and T2, especially for T2, was observed. These new multifunctional materials are the first series of fluorinated polymers with AIE fluorophores for bioapplications.

A Highly Selective Mitochondria-Targeting Fluorescent K+ Sensor

Regulation of intracellular potassium (K(+) ) concentration plays a key role in metabolic processes. So far, only a few intracellular K(+) sensors have been developed. The highly selective fluorescent K(+) sensor KS6 for monitoring K(+) ion dynamics in mitochondria was produced by coupling triphenylphosphonium, borondipyrromethene (BODIPY), and triazacryptand (TAC). KS6 shows a good response to K(+) in the range 30-500 mM, a large dynamic range (Fmax /F0 ≈130), high brightness (ϕf =14.4 % at 150 mM of K(+) ), and insensitivity to both pH in the range 5.5-9.0 and other metal ions under physiological conditions. Colocalization tests of KS6 with MitoTracker Green confirmed its predominant localization in the mitochondria of HeLa and U87MG cells. K(+) efflux/influx in the mitochondria was observed upon stimulation with ionophores, nigericin, or ionomycin. KS6 is thus a highly selective semiquantitative K(+) sensor suitable for the study of mitochondrial potassium flux in live cells.

K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells.

P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the mitochondrial potassium pool was mobilized in a P2X7 signaling, and ATP dose-dependent manner, suggesting a role for mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further, intracellular calcium chelation with BAPTA-AM indicated that P2X7-induced potassium depletion was independent of calcium mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.