An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

UNLABELLED: Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. IMPORTANCE: The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.

Femtomole SHAPE reveals regulatory structures in the authentic XMRV RNA genome.

Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and subfemtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between the packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.

Separations in poly(dimethylsiloxane) microchips coated with supported bilayer membranes.

Hybrid microchannels composed of poly(dimethylsiloxane) and glass were coated with supported bilayer membranes (SBMs) by the process of vesicle fusion. The electroosmotic mobility (mu(eo)) of zwitterionic, positively charged, and negatively charged phospholipid membranes was measured over a 4 h time to evaluate the stability of the coatings in an electric field. Coated microchips with a simple cross design were used to separate the fluorescent dyes fluorescein and Oregon Green. Migration time reproducibility was better than 5% RSD over 70 min of continuous separations. Separation of Oregon Green and fluorescein in channels coated with zwitterionic phosphatidylcholine (PC) membranes yielded efficiencies of 611,000 and 499,000 plates/m and a resolution of 2.4 within 2 s. Both zwitterionic and negatively charged membranes were used to separate peptide substrates from their phosphorylated analogues with efficiencies of 200,000-400,000 plates/m. Notably, separations of fluorescently labeled ABL substrate peptide from its phosphorylated counterpart were achieved using a high-salt physiological buffer with near-baseline resolution in 10 s. PC-coated devices were used to successfully separate enhanced green fluorescent protein (eGFP) from a fusion protein (eGFP-Crakl) with an efficiency of 358,000 and 278,000 plates/m respectively in less than 12 s. These SBM-based coatings may enable the separation of a broad range of analytes and may be ideal in biological applications for microfluidics.

Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis.

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.

Determination of sphingosine kinase activity for cellular signaling studies.

Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The KM of sphingosine kinase for its fluorescently labeled substrate was 38 +/- 18 microM with a Vmax of 0.4 +/- 0.2 microM/min and a kcat of 3900 s-1. Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.

Chemical analysis of single cells.

Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

Demonstration and use of nanoliter sampling of in vivo rat vitreous and vitreoretinal interface.

PURPOSE: An understanding of the chemical microenvironments at different locations on the retina can provide unique insights into retinal neurochemistry and pathology. The anatomical shape and the small volumes available from a spatially restricted volume greatly complicate these types of measurements. The aim of this study was to demonstrate an in vivo sampling system to probe different regions of the rat retina. METHODS: A low-flow push-pull perfusion probe was developed with concentric fused-silica capillaries. It was designed to fit through a 29-gauge needle for placement in the vitreous and at the vitreoretinal interface of the rat eye. Physiological saline was perfused and withdrawn through outer and inner capillaries, respectively, at flow rates between 10 and 50 nl/min. Samples of 500 nl were collected for amino acid analysis by capillary electrophoresis. Perfusion of a potent and selective inhibitor of the excitatory amino acid transporters was performed through the probe with the tip located 1-2 mm away from the optic nerve head. RESULTS: Ten amino acids were quantified from the perfusates of vitreous and the vitreoretinal interface. Sampling through time showed the use of this system to monitor retinal changes in these amino acids. The infusion of a transport protein antagonist shows a statistically significant increase in the glutamate concentration in collected samples when the probe tip is placed peripheral to but not over the optic nerve head. CONCLUSIONS: We demonstrate a new method for following neurochemical changes at the retina with spatial resolution. This in vivo method is widely applicable to the site-specific study of states of normal and dysfunctional retinal neurochemistry.

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis.

In vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates. Low-flow push-pull perfusion sampling is compatible with collecting small volume samples in a complicated matrix that are potentially difficult to separate. An efficient, sensitive, and rapid analysis of amino acids from in vivo perfusates of the vitreous is presented with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde (CBQCA) derivatitation and capillary electrophoresis (CE) separation with laser-induced fluorescence detection (LIF). Derivatization with CBQCA for up to 2 h provided high sensitivity and low detection limits at the nM level. Seventeen amino acids including D-serine (D-Ser) and D-aspartate (D-Asp) were resolved in less than 10 min. Importantly, D-Ser is separated from its enantiomeric pair. Characterization of vitreal amino acids with this assay technique will be useful for understanding ocular diseases and physiological mechanisms in vision.

Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis.

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.

Solid-phase immunoassay detection of peptides from complex matrices without a separation.

A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps. Both direct and indirect immunoassay methods are demonstrated; the indirect is more sensitive and features a 1 pmol detection limit of neat dynorphin A solutions. To further increase the immunoassay sensitivity, a novel capillary blotting system with hydrophilic membranes was designed where optimized sample volumes of 167 nL were deposited for each spot. The area-reduced blotting method shows a 1000-fold improved, 1.3 fmol spot(-1) detection limit of a dynorphin A diluted in a buffered solution of 150 mg L(-1) of casein. Low-flow push-pull perfusates with volumes of 1 microL sampled from the striatum of the rat were assayed for dynorphin A by the method of standard addition. The detection limit was estimated to be 1.9 fmol in the low-flow push-pull perfusates. These data demonstrate a solid-phase near infrared immunofluorescence strategy for the study of peptides directly blotted from chemically complex biological fluid matrices.

Demonstration of low flow push-pull perfusion.

Methods to follow in vivo chemical composition provide information regarding the processes of intercellular communication. There is a need for methods that provide chemical information from small volumes of the central nervous system (CNS) without sacrificing neurochemical recovery. One method that offers potential for providing such information is push-pull perfusion. In this study a low flow push-pull perfusion system is introduced that provides high (70-80%) in vitro recoveries. A concentric probe design is used with a 27-gauge stainless steel outer cannula for saline infusion and an inner fused silica capillary for fluid withdrawal. Flow rates of 10-50 nl/min were reliably generated and were well matched in vitro. Sampling was performed in the striatum of an anesthetized rat generating a 0.5 microl sample every 12 min. Capillary electrophoresis was used to determine glutamate levels in each sample; the basal level was found to be 1.97+/-0.70 microM. The method described was also demonstrated to deliver L-trans-pyrrolidine-2,4-dicarboxylic acid through the perfusion solution while sampling. Post-sampling histological analysis demonstrates little tissue disturbance to the sampled region. These data provide evidence that low flow push-pull method is a viable alternative for studying neurochemical signaling in the CNS.