Trapping of proteins under physiological conditions in a nanopipette.
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DNA↗
Biomedical subjects
David Klenerman
Publications and source records attributed to David Klenerman.
Scanning ion conductance microscopy reveals how a functional renal epithelial monolayer maintains its integrity.
BACKGROUND: To function as a transport barrier a renal tubule epithelial monolayer needs to maintain its integrity when, sudden hypertonic stress causes cell shrinkage, new cells are added, or cells in the monolayer die. However, the mechanism used to achieve this is largely unknown. Scanning ion conductance microscopy (SICM) has been shown to be suitable for imaging the surface of live renal cells with high topographic resolution, and can be used to elucidate how a functional renal epithelial monolayer maintains its integrity. METHODS: SICM was used for high spatial resolution topographic imaging of Xenopuslaevis renal epithelial A6 cells cultured on membrane filter inserts. RESULTS: The SICM images of A6 cells showed that the epithelial monolayer maintains its integrity under hypertonic stress, and during cell division and death. Sequential SICM topographic images revealed detailed structural changes and their time course for these protective processes, which involve highly cooperative cell movement. Some "balloon-like" structures were observed at susceptible tight junction regions, which were proposed to help cell maintaining the monolayer permeability integrity. CONCLUSION: SICM is a powerful tool for research on living renal epithelial cells, and has been used to elucidate how a functional epithelial monolayer maintains its integrity. Using this technique we have observed that during hypertonic stress and regeneration, an organized sequence of events protect the loss of integrity of monolayer so that tight junctions and cell-cell contact are maintained and disruption to the function of whole monolayer is prevented.
Animals↗
Individual molecules of dye-labeled DNA act as a reversible two-color switch upon application of an electric field.
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Color↗
Influence of the foundation layer on the layer-by-layer assembly of poly-L-lysine and poly(styrenesulfonate) and its usage in the fabrication of 3D microscale features.
The layer-by-layer (LBL) assembly of a polypeptide, poly-L-lysine (PLL), with poly(styrenesulfonate) sodium salt (PSS) on flat template-stripped gold (TSG) surfaces precoated with a self-assembled monolayer of alkanethiols terminated with positive (pyridinium), negative (carboxylic acid), and neutral [hexa(ethylene glycol)] groups is investigated. Both the topography and the rate of film thickness growth are found to be strongly dependent on the initial surface foundation layer. LBL assembly of PLL and PSS on patterned TSG surfaces produced by micro contact printing leads to structurally distinct microscale features, including pillars, ridges, and wells, whose height can be controlled with nanometer precision.
Membranes, Artificial↗
Measuring single-molecule nucleic acid dynamics in solution by two-color filtered ratiometric fluorescence correlation spectroscopy.
This work presents a general method for determining single-molecule intramolecular dynamics in biomolecules by using a reporter fluorophore, whose fluorescence is quenched or partially quenched as a result of intramolecular motion, and a remote observer fluorophore. These fluorophores were excited independently with two different lasers, and the ratio of the two fluorophores' fluorescence was calculated. The time-varying ratio was then filtered to reduce contributions from molecules outside the overlapped laser volume and then correlated. The rates of opening and closing of a DNA hairpin were measured by using both fluorescence correlation spectroscopy and this method for comparison. We found at 50 pM, where molecules were studied one by one as they diffused through the probe volume, we obtained accurate opening and closing rates and could also measure dynamic heterogeneity. To demonstrate applicability to a more complex biological molecule we then probed intramolecular motions in the dimer of a human telomerase RNA fragment (hTR(380-444)), in the presence of an excess of monomer. The motion was found to occur on the time scale of 180-750 micros and slowed with increasing magnesium ion concentration. Blocking experiments using complementary oligonucleotides suggested that the motion involves substantial changes in dimer tertiary structure. This method appears to be a general method for selectively studying intramolecular motion in large biomolecules or complexes.
Base Sequence↗
Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution.
We have used two-color fluorescence coincidence detection to directly count individual protein-antibody complexes of protein G or herpes simplex virus labeled with one or more red- and blue-excited antibodies. This allowed quantitative measurement of the concentration of the protein-antibody complexes over 3 orders of magnitude down to the femtomolar level. Single molecule measurements in diluted serum are also possible. The sample preparation is simple, takes place in solution, and requires no separation. Both the antibody affinity and complex dissociation rate are important in determining the sensitivity of the method. At present, the sensitivity limit of 50 fM is determined by the encounter rate of the labeled analyte with the probe volume. This method can be used to detect and quantitate proteins and to measure the stoichiometry, equilibrium constant, and dissociation rate of protein-protein complexes at low concentrations.
Animals↗
An addressable antibody nanoarray produced on a nanostructured surface.
The ability to address specific nanoscale features is required to produce diverse biological nanoarrays or perform local assembly using biological building blocks and is an important unsolved problem in nanotechnology. In this work, we describe the use of a novel nanofabricated gold surface, with regions of distinct topography and chemical functionalities, to solve this problem. First, nanoarrays of IgG antibodies were produced by selective immobilization in nanoholes on the surface. The smallest feature size was determined by the hole size (fwhm 90 nm) and not surface diffusion. Using holes of 300 nm diameter, we selectively addressed specific features in the array by nanopipet delivery of a functional antibody, anti-IgG. To our knowledge, this is the first example of addressing specific biologically functional features on a surface at the nanoscale.
3-Mercaptopropionic Acid↗
The use of scanning ion conductance microscopy to image A6 cells.
BACKGROUND: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.
Animals↗
Frequency and voltage dependence of the dielectrophoretic trapping of short lengths of DNA and dCTP in a nanopipette.
The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.
DNA↗
Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.
We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 +/- 0.4 kcal.mol-1 and -52.3 +/- 1.4 cal.mol-1.K-1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 +/- 0.2 kcal.mol-1 and -23.0 +/- 0.8 cal.mol-1.K-1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 kBT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.
Base Composition↗
Identification of a new RNA.RNA interaction site for human telomerase RNA (hTR): structural implications for hTR accumulation and a dyskeratosis congenita point mutation.
The enzyme telomerase is a ribonucleoprotein that has a critical role in the maintenance of stable telomeres in organisms that possess linear chromosomes. Using a recently developed single molecule fluorescence coincidence method, we have studied the RNA component of telomerase (hTR) and directly observed multimerisation of hTR in solution. RNA mutagenesis and blocking oligonucleotides were employed to identify the single-stranded internal loop J7b/8a as an important and specific hTR.hTR interaction site. This observation was confirmed by studies on a model RNA fragment (hTR(380-444)), comprising part of the H/ACA domain, the internal loop J7b/8a and the CR7 domain, that was found to dimerise. Substitution mutagenesis within the proposed RNA.RNA interaction site of hTR(380-444) resulted in a loss of dimerisation potential and insertion of the dyskeratosis congenita mutation C408G led to a significant reduction in dimer formation. Together, these results suggest that this RNA.RNA interaction site may be functionally relevant.
Binding Sites↗
Multicomponent submicron features of biomolecules created by voltage controlled deposition from a nanopipet.
We have used a nanopipet as a nanopen to locally and controllably deposit complex biomolecules, including antibodies and DNA, onto a surface to create multicomponent and functional submicron features. Key advantages of this method are that the biomolecules are always in solution and the applied voltage provides fine control of the delivery down to the single molecule level. Two consecutive cycles of deposition, to produce spatially varying features with different biological properties, were demonstrated with fluorescently labeled antibodies or biotin. This approach combines "top-down" fabrication, using the nanopen for local application, and "bottom-up" fabrication, using molecular recognition for self-assembly at defined positions, and opens up new possibilities in nanotechnology.
Animals↗
Vibrational spectroscopy of mechanically compressed monolayers.
Sum-frequency spectroscopy has been used to investigate the behavior of self-assembled monolayers in a solid-solid contact. Various alkanethiol layers on gold were observed before, during, and after compression to 660 MPa against a sapphire counterface. Well-ordered layers that differ only in the length of their alkane tails (C(8) versus C(18)) behave similarly. In contrast, defective and partly melted monolayers are more sensitive to stress than are their well-ordered analogues, and they are more prone to irreversible changes. In all cases, the intensity of methyl C-H stretching modes decreases with applied pressure, indicating a loss of net orientational order among the terminal methyl groups. The magnitude of this effect in well-ordered layers can be compared with the theoretical sensitivity of the resonant sum-frequency signal to molecular orientation. On these grounds, an increased population of terminal gauche conformers is identified as the disordering mechanism under pressure.
Journal Article↗
Dynamic assembly of surface structures in living cells.
Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary "building blocks" for the assembly of specialized structures on the cell surface.
Animals↗
Kinetics of unfolding the human telomeric DNA quadruplex using a PNA trap.
The kinetics of opening of the DNA quadruplex formed by the human telomeric repeat have been investigated using real-time fluorescence resonance energy transfer (FRET) measurements with a peptide nucleic acid (PNA) trap. It has been found that this opening is zero-order with respect to PNA, indicating that the initial step is a rate-limiting internal rearrangement of the quadruplex. A study of the temperature dependence of the rate of quadruplex opening was performed and the activation energy of the process estimated to be 98 +/- 8 kJ mol(-1).
Circular Dichroism↗
Ultrasensitive coincidence fluorescence detection of single DNA molecules.
We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.
DNA↗
Surface-sensitive molecular information on polymers from UV-resonance Raman spectroscopy.
Ultraviolet resonance Raman spectroscopy (UV-RRS) has been applied to a series of samples of poly(ethylene 2,6-naphthalene dicarboxylate) (PEN) and poly(ethylene terephthalate) (PET) of varying thickness. The spectra demonstrate that under resonance conditions, when absorption is very strong, only a very thin top layer of the sample is probed (hundreds of nanometers range). This allows probing molecular vibrational spectra of the top layer of the sample, with a surface-resolution at least an order of magnitude better than in the case of normal non-resonance Raman spectroscopy and using a microscope.
Crystallography↗
Writing with DNA and protein using a nanopipet for controlled delivery.
We present a new, general method for the controlled deposition of biological molecules on surfaces, based on a nanopipet operating in ionic solution. The potential applied to the pipet tip controls the flux of biological molecules from the pipet, allowing fine control of the delivery rate. We used the ion current to control the distance of the pipet from the surface of a glass slide and deposited the fluorescently labeled DNA or protein G at a defined location onto the surface. Features of 830 nm size were obtained by depositing the biotinylated DNA onto a streptavidin surface; 1.3 mum size spots were obtained by depositing protein G onto a positively charged glass surface.
DNA, Single-Stranded↗