The Transition from Unfolded to Folded G-Quadruplex DNA Analyzed and Interpreted by Two-Dimensional Infrared Spectroscopy.

A class of DNA folds/structures known collectively as G-quadruplexes (G4) commonly forms in guanine-rich areas of genomes. G4-DNA is thought to have a functional role in the regulation of gene transcription and telomerase-mediated telomere maintenance and, therefore, is a target for drugs. The details of the molecular interactions that cause stacking of the guanine-tetrads are not well-understood, which limits a rational approach to the drugability of G4 sequences. To explore these interactions, we employed electron-vibration-vibration two-dimensional infrared (EVV 2DIR) spectroscopy to measure extended vibrational coupling spectra for a parallel-stranded G4-DNA formed by the Myc2345 nucleotide sequence. We also tracked the structural changes associated with G4-folding as a function of K+-ion concentration. To classify the structural elements that the folding process generates in terms of vibrational coupling characteristics, we used quantum-chemical calculations utilizing density functional theory to predict the coupling spectra associated with given structures, which are compared against the experimental data. Overall, 102 coupling peaks are experimentally identified and followed during the folding process. Several phenomena are noted and associated with formation of the folded form. This includes frequency shifting, changes in cross-peak intensity, and the appearance of new coupling peaks. We used these observations to propose a folding sequence for this particular type of G4 under our experimental conditions. Overall, the combination of experimental 2DIR data and DFT calculations suggests that guanine-quartets may already be present before the addition of K+-ions, but that these quartets are unstacked until K+-ions are added, at which point the full G4 structure is formed.

Single cell quantification of microRNA from small numbers of non-invasively sampled primary human cells.

Expression levels of microRNAs (miRNAs) in single cells are low and conventional miRNA detection methods require amplification that can be complex, time-consuming, costly and may bias results. Single cell microfluidic platforms have been developed; however, current approaches are unable to absolutely quantify single miRNA molecules expressed in single cells. Herein, we present an amplification-free sandwich hybridisation assay to detect single miRNA molecules in single cells using a microfluidic platform that optically traps and lyses individual cells. Absolute quantification of miR-21 and miR-34a molecules was achieved at a single cell level in human cell lines and validated using real-time qPCR. The sensitivity of the assay was demonstrated by quantifying single miRNA molecules in nasal epithelial cells and CD3+ T-cells, as well as nasal fluid collected non-invasively from healthy individuals. This platform requires ~50 cells or ~30 µL biofluid and can be extended for other miRNA targets therefore it could monitor miRNA levels in disease progression or clinical studies.

Toward high-throughput oligomer detection and classification for early-stage aggregation of amyloidogenic protein.

Aggregation kinetics of proteins and peptides have been studied extensively due to their significance in many human diseases, including neurodegenerative disorders, and the roles they play in some key physiological processes. However, most of these studies have been performed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents as indicators of fibrillization. Such techniques are highly successful in making inferences about the nucleation and growth mechanism of fibrils, yet cannot directly measure assembly reactions at low protein concentrations which is the case for amyloid-ß (Aß) peptide under physiological conditions. In particular, the evolution from monomer to low-order oligomer in early stages of aggregation cannot be detected. Single-molecule methods allow direct access to such fundamental information. We developed a high-throughput protocol for single-molecule photobleaching experiments using an automated fluorescence microscope. Stepwise photobleaching analysis of the time profiles of individual foci allowed us to determine stoichiometry of protein oligomers and probe protein aggregation kinetics. Furthermore, we investigated the potential application of supervised machine learning with support vector machines (SVMs) as well as multilayer perceptron (MLP) artificial neural networks to classify bleaching traces into stoichiometric categories based on an ensemble of measurable quantities derivable from individual traces. Both SVM and MLP models achieved a comparable accuracy of more than 80% against simulated traces up to 19-mer, although MLP offered considerable speed advantages, thus making it suitable for application to high-throughput experimental data. We used our high-throughput method to study the aggregation of Aß40 in the presence of metal ions and the aggregation of α-synuclein in the presence of gold nanoparticles.

Evaluation of FOXO1 Target Engagement Using a Single-Cell Microfluidic Platform.

The cellular thermal shift assay (CETSA) has been used extensively since its introduction to study drug-target engagement within both live cells and cellular lysate. This has proven to be a useful tool in early stage drug discovery and is used to study a wide range of protein classes. We describe the application of a single-cell CETSA workflow within a microfluidic affinity capture (MAC) chip. This has enabled us to quantitatively determine the active FOXO1 single-molecule count and observe FOXO1 stabilization and destabilization in the presence of three small molecule inhibitors, including demonstrating the determination of EC50. The successful use of the MAC chip for single-cell CETSA paves the way for the study of precious clinical samples owing to the low number of cells needed by the chip. It also provides a useful tool for studying any underlying population heterogeneity that exists within a cellular system, a feature that is usually masked when conducting ensemble measurements.

Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer.

Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4's hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.

A Novel Aß40 Assembly at Physiological Concentration.

Aggregates of amyloid-ß (Aß) are characteristic of Alzheimer's disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aß. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aß to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration.

Detection of Drug Binding to a Target Protein Using EVV 2DIR Spectroscopy.

We demonstrate that electron-vibration-vibration two-dimensional infrared spectroscopy (EVV 2DIR) can be used to detect the binding of a drug to a target protein-active site. The EVV 2DIR spectrum of the FGFR1 kinase target protein is found to have ∼200 detectable cross-peaks in the spectral region 1250-1750 cm-1/2600-3400 cm-1, with additional 63 peaks caused by the addition of a drug, SU5402. Of these 63 new peaks, it is shown that only six are due to protein-drug interactions, with the other 57 being due to vibrational coupling within the drug itself. Quantum mechanical calculations employing density functional theory are used to support assignment of the six binding-dependent peaks, with one being assigned to a known interaction between the drug and a backbone carbonyl group which forms part of the binding site. None of the 57 intramolecular coupling peaks associated with the drug molecule change substantially in either intensity or frequency when the drug binds to the target protein. This strongly suggests that the structure of the drug in the target binding site is essentially identical to that when it is not bound.

Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

Acoustic suppression of the coffee-ring effect.

We study the influence of acoustic fields on the evaporative self-assembly of solute particles suspended inside sessile droplets of complex fluids. The self-assembly process often results in an undesirable ring-like heterogeneous residue, a phenomenon known as the coffee-ring effect. Here we show that this ring-like self-assembly can be controlled acoustically to form homogeneous disc-like or concentrated spot-like residues. The principle of our method lies in the formation of dynamic patterns of particles in acoustically excited droplets, which inhibits the evaporation-driven convective transport of particles towards the contact line. We elucidate the mechanisms of this pattern formation and also obtain conditions for the suppression of the coffee-ring effect. Our results provide a more general solution to suppress the coffee-ring effect without any physiochemical modification of the fluids, the particles or the surface, thus potentially useful in a broad range of industrial and analytical applications that require homogenous solute depositions.

Oxygen deficient α-Fe2O3 photoelectrodes: a balance between enhanced electrical properties and trap-mediated losses.

Intrinsic doping of hematite through the inclusion of oxygen vacancies (VO) is being increasingly explored as a simple, low temperature route to preparing active water splitting α-Fe2O3-x photoelectrodes. Whilst it is widely accepted that the introduction of VO leads to improved conductivities, little else is verified regarding the actual mechanism of enhancement. Here we employ transient absorption (TA) spectroscopy to build a comprehensive kinetic model for water oxidation on α-Fe2O3-x . In contrast to previous suggestions, the primary effect of introducing VO is to block very slow (ms) surface hole - bulk electron recombination pathways. In light of our mechanistic research we are also able to identify and address a cause of the high photocurrent onset potential, a common issue with this class of electrodes. Atomic layer deposition (ALD) of Al2O3 is found to be particularly effective with α-Fe2O3-x , leading to the photocurrent onset potential shifting by ca. 200 mV. Significantly TA measurements on these ALD passivated electrodes also provide important insights into the role of passivating layers, that are relevant to the wider development of α-Fe2O3 photoelectrodes.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 µm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy.

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy.

Addressable droplet microarrays for single cell protein analysis.

Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells.

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.

Absolute quantification of protein copy number using a single-molecule-sensitive microarray.

We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6 × 10(6) molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to calibrate data retrospectively to determine the absolute protein copy number at the single cell level in two human cancer cell lines.

Interfacial charge separation in Cu2O/RuO(x) as a visible light driven CO2 reduction catalyst.

We employ transient absorption spectroscopy to record the absorption spectrum of photogenerated charge carriers in Cu2O. We have found that CO2 reduction in Cu2O is limited by fast electron-hole recombination. The deposition of RuOx nanoparticles on Cu2O results in a twofold increased yield of long-lived electrons, indicating partially reduced electron-hole recombination losses. This observation correlates with an approximately sixfold increase in the yield of CO2 reduction to CO.

Efficient Suppression of Electron-Hole Recombination in Oxygen-Deficient Hydrogen-Treated TiO2 Nanowires for Photoelectrochemical Water Splitting.

There is an increasing level of interest in the use of black TiO2 prepared by thermal hydrogen treatments (H:TiO2) due to the potential to enhance both the photocatalytic and the light-harvesting properties of TiO2. Here, we examine oxygen-deficient H:TiO2 nanotube arrays that have previously achieved very high solar-to-hydrogen (STH) efficiencies due to incident photon-to-current efficiency (IPCE) values of >90% for photoelectrochemical water splitting at only 0.4 V vs RHE under UV illumination. Our transient absorption (TA) mechanistic study provides strong evidence that the improved electrical properties of oxygen-deficient TiO2 enables remarkably efficient spatial separation of electron-hole pairs on the submicrosecond time scale at moderate applied bias, and this coupled to effective suppression of microsecond to seconds charge carrier recombination is the primary factor behind the dramatically improved photoelectrochemical activity.

Quantitative single cell and single molecule proteomics for clinical studies.

A central aspect of cellular systems biology is the study of cell-to-cell variability driven by network control of molecular noise. Proteins are produced in stochastic bursts and, although time averaging smoothes their accumulated levels, variation in their copy number is substantial in members of environmental sensing and signalling networks. We have developed a label-free, microfluidic antibody capture chip platform called the MAC chip, to quantify precisely the copy numbers of many proteins from a single cell in a multiplexed single assay format. We intend to investigate protein noise in circulating tumour cells (CTCs) isolated from biopsies of cancer patients through the identification of biomolecular signatures, such as p53 tumour suppressor protein, which correlate with biological properties and clinical outcomes during treatment.

Charge carrier separation in nanostructured TiO2 photoelectrodes for water splitting.

There is intense interest in developing new novel nanostructured photoanodes for water splitting. It is therefore important that methods to analyze the effect of nanostructuring on water splitting yields are developed in order to rationalize the relative merits of this approach for different materials. In this study the dependence of charge separation efficiency (η(sep)) on potential during photoelectrochemical water splitting at pH 2 has been quantified in a model electrode system (nanocrystalline, mesoporous TiO2) using two independent methods. These are (i) analysis of incident photon conversion efficiency (IPCE) measurements and (ii) transient absorption (TA) spectroscopy measurements. The techniques provide good agreement with each other and show that a low maximum value of η(sep) (~0.18) is the primary cause of the low IPCE for water oxidation on these nc-TiO2 electrodes.

Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis.

Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 µm to 15 µm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.