Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

Proteomics-Based Regression Model for Assessing the Development of Chronic Lymphocytic Leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is very ambiguous, showing either an indolent nature of the disease or having latent dangerous progression, which, if diagnosed, will require an urgent therapy. The prognosis of the course of the disease and the estimation of the time of therapy initiation are crucial for the selection of a successful treatment strategy. A reliable estimating index is needed to assign newly diagnosed CLL patients to the prognostic groups. In this work, we evaluated the comparative expressions of proteins in CLL blood cells using a label-free quantification by mass spectrometry and calculated the integrated proteomic indexes for a group of patients who received therapy after the blood sampling over different periods of time. Using a two-factor linear regression analysis based on these data, we propose a new pipeline for evaluating model development for estimation of the moment of therapy initiation for newly diagnosed CLL patients.

Aptamer-facilitated mass cytometry.

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.

DNA Aptamers for the Characterization of Histological Structure of Lung Adenocarcinoma.

Nucleic acid aptamers are becoming popular as molecular probes for identification and imaging pathology and, at the same time, as a convenient platform for targeted therapy. Recent studies have shown that aptamers may be effectively used for tumor characterization and as commercially available monoclonal antibodies. Here we present three DNA aptamers binding to whole transformed lung cancer tissues, including tumor cells, connective tissues, and blood vessels. Protein targets have been revealed using affinity purification followed by mass spectrometry analyses, and they have been validated using a panel of correspondent antibodies and 3D imaging of tumor tissues. Each of the proteins targeted by the aptamers is involved in cancer progression and most of them are crucial for lung adenocarcinoma. We propose the use of these aptamers in aptahistochemistry for the characterization of the histological structure of lung adenocarcinoma. The value of the presented aptamers is their application together or separately for indicating the spread of neoplastic transformation, for complex differential diagnostics, and for targeted therapy of the tumor itself as well as all transformed structures of the adjacent tissues. Moreover, it has been demonstrated that these aptamers could be used for intraoperative tumor visualization and margin assessment.

Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS.

To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE-UV-IM-MS). Kinetic CE-UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products. This coupled CE-UV-IM-MS system allowed the simultaneous, real-time observation of the effect of small-molecule inhibitors on both the conformational distribution and enzymatic activity of the human tissue transglutaminase TG2. By expanding mass spectrometry profiling of enzymatic reactions beyond proteins and substrates to include protein dynamics, CE-UV-IM-MS opens a new avenue for the modulation and regulation of cellular functions, drug development and protein engineering.

Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Carbohydrate-based ice recrystallization inhibitors increase infectivity and thermostability of viral vectors.

The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL(-1) during 40 days, and HSV-1, 2.7 Log10 PFU mL(-1) during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL(-1) after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry.

G-quadruplex-forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we couple on-line kinetic capillary electrophoresis with mass spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes in solution. We show that peaks shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA-metal affinity interactions and G-quadruplex formation; and ion mobility mass spectrometry (IM-MS) provides information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes. KCE-MS separates a thrombin-binding aptamer d[GGTTGGTGTGGTTGG] from mutated sequences based on affinity to potassium, and reveals the apparent equilibrium folding constant (K F≈150 µm), folding rate constant (k on≈1.70×10(3) s(-1) m(-1)), unfolding rate constant (k off≈0.25 s(-1)), half-life time of the G-quadruplex (t 1/2≈2.8 s), and relaxation time (τ≈3.9 ms at physiological 150 mm [K(+)]). In addition, KCE-MS screens for a GQ-stabilizing/-destabilizing effect of DNA binding dyes and an anticancer drug, cisplatin.

Bioanalysis for biocatalysis: multiplexed capillary electrophoresis-mass spectrometry assay for aminotransferase substrate discovery and specificity profiling.

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections-incubation-separation-enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (E. coli aspartate aminotransferase, E. coli branched-chain amino acid aminotransferase, and Bacillus sp. YM-1 D-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent K(M) and k(cat) parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications.

Assessment of energetic costs of AhR activation by ß-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis.

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist ß-naphthoflavone (ßNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na(+)/K(+)-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1µM ßNF for 48h did not alter hepatocyte AEC, protein synthesis, or Na(+)/K(+)-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies.

Real-time monitoring of protein conformational dynamics in solution using kinetic capillary electrophoresis.

Conformational analysis: Capillary electrophoresis (CE) allows for the rapid separation of slowly interconverting protein conformers. Kinetic analysis (k(open), k(closed), and K(d)) of electropherograms in the presence and absence of effector ligands allows the measurement of kinetic and thermodynamic constants associated with conformational changes and ligand binding.

Viral quantitative capillary electrophoresis for counting and quality control of RNA viruses.

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze-thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10(8) to 10(13) ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation.

Comparative study of three methods for affinity measurements: capillary electrophoresis coupled with UV detection and mass spectrometry, and direct infusion mass spectrometry.

We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and ß-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

The collaborative role of molecular conformation and energetics in the binding of gas-phase non-covalent polymer/amine complexes.

A large series of similar non-covalent complexes were probed using ion mobility spectrometry, molecular mechanics/molecular dynamics (MM/MD), electrospray-tandem mass spectrometry (ESI-MS/MS) and RRKM theory in order to determine the effects of charge state and charge location upon the conformation, the 0 K activation energy (E(0)) and the entropy of activation (ΔS(‡)) of the dissociation of these complexes. The non-covalent complexes consisted of poly(methylmethacrylate) oligomers and singly and doubly charged diaminoalkanes of varying length. This allowed for control of the charge separation within the complexes, as well as the size of the complex. A destabilizing effect was observed in complexes containing protons in close proximity, and/or short oligomers. Interestingly, a multiple charge stabilizing effect was observed when charge sites were sufficiently separated and/or when the polymer moiety of the complex was large. ΔS(‡) values of doubly charged complexes showed a greater increase with increasing polymer size in comparison to singly charged complexes. This entropic observation is explained by structure, where IMS and MM/MD determined that the charge location was the determining factor of the overall conformation of these complexes and multiple charging resulted in more rigid arrangements. Dissociation of a tightly bound complex is more entropically favorable than a loosely bound complex. Also presented is a MM/MD refinement regime derived from IMS measurements.

Viral quantitative capillary electrophoresis for counting intact viruses.

The quantification of a virus plays an important role in vaccine development, clinical diagnostics, and environmental contamination assays. In all these cases, it is essential to calculate the concentration or number of intact virus particles (ivp) and estimate the degree of degradation and contamination of virus samples. In this work, we propose a cost-efficient, robust method for the quantification and characterization of intact viruses based on capillary zone electrophoresis. This separation method is demonstrated on vaccinia virus (VV) with oncolytic properties. After virus sample preparation, the solution contains intact VV as well as broken viruses and residual DNA from the host cell used for preparation. Regulatory requirements limit the amount of the host cell DNA that can be present in vaccines or human therapeutics. We apply capillary electrophoresis to separate intact virus particles and the residual DNA and to measure the level of virus contamination with DNA impurities. Intercalating YOYO-1 dye is used to detect the encapsulated and free DNA by laser-induced fluorescence. After soft lysis of VV with proteinase K, all encapsulated DNA is dissolved to the free DNA. The change in peak areas and a DNA calibration curve help determine the initial concentration of intact viruses. This viral quantitative capillary electrophoresis (Viral qCE) is able to quantify the oncolytic vaccinia virus in the range of 10(6) to 10(12) ivp/mL.

Revealing equilibrium and rate constants of weak and fast noncovalent interactions.

Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k(on), k(off), and K(d) of weak (K(d) > 1 µM) and fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor. Conceptually, an equilibrium mixture (EM) of a ligand (L), target (T), and a complex (C) is prepared. The mixture is introduced into the beginning of a capillary reactor with aspect ratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > K(d) > 80 µM) and fast (0.25 s ≥ τ ≥ 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and α- and ß-cyclodextrins. The affinity of the drugs was significantly higher for ß-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation.