MASKE: Macroscopic Approach to Studying Kinetics at Equilibrium

The kinetics of biomolecular interactions at equilibrium is typically studied by “microscopic” methods that monitor concentration fluctuations of molecules in an “observation” volume in which the number of molecules is so small that the equilibrium is statistically impossible. Here, we introduce a “macroscopic” method for studying kinetics of biomolecular interactions at equilibrium which does not rely on monitoring the fluctuation of concentrations. We termed this method MASKE: a “macroscopic approach to studying kinetics at equilibrium”. Conceptually, in MASKE, two equilibrium reaction mixtures, “unlabeled” and “labeled”, are both prepared with two reactants and their complex; in the labeled mixture, one reactant is labeled for detection. A “macroscopic” amount of the labeled mixture is introduced into a long and narrow reactor filled with the unlabeled mixture, and a differential mobility of the reactant versus the complex is then induced by an external action along the reactor. The kinetics of complex formation and dissociation is then studied from the label-propagation pattern. In this work, we developed the theory of MASKE and experimentally proved it with a capillary as a reactor, a fluorophore as a label, and an electric field as a differential mobility inducer. Two pairs of molecules interacting with significantly different rate constants were used in this proof-of-principle work

MASKE: Macroscopic Approach to Studying Kinetics at Equilibrium

The kinetics of biomolecular interactions at equilibrium is typically studied by “microscopic” methods that monitor concentration fluctuations of molecules in an “observation” volume in which the number of molecules is so small that the equilibrium is statistically impossible. Here, we introduce a “macroscopic” method for studying kinetics of biomolecular interactions at equilibrium which does not rely on monitoring the fluctuation of concentrations. We termed this method MASKE: a “macroscopic approach to studying kinetics at equilibrium”. Conceptually, in MASKE, two equilibrium reaction mixtures, “unlabeled” and “labeled”, are both prepared with two reactants and their complex; in the labeled mixture, one reactant is labeled for detection. A “macroscopic” amount of the labeled mixture is introduced into a long and narrow reactor filled with the unlabeled mixture, and a differential mobility of the reactant versus the complex is then induced by an external action along the reactor. The kinetics of complex formation and dissociation is then studied from the label-propagation pattern. In this work, we developed the theory of MASKE and experimentally proved it with a capillary as a reactor, a fluorophore as a label, and an electric field as a differential mobility inducer. Two pairs of molecules interacting with significantly different rate constants were used in this proof-of-principle work

MASKE: Macroscopic Approach to Studying Kinetics at Equilibrium

The kinetics of biomolecular interactions at equilibrium is typically studied by “microscopic” methods that monitor concentration fluctuations of molecules in an “observation” volume in which the number of molecules is so small that the equilibrium is statistically impossible. Here, we introduce a “macroscopic” method for studying kinetics of biomolecular interactions at equilibrium which does not rely on monitoring the fluctuation of concentrations. We termed this method MASKE: a “macroscopic approach to studying kinetics at equilibrium”. Conceptually, in MASKE, two equilibrium reaction mixtures, “unlabeled” and “labeled”, are both prepared with two reactants and their complex; in the labeled mixture, one reactant is labeled for detection. A “macroscopic” amount of the labeled mixture is introduced into a long and narrow reactor filled with the unlabeled mixture, and a differential mobility of the reactant versus the complex is then induced by an external action along the reactor. The kinetics of complex formation and dissociation is then studied from the label-propagation pattern. In this work, we developed the theory of MASKE and experimentally proved it with a capillary as a reactor, a fluorophore as a label, and an electric field as a differential mobility inducer. Two pairs of molecules interacting with significantly different rate constants were used in this proof-of-principle work

Protein Electrocatalysis for Direct Sensing of Circulating MicroRNAs

MicroRNAs (miRNAs) are potentially useful biomarkers for diagnosis, classification, and prognosis of many diseases, including cancer. Herein, we developed a protein-facilitated electrocatalytic quadroprobe sensor (Sens^PEQ) for detection of miRNA signature of chronic lymphocytic leukemia (CLL) in human serum. The developed signal-ON sensor provides a compatible combination of two DNA adaptor strands modified with four methylene blue molecules and electrocatalysis using glucose oxidase in order to enhance the overall signal gain. This enhanced sensitivity provided the response necessary to detect the low-abundant serum miRNAs without preamplification. The developed Sens^PEQ is exquisitely sensitive to subtle π-stack perturbations and capable of distinguishing single base mismatches in the target miRNA. Furthermore, the developed sensor was employed for profiling of three endogenous miRNAs characteristic to CLL, including hsa-miR-16-5p, hsa-miR-21-5p, and hsa-miR-150-5p in normal healthy serum, chronic lymphocytic leukemia Rai stage 1 (CLL-1), and stage 3 (CLL-3) sera, using a non-human cel-miR-39-3p as an internal standard. The sensor results were verified by conventional SYBR green-based quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis

DFT-Enabled Development of Hemilabile (P^∧N) Ligands for Gold(I/III) RedOx Catalysis: Application to the Thiotosylation of Aryl Iodides

Ligand-enabled oxidative addition of Csp2-X bonds to Au(I) centers has recently appeared as a valuable strategy for the development of catalytic RedOx processes. Several cross-coupling reactions that were previously considered difficult to achieve were reported lately, thus expanding the synthetic potential of gold(I) complexes beyond the traditional nucleophilic functionalization of π-systems. MeDalPhos has played an important role in this development and, despite several studies on alternative structures, remains, so far, the only general ligand for such process. We report herein the discovery and DFT-enabled structural optimization of a new family of hemilabile (P∧N) ligands that can promote the oxidative addition of aryl iodides to gold(I). These flexible ligands, which possess a common 2-methylamino heteroaromatic N-donor motif, are structurally and electronically tunable, beyond being easily accessible and affordable. The corresponding Au(I) complexes were shown to outperform the reactivity of (MeDalPhos)Au(I) in a series of alkoxy- and amidoarylations of alkenes. Their synthetic potential and comparatively higher reactivity were further highlighted in the thiotosylation of aryl iodides, a challenging unreported C–S cross-coupling reaction that could not be achieved under classical Pd(0/II) catalysis and that allows for general and divergent access to aryl sulfur derivatives

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 kon, koff, and Kd of weak (Kd > 1 μM) and fast kinetics (relaxation time, τ 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 > Kd > 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 β-cyclodextrins. The affinity of the drugs was significantly higher for β-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation

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 106 to 1012 ivp/mL

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

In this paper, an on-line solid-phase extraction capillary electrophoresis–mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol·L–1, and the limit of detection (LOD) was around 10 nmol·L–1 (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-5p and a 23 nucleotide long 5′-phosphorylated miRNA with 3′-uridylation (iso-miR-16-5p), were only detected in the CLL patients

On-line Aptamer Affinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Blood α‑Synuclein

In this paper, an on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry method is described for the purification, preconcentration, separation, and characterization of α-synuclein (α-syn) in blood at the intact protein level. A single-stranded DNA aptamer is used to bind with high affinity and selectivity α-syn, which is a major component of Lewy bodies, the typical aggregated protein deposits found in Parkinson’s disease (PD). Under the conditions optimized with recombinant α-syn, repeatability (2.1 and 5.4% percent relative standard deviation for migration times and peak areas, respectively) and microcartridge lifetime (around 20 analyses/microcartridge) were good, the method was linear between 0.5 and 10 μg·mL–1, and limit of detection was 0.2 μg·mL–1 (100 times lower than by CE-MS, 20 μg·mL–1). The method was subsequently applied to the analysis of endogenous α-syn from red blood cells lysate of healthy controls and PD patients

On-line Aptamer Affinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Blood α‑Synuclein

In this paper, an on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry method is described for the purification, preconcentration, separation, and characterization of α-synuclein (α-syn) in blood at the intact protein level. A single-stranded DNA aptamer is used to bind with high affinity and selectivity α-syn, which is a major component of Lewy bodies, the typical aggregated protein deposits found in Parkinson’s disease (PD). Under the conditions optimized with recombinant α-syn, repeatability (2.1 and 5.4% percent relative standard deviation for migration times and peak areas, respectively) and microcartridge lifetime (around 20 analyses/microcartridge) were good, the method was linear between 0.5 and 10 μg·mL–1, and limit of detection was 0.2 μg·mL–1 (100 times lower than by CE-MS, 20 μg·mL–1). The method was subsequently applied to the analysis of endogenous α-syn from red blood cells lysate of healthy controls and PD patients