Germ Cell-Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multistep gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.

Ideal-Filter Capillary Electrophoresis (IFCE) Facilitates the One-Step Selection of Aptamers.

Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one-step selection of aptamers. We introduce partitioning by ideal-filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE-based partitioning reaches 109 , which is ten million times higher than that of typical solid-phase partitioning methods. One step of IFCE-based partitioning is sufficient for the selection of a high-affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.

Systematic Approach to Optimization of Experimental Conditions in Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is an efficient method for studying intermolecular interactions. Optimization of NECEEM experiments is not a trivial task, due to the complex interrelation between numerous experimental parameters and their combined effects on the accuracy and precision of measurements. Here we present an "algorithmic" approach for NECEEM optimization, which eliminates all of the guesswork out of this process and allows researchers to approach it in a systematic manner. We have fully tested our approach using comprehensive in silico analysis and have showed its utility within a real experimental study. The new approach makes NECEEM more robust, resilient to errors, and easily approachable for researchers with varying experience in CE.

Analysis of DNA in Phosphate Buffered Saline Using Kinetic Capillary Electrophoresis.

Kinetic capillary electrophoresis (KCE) methods are useful in the study of kinetics and equilibrium properties of interactions between DNA and its binding partners (ligands). KCE experiments are typically performed in a narrow set of "conventional" low-conductivity run buffers while DNA-ligand interactions in biological systems occur in physiological fluids, characterized by high ionic strengths. The nature and ionic strength of the buffer, in which DNA-ligand interaction occurs, can significantly influence the binding. Therefore, KCE experiments meant to study such interactions would greatly benefit if they could be performed in physiological buffers, such as phosphate buffered saline (PBS). No previous KCE studies of DNA used PBS as the run buffer. Here, we test the feasibility of using PBS as a KCE run buffer for analysis of DNA and show that its usage under standard KCE conditions renders DNA undetectable. We uncover the causes of this previously unreported detrimental effect and come up with a modification of KCE which allows one to overcome it. We apply the modified KCE method to an experimental model of a platelet-derived growth factor (PDGF) protein and its DNA aptamer, which was selected in PBS, and show that the results obtained in PBS run buffer are much closer to previously reported values than those which were obtained with a conventional low-conductivity capillary electrophoresis (CE) buffer.

Using nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) for simultaneous determination of concentration and equilibrium constant.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the equilibrium constant, K(d), and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both K(d) and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and K(d) of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at equilibrium.

Reducing pH gradients in free-flow electrophoresis.

Small-volume continuous-flow synthesis (small-volume CFS) offers a number of benefits for use in small-scale chemical production and exploratory chemistry. Typically, small-volume CFS is followed by discontinuous purification; however, a fully continuous synthesis-purification combination is more attractive. Milli free-flow electrophoresis (mFFE) is a promising continuous-flow purification technique that is well suited for integration with small-volume CFS. The purification stability of mFFE, however, needs to be significantly improved before it can be feasible for this combination. One of the major sources of instability of mFFE is attributed to the ions produced as a result of electrolysis. These ions can form pH and conductivity gradients in mFFE, which are detrimental to separation quality. The severity of these gradients has not been thoroughly studied in mFFE. In this paper, we have experimentally demonstrated that detrimental pH gradients occur at flow rates of 8 mL/min and less, and electric field strengths of 25 V/cm and greater. To decrease the pH gradients, it is necessary to evacuate H(+) and OH(-) as soon as they are generated; this can be done by increasing local hydrodynamic flow rates. We calculated the necessary flow rate, to be applied at the electrode, which can effectively wash away both ions before they can cause a detrimental pH gradient. These optimized flow rates can be attained by designing a device that incorporates deep channels. We have confirmed the effectiveness of these channels using a prototyped device. The new design allows mFFE users to work over a wider range of flow-rate and electric-field conditions without experiencing significant changes in pH.

Extracting kinetics from affinity capillary electrophoresis (ACE) data: a new blade for the old tool.

We describe a mathematical approach that enables extraction of kinetic rate constants from thousands of studies conducted over the past two decades with affinity capillary electrophoresis (ACE). Previously, ACE has been used almost exclusively for obtaining equilibrium constants of intermolecular interactions. In this article, we prove that there exists an analytical solution of partial differential equations describing mass transfer in ACE. By using an in silico study, we demonstrate that the solution is applicable to experimental conditions that are typically used in ACE and found in most historical ACE experiments. The solution was validated by extracting rate constants from previously published ACE data and closely matching independently obtained results. Lastly, it was used to obtain previously unknown rate constants from historical ACE data. The new mathematical approach expands the applicability of ACE to a wider range of biomolecular interactions and enables both prospective and retrospective data analysis. The obtained kinetic information will be of significant practical value to the fields of pharmacology and molecular biology.

Improvements to direct quantitative analysis of multiple microRNAs facilitating faster analysis.

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.

Stable DNA aggregation by removal of counterions.

Negatively charged DNA can form extremely stable complexes with positively charged ions. These counterions are very difficult to remove from DNA; therefore, little is known about DNA behavior in their deficiency. We investigated whether removal of counterions from the strongly bound counterion layer would elicit any novel DNA properties or behaviors. In order to remove the tightly bound counterions, we used dialysis against deionized water in the presence of a strong (0.6 kV/cm) electric field. The electric field promoted the dissociation of the DNA-counterion complexes, while dialysis facilitated irreversible partitioning of counterions and DNA. Counterintuitively, when deprived of counterions, DNA precipitated from the solution into amorphous aggregates. The aggregates remained stable even when the electric field was turned off but readily redissolved when counterions were reintroduced. The phenomenon is likely explained by attraction of like-charged DNA polyions due to entropic-stabilization of condensed counterion layers.

Non-uniform velocity of homogeneous DNA in a uniform electric field: consequence of electric-field-induced slow dissociation of highly stable DNA-counterion complexes.

Identical molecules move with identical velocities when placed in a uniform electric field within a uniform electrolyte. Here we report that homogeneous DNA does not obey this fundamental rule. While most DNA moves with similar velocities, a fraction of DNA moves with velocities that vary within a multiple-fold range. The size of this irregular fraction increases several orders of magnitude when exogenous counterions are added to DNA. The irregular fraction decreases several orders of magnitude when DNA counterions are removed by dialysis against deionized water in the presence of a strong electric field (0.6 kV/cm). Dialysis without the field is ineffective in decreasing the size of irregular fraction. These results suggest that (i) DNA can form very stable complexes with counterions, (ii) these complexes can be dissociated by an electric field, and (iii) the observed non-uniform velocity of DNA is caused by electric-field-induced slow dissociation of these stable complexes. Our findings help to better understand a fundamental property of DNA: its interaction with counterions. In addition, these findings suggest a practical way of making electromigration of DNA more uniform: removal of strongly bound DNA counterions by electro-dialysis against deionized water.

Peak-shape correction to symmetry for pressure-driven sample injection in capillary electrophoresis.

Pressure-driven sample injection in capillary electrophoresis results in asymmetric peaks due to difference in shapes between the front and the back boundaries of the sample plug. Uneven velocity profile of fluid flow across the capillary gives the front boundary a parabolic shape. The back side, on the other hand, has a flat interface with the electrophoresis run buffer. Here, we propose a simple means of correcting this asymmetry by pressure-driven "propagation" of the injected plug, with the parabolic sample-buffer interface established at the back. We prove experimentally that such a propagation procedure corrects peak asymmetry to the level comparable to injection through electroosmosis. Importantly, the propagation-based correction procedure also solves a problem of transferring the sample into the efficiently cooled zone of the capillary for capillary electrophoresis (CE) instruments with active cooling. The suggested peak correction procedure will find applications in all CE methods that rely on peak shape analysis, e.g., nonequilibrium capillary electrophoresis of equilibrium mixtures.

Method for determination of peak areas in nonequilibrium capillary electrophoresis of equilibrium mixtures.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) facilitates determination of both the kinetic constants (k(off)) and the equilibrium constants (K(d)) of complex dissociation from a single experiment. A typical NECEEM electropherogram consists of two peaks and an "exponential bridge" between them, smoothly merging into the peaks. The values of k(off) and K(d) are usually calculated with simple algebraic formulas, by utilizing the areas of the peaks and the bridge. Accurate determination of the two constants requires accurate positioning of the two boundaries separating the bridge from the peaks. Here, we propose a more systematic method for the determination of boundaries between the peaks and the bridge. The method involves a simple geometrical analysis of a NECEEM electropherogram based on an assumption of symmetry in ordinary electrophoretic peaks. To test the method, we (i) constructed a series of computer-simulated NECEEM electropherograms, (ii) determined the two boundaries with our method, and (iii) calculated the values of k(off) and K(d). We found that the deviation of the calculated values from those used to simulate the electropherograms did not exceed 15% for k(off) and 25% for K(d), as long as the peaks and the bridge were visually identifiable. We finally applied the method to the determination of K(d) and k(off) values for the interaction between AlkB protein and its DNA aptamer. The developed method for rational boundary determination in NECEEM will facilitate accurate data analysis in a simple and efficient manner.

DNA adsorption to the reservoir walls causing irreproducibility in studies of protein-DNA interactions by methods of kinetic capillary electrophoresis.

Methods of kinetic capillary electrophoresis (KCE) facilitate kinetic studies of protein-DNA interactions and highly efficient selection of DNA aptamers for protein targets. Here, we report a previously unnoticed source of error that affects the precision and accuracy of KCE-based measurements. The error manifests itself in cases that require the use of low concentrations of DNA. In such measurements, the reproducibility of the signal generated by the same fluorescently labeled DNA sample can have a relative standard deviation (RSD) as high as 40%. We have investigated the cause of the irreproducibility and found that it is attributed to DNA adsorption to the surface of the sample vials, in which protein-DNA mixtures are prepared prior to a KCE experiment. The use of commercially available "high DNA recovery" sample vials does not resolve the problem. We have found that the problem can be significantly alleviated by the passivation of the vial surface with blocking agents, such as masking DNA or bovine serum albumin (BSA). The described adsorption of DNA to the surface of sample vials may also be important in other procedures that deal with low DNA concentrations, such as aptamer selection and quantitative PCR.

Slow-dissociation and slow-recombination assumptions in nonequilibrium capillary electrophoresis of equilibrium mixtures.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a kinetic affinity method with both analytical and preparative applications. NECEEM requires that the dissociation of the complexes be negligible in its first phase and the recombination of the dissociated complexes be negligible in its second phase. Here, we introduce a method, which facilitates easy examination of whether or not these requirements are satisfied. We derived expressions for two parameters, termed the slow-dissociation parameter (SDP) and slow-recombination parameter (SRP), which can be used to assess the assumptions. Both parameters should be much less than 1 for the assumptions to be satisfied. We calculated the two parameters for new and previously published NECEEM experiments and found that the assumptions were satisfied in all of them. Finally, we discuss changes to NECEEM conditions that should be done if the assumptions are found not to be satisfied. The SDP/SRP assessment helps to easily validate the results of NECEEM-based analyses and thus makes the NECEEM method more robust.

Selection of aptamers for a non-DNA binding protein in the context of cell lysate.

Aptamer-facilitated Protein Isolation from Cells (AptaPIC) is a recently introduced method that allows, in particular, generation of aptamers for a protein target in a context of a crude cell lysate. The approach enables efficient, tag-free, affinity purification of target proteins which are not available in a pure form a priori, and for which no affinity ligands are available. In the proof-of-principle work, AptaPIC was used to develop aptamers for and purify MutS, a DNA mismatch repair protein. The DNA-binding nature of MutS raised concerns that AptaPIC was not a generic technique and could be inapplicable to protein targets that do not possess native nucleic acid-binding properties. Here we prove that these concerns are invalid. We used AptaPIC to generate pools of aptamers for human Platelet-Derived Growth Factor chain B (PDGF-B) protein, a non-DNA binding protein, in the context of a bacterial cell lysate, and subsequently purify it from the same lysate. Within a small number of rounds, the efficiencies of aptamer selection were similar in conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for pure protein and in AptaPIC for protein in the cell lysate. The conventional selection approach resulted in an aptamer pool with an EC(50) value of 2.0±0.1 µM, while the AptaPIC selection approach resulted in a pool with an EC(50) value of 3.9±0.4 µM. Our results clearly demonstrate that selection of aptamers for proteins in the cell lysate is not only realistic but also efficient.

Selection of aptamers for a protein target in cell lysate and their application to protein purification.

Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.