Biomedical subjects

Sergey N Krylov

Publications and source records attributed to Sergey N Krylov.

At least 19 recordsLinked to original sources

PubMed2006

Cell lysis inside the capillary facilitated by transverse diffusion of laminar flow profiles (TDLFP).

Chemical cytometry studies the molecular composition of individual cells by means of capillary electrophoresis or capillary chromatography. In one of its realizations an intact cell is injected inside the capillary, the plasma membrane is disrupted to release the cellular contents into the separation buffer, and, finally, the molecules of interest are separated and detected. The solubilization of the plasma membrane with a surfactant is a simple and efficient way of achieving cell lysis inside the capillary. To facilitate cell lysis by a surfactant the cell has to be contacted with the surfactant inside the capillary. We recently introduced a generic method for mixing solutions inside the capillary termed transverse diffusion of laminar flow profiles (TDLFP). In this work, we propose that TDLFP can facilitate efficient cell lysis inside the capillary. Conceptually, a short plug of the surfactant is injected by pressure prior to cell injection. The cell is then injected by pressure within a plug of the physiological buffer. Due to the parabolic profiles of pressure-driven laminar flows the interface between the plug of the surfactant and that of the physiological buffer is predominantly longitudinal. Transverse diffusion mixes the surfactant with the physiological buffer, which leads to surfactant's contact with the cell and subsequent cell lysis. Here, we demonstrate that the proposed concept is valid. TDLFP-facilitated cell lysis by a short plug of the surfactant allows us to exclude the surfactant from the run buffer, and, hence, facilitates modes of separation, which are incompatible with the surfactant's presence in the run buffer. In addition to cell lysis, TDLFP will be used to mix the cellular components with labeling reactants, affinity probes, inhibitors, etc. We foresee that the generic nature and enabling capabilities of TDLFP will speed up the maturation of chemical cytometry into a practical bioanalytical tool.

Animals↗

PubMed2006

AID associates with single-stranded DNA with high affinity and a long complex half-life in a sequence-independent manner.

Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and single-stranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.

Amino Acid Motifs↗

PubMed2006

Plug-plug kinetic capillary electrophoresis: method for direct determination of rate constants of complex formation and dissociation.

We present a method for direct determination of rate constants of complex formation, k(on), and dissociation, k(off). The method is termed plug-plug kinetic capillary electrophoresis (ppKCE). To explain the concept of the method, we consider the formation of a noncovalent complex C between molecules A and B; A is assumed to migrate slower in electrophoresis than B. In ppKCE, a short plug of A is injected into a capillary, followed by a short plug of B. When a high voltage is applied, the electrophoretic zone of B moves through that of A, allowing for the formation of C. When the zones of A and B are separated, C starts dissociating. The features of the resulting electropherogram are defined by both binding and dissociation. We developed a unique mathematical approach that allows finding k(on) and k(off) from a single electropherogram without nonlinear regression analysis. The approach uses algebraic functions with the only input parameters from electropherograms being areas and migration times of electrophoretic peaks. In this work, we explain theoretical bases of ppKCE and prove the principle of the method by finding k(on) and k(off) for a protein-ligand complex. The unique capability of the method to directly determine both k(on) and k(off) along with its simplicity make ppKCE highly attractive to a broad community of molecular scientists.

Computer Simulation↗

PubMed2006

Reversible photocontrol of DNA binding by a designed GCN4-bZIP protein.

Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain. As predicted, the trans form of the chromophore destabilizes the helical structure of the coiled-coil region of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis photoisomerization of the chromophore increases helical content and substantially enhances DNA binding. The system is observed to be readily reversible; thermal relaxation of the chromophore to the trans state and concomitant dissociation of the protein-DNA complex occurs with tau(1/2) approximately 10 min at 37 degrees C. It appears that conformational dynamics in the zipper domain make the transition state for isomerization readily available so that retention of reversible switching is observed.

Amino Acid Sequence↗

PubMed2006

Selection of smart aptamers by methods of kinetic capillary electrophoresis.

We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.

Aptamers, Nucleotide↗

PubMed2006

Identification of base pairs in single-nucleotide polymorphisms by MutS protein-mediated capillary electrophoresis.

Single-nucleotide polymorphisms (SNPs) are widespread genomic variations, which are associated with serious health disorders and drug resistance. Multiple clinical applications and studies of global population genetics require fast and informative analysis of SNPs. Most of conventional methods sense the presence of the SNP but cannot identify the base pair in it. Here we report simple identification of base pairs in SNPs without DNA sequencing. Our approach is based on the unique ability of MutS protein to bind different single-nucleotide mismatches in DNA with different affinities. Conceptually, the DNA in question is mixed with reference DNA, melted, and reannealed. If the DNA in question has an SNP, the products of reannealing will have two different single-nucleotide mismatches, which provide a base-pair-specific signature of the SNP. The products of reannealing are mixed with MutS, equilibrated, and separated by equilibrium capillary electrophoresis of equilibrium mixtures with MutS in the run buffer. The pattern of migration times of DNAs with mismatches is used for unequivocal identification of the base pair in the SNP. In addition to its ability to identify base pairs in SNPs, the new analytical approach is fast, simple, highly sensitive, and requires no quantitation. It will find applications in studies of heterogeneity of base pairs in known SNPs in large human populations.

Base Pairing↗

PubMed2006

Non-SELEX selection of aptamers.

Aptamers are typically selected from libraries of random DNA (or RNA) sequences by SELEX, which involves multiple rounds of alternating steps of partitioning and PCR amplification. Here we report, for the first time, non-SELEX selection of aptamers-a process that involves repetitive steps of partitioning with no amplification between them. A highly efficient affinity method, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), was used for partitioning. We found that three steps of NECEEM-based partitioning in the non-SELEX approach were sufficient to improve the affinity of a DNA library to a target protein by more than 4 orders of magnitude. The resulting affinity was higher than that of the enriched library obtained in three rounds of NECEEM-based SELEX. Remarkably, NECEEM-based non-SELEX selection took only 1 h in contrast to several days or several weeks required for a typical SELEX procedure by conventional partitioning methods. In addition, NECEEM-based non-SELEX allowed us to accurately measure the abundance of aptamers in the library. Not only does this work introduce an extremely fast and economical method for aptamer selection, but it also suggests that aptamers may be much more abundant than they are thought to be. Finally, this work opens the opportunity for selection of drug candidates from libraries of small molecules, which cannot be PCR-amplified and thus are not approachable by SELEX.

Aptamers, Nucleotide↗

PubMed2006

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM): A novel method for biomolecular screening.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a new separation-based affinity method. It has kinetic capabilities exceeding those of surface plasmon resonance (SPR) and does not require immobilization of molecules on the surface. Another distinctive feature of NECEEM is that--if it is combined with an advanced method for the mixing solutions inside a capillary, termed transverse diffusion of laminar flow profiles (TDLFP)--it requires only nanoliter volumes of solutions. The proven applications of NECEEM to biomolecular screening include 1) measuring kinetic and thermodynamic parameters of protein-ligand interactions, 2) quantitative affinity analyses of proteins and hybridization analyses of DNA and RNA, and 3) selection of binding ligands from combinatorial libraries. NECEEM is easy to automate and parallelize. Because of its simplicity and analytical power, NECEEM has the potential to become a workhorse in studies of biomolecular interactions. The author reviews theoretical bases of NECEEM and its applications to biomolecular screening.

Affinity Labels↗

PubMed2006

Selection of surfactants for cell lysis in chemical cytometry to study protein-DNA interactions.

Protein-DNA interactions play a defining role in many cellular processes. Studying such interactions at the single-cell level is important and challenging. Here we make the first step toward achieving this goal with chemical cytometry. Chemical cytometry utilizes capillary separation for detailed chemical analyses of single cells. The cell is injected into a capillary, lysed, and its components are analyzed by CE or capillary chromatography with highly sensitive detection. In order to apply chemical cytometry to studies of protein-DNA interactions, cell lysis must not destroy protein-DNA complexes. Surfactants represent the most practical means of cell lysis inside the capillary. This work aimed at finding surfactants and lysis conditions that do not destroy protein-DNA complexes. We studied three groups of surfactants--ionic, zwitterionic, and nonionic--with respect to their ability to lyse the cell membrane without significantly influencing the stability of protein-DNA complexes. Nonequilibrium CE of equilibrium mixtures with surfactants in the equilibrium mixtures and in the run buffer was used to measure the equilibrium constant, K(d), and rate constant, k(off), of protein-DNA complex dissociation. We found that nonionic surfactants worked best: they lyse the plasma membrane without significantly influencing K(d), k(off), or the EOF. This work creates the foundation for studies of protein-DNA interactions in single cells by chemical cytometry.

Cell Separation↗

PubMed2005

Calibration-free quantitative analysis of mRNA.

Here we introduce a method for accurate and sensitive quantitative analysis of mRNA, which does not require calibration with mRNA. The method uses a fluorescently labeled hybridization probe as a reference standard. It involves the following: (i) annealing mRNA to the excess of the fluorescently labeled ssDNA hybridization probe, (ii) separation of the mRNA-probe hybrid from the excess of the probe by gel-free capillary electrophoresis mediated by ssDNA-binding protein, (iii) fluorescence detection of the hybrid and the excess probe, and (iv) quantification of mRNA using a simple algebraic formula. The method also overcomes a number of other limitations of conventional methods: the entire procedure currently takes only 2 h and accurately quantifies 10(5) copies of mRNA. With further improvements to the method, the procedure can be potentially shortened to 10 min, and the limit of quantification can be decreased to as few as 100 copies of mRNA. In this work, we prove the principle of the method by quantifying mRNA of green fluorescent protein in the matrix of total cellular RNA. The developed method is quantitative, simple, fast, and highly sensitive. It requires commercially available instrumentation only. The method will be an indispensable tool for molecular and cell biology studies.

Electrophoresis, Capillary↗

PubMed2005

Kinetic capillary electrophoresis (KCE): a conceptual platform for kinetic homogeneous affinity methods.

We propose kinetic capillary electrophoresis (KCE) as a conceptual platform for the development of kinetic homogeneous affinity methods. KCE is defined as the CE separation of species that interact during electrophoresis. Depending on how the interaction is arranged, different KCE methods can be designed. All KCE methods are described by the same mathematics: the same system of partial differential equations with only initial and boundary conditions being different. Every qualitatively unique set of initial and boundary conditions defines a unique KCE method. Here, we (i) present the theoretical bases of KCE, (ii) define four new KCE methods, and (iii) propose a multimethod KCE toolbox as an integrated kinetic technique. Using the KCE toolbox, we were able to, for the first time, observe high-affinity (specific) and low-affinity (nonspecific) interactions within the same protein-ligand pair. The concept of KCE allows for the creation of an expanding toolset of powerful kinetic homogeneous affinity methods, which will find their applications in studies of biomolecular interactions, quantitative analyses, and selecting affinity probes and drug candidates from complex mixtures.

DNA, Single-Stranded↗

PubMed2005

Transverse diffusion of laminar flow profiles to produce capillary nanoreactors.

We introduce transverse diffusion of laminar flow profiles (TDLFP), the first generic method for mixing two or more reactants inside capillaries. Conceptually, solutions of reactants are injected inside the capillary by pressure as a series of consecutive plugs. Due to the laminar nature of flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between them. After injection, the plugs are mixed by transverse diffusion; longitudinal diffusion does not contribute to mixing. To prove the principle, we used TDLFP to mix two reactants-an enzyme and its substrate. After mixing the reactants by TDLFP, we incubated reaction mixtures for different periods of time and measured the reaction kinetics. We found that the reaction proceeded in time- and concentration-dependent fashion, thus confirming that the reactants were mixed by TDLFP. Remarkably, the experimental reaction kinetics were not only in qualitative agreement but also in good quantitative agreement with theoretically predicted ones. TDLFP has a number of enabling features. By facilitating the preparation of reaction mixtures inside the capillary, TDLFP lowers reagent consumption to nanoliters (microliters are required for conventionally mixing reagents in a vial). The reaction products can be then analyzed "on-line" by capillary separation coupled with optical, electrochemical, or mass spectrometric detection. The combination of TDLFP with capillary separation will be an indispensable tool in screening large combinatorial libraries for affinity probes and drug candidates: a few microliters of a target protein will be sufficient to screen thousands of compounds. The new method paves the road to a wide use of capillary nanoreactors in different areas of physical and life sciences.

Capillaries↗

PubMed2005

Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM).

We coin a term of "smart aptamers", which describes aptamers with predefined binding parameters of their interaction with the target. Here, we introduce a method for selection of smart aptamers with predefined values of Kd: equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). Conceptually, a mixture of a target with a DNA (RNA) library is prepared and equilibrated. A plug of the equilibrium mixture is injected into a capillary prefilled with a run buffer containing the target at the concentration identical to the target concentration in the equilibrium mixture. The components of the equilibrium mixture are separated by capillary electrophoresis while equilibrium is maintained between the target and aptamers. The unique feature of ECEEM is that aptamers with different Kd values migrate with different and predictable mobilities. Thus, collecting fractions with different mobilities results in smart aptamers with different and predefined Kd values. In this proof-of-principle work, we used ECEEM to select smart aptamers for MutS protein, for which aptamers have never been previously selected. Three rounds of ECEEM-based selection were sufficient to obtain smart aptamers with Kd values approaching theoretically predicted ones. ECEEM is the first method for aptamer selection whose ability to generate smart aptamers has been experimentally proven.

Base Sequence↗

PubMed2005

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers.

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.

Alkyl and Aryl Transferases↗

PubMed2005

Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures.

We introduce temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and demonstrate its use to study thermochemistry of protein-DNA interactions. Being a homogeneous kinetic method, temperature-controlled NECEEM uniquely allows finding temperature dependencies of equilibrium and kinetic parameters of complex formation without the immobilization of the interacting molecules on the surface of a solid substrate. In this work, we applied temperature-controlled NECEEM to study the thermochemistry of two protein-DNA pairs: (i) Taq DNA polymerase with its DNA aptamer and (ii) E. coli single-stranded DNA binding protein with a 20-base-long single-stranded DNA. We determined temperature dependencies of three parameters: the equilibrium binding constant (Kb), the rate constant of complex dissociation (k(off)), and the rate constant of complex formation (k(on)). The Kb(T) functions for both protein-DNA pairs had phase-transition-like points suggesting temperature-dependent conformational changes in structures of the interacting macromolecules. Temperature dependencies of k(on) and k(off) provided insights into how the conformational changes affected two opposite processes: binding and dissociation. Finally, thermodynamic parameters, DeltaH and DeltaS, for complex formation were found for different conformations. With its unique features and potential applicability to other macromolecular interactions, temperature-controlled NECEEM establishes a valuable addition to the arsenal of analytical methods used to study dynamic molecular complexes.

Aptamers, Nucleotide↗

PubMed2005

Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures.

Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double-stranded DNA (dsDNA) but not to bind single-stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413-421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity-mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence-specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau-DNA complexes studied.

Animals↗

PubMed2005

Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.

BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.

Alkyl and Aryl Transferases↗

PubMed2004

Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis.

Until now, all methods for temperature sensing in capillary electrophoresis (CE) relied on molecular probes with temperature-dependent spectral/optical properties. Here we introduce a nonspectroscopic approach to determining temperature in CE. It is based on measuring a temperature-dependent rate constant of complex dissociation by means of a kinetic CE method known as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Conceptually, a calibration curve of "the rate constant versus temperature" is built using NECEEM and a CE instrument with a reliable temperature control or, alternatively, a nonelectrophoretic method, such as surface plasmon resonance. The calibration curve is then used to find the temperature during CE in the same buffer but with another CE apparatus or under otherwise different conditions (cooling efficiency, length and diameter of the capillary, electrical field, etc.). In this proof-of-principle work, we used the dissociation of a protein-DNA complex to demonstrate that the NECEEM-based temperature determination method allows for temperature determination in CE with a precision of 2 degrees C. Then, we applied the NECEEM-based temperature determination method to study heat dissipation efficiency in CE instruments with active and passive cooling of the capillary. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, e.g. electrochemical detection. A "kinetic probe" can be coloaded into the capillary along with a sample for in situ temperature measurements. Higher order chemical reactions can also be used for temperature sensing, provided a kinetic CE method for measuring a corresponding rate constant is available.

DNA↗