Svetlana M. Krylova

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.

Transverse diffusion of laminar flow profiles – a generic method for mixing reactants in capillary microreactor

The capillary is an attractive format for integrated microanalyses, which start with the injection of separate reactants into the capillary and their mixing inside the capillary. Due to the nonturbulent nature of flow inside the capillary, mixing reactants in a generic way is a challenging task. Three approaches have been suggested as a solution: mixing by electrophoresis, mixing by longitudinal diffusion, and, most recently, mixing by transverse diffusion of laminar flow profiles (TDLFP). This is the first review on TDLFP, describing: (i) the physical basis of the method, (ii) its theory, (iii) analytical and numerical solutions for the calculation of concentration profiles of mixed reactants, (iv) up-to-date applications, and (v) problems to be solved and future directions.

Label-Free Solution-Based Kinetic Study of Aptamer–Small Molecule Interactions by Kinetic Capillary Electrophoresis with UV Detection Revealing How Kinetics Control Equilibrium

Here we demonstrate a label-free solution-based approach for studying the kinetics of biopolymer-small molecule interactions. The approach utilizes kinetic capillary electrophoresis (KCE) separation and UV light absorption detection of the unlabeled small molecule. In this proof-of-concept work, we applied KCE-UV to study kinetics of interaction between a small molecule and a DNA aptamer. From the kinetic analysis of a series of aptamers, we found that dissociation rather than binding controls the stability of the complex. Because of its label-free features and generic nature, KCE-UV promises to become a practical tool for challenging kinetic studies of biopolymer-small molecule interactions.

Emulsion PCR significantly improves nonequilibrium capillary electrophoresis of equilibrium mixtures-based aptamer selection: allowing for efficient and rapid selection of aptamer to unmodified ABH2 protein.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), a homogeneous approach to select DNA aptamers, is among the most efficient partitioning techniques. In contrast with surface-based systematic evolution of ligands by exponential enrichment (SELEX) approaches, the ability of NECEEM to select aptamers to unmodified proteins in solution is preferable for identifying aptamers for eventual in vivo use. The high stringency and low sample volumes of NECEEM, although generally beneficial, can result in binding of very few aptamers, requiring highly efficient amplification to propagate them. When amplified with standard PCR, detectable library enrichment can fail due to the fast conversion of the aptamers into byproducts and preferential amplification of nonbinders. As an alternative, we proposed the use of emulsion PCR (ePCR), which is known to reduce byproduct formation, as a PCR mode for coupling with NECEEM partitioning. For the first time, we tested the advantages of ePCR in NECEEM-based aptamer selection to a medically relevant DNA repair enzyme, ABH2. We report that the combination of ePCR with NECEEM allowed for the selection of aptamers in the first three rounds of SELEX, while SELEX with conventional PCR failed in a number of attempts. Selected aptamers to an unmodified ABH2 protein have potential use in diagnostics and as leads for anticancer cotherapies, used as enhancements of alkylating agents in chemotherapy.

Dynamic combinatorial mass spectrometry leads to inhibitors of a 2-oxoglutarate-dependent nucleic acid demethylase.

2-Oxoglutarate-dependent nucleic acid demethylases are of biological interest because of their roles in nucleic acid repair and modification. Although some of these enzymes are linked to physiology, their regulatory roles are unclear. Hence, there is a desire to develop selective inhibitors for them; we report studies on AlkB, which reveal it as being amenable to selective inhibition by small molecules. Dynamic combinatorial chemistry linked to mass spectrometric analyses (DCMS) led to the identification of lead compounds, one of which was analyzed by crystallography. Subsequent structure-guided studies led to the identification of inhibitors of improved potency, some of which were shown to be selective over two other 2OG oxygenases. The work further validates the use of the DCMS method and will help to enable the development of inhibitors of nucleic acid modifying 2OG oxygenases both for use as functional probes and, in the longer term, for potential therapeutic use.

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes.

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K(d) values between 20 and 240nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.

Direct Analysis of Enzyme-Catalyzed DNA Demethylation

N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.

Kinetic Capillary Electrophoresis with Mass-Spectrometry Detection (KCE- MS) Facilitates Label-Free Solution-Based Kinetic Analysis of Protein-Small Molecule Binding

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.

Prediction of protein-DNA complex mobility in gel-free capillary electrophoresis.

Selection of protein binders from highly diverse combinatorial libraries of DNA-encoded small molecules is a highly promising approach for discovery of small-molecule drug leads. Methods of kinetic capillary electrophoresis provide the high efficiency of partitioning required for such selection but require the knowledge of electrophoretic mobility of the protein-ligand complex. Here we present a theoretical approach for an accurate estimate of the electrophoretic mobility of such complexes. The model is based on a theory of the thin double layer and corresponding expressions used for the mobilities of a rod-like short oligonucleotide and a sphere-like globular protein. The model uses empirical values of mobilities of free protein, free ligand, and electroosmotic flow. The model was tested with a streptavidin-dsDNA complex linked through biotin (small molecule). The deviation of the prediction from the experimental mobility did not exceed 4%, thus confirming that not only is the model adequate but it is also accurate. This model will facilitate reliable use of KCE methods for selection of drug leads from libraries of DNA-encoded small molecules.

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.