Maxim V. Berezovski

Aptamer-Facilitated Biomarker Discovery (AptaBiD)

Here we introduce a technology for biomarker discovery in which (i) DNA aptamers to biomarkers differentially expressed on the surfaces of cells being in different states are selected; (ii) aptamers are used to isolate biomarkers from the cells; and (iii) the isolated biomarkers are identified by means of mass spectrometry. The technology is termed aptamer-facilitated biomarker discovery (AptaBiD). AptaBiD was used to discover surface biomarkers that distinguish live mature and immature dendritic cells. We selected in vitro two DNA aptamer pools that specifically bind to mature and immature dendritic cells with a difference in strength of approximately 100 times. The aptamer pools were proven to be highly efficient in flow- and magnetic-bead-assisted separation of mature cells from immature cells. The two aptamer pools were then used to isolate biomarkers from the cells. The subsequent mass spectrometry analysis of the isolated proteins revealed unknown biomarkers of immature and mature dendritic cells.

Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides

Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers — a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.

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.

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

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.

Kinetic capillary electrophoresis-based affinity screening of aptamer clones.

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K(d) values for the in-capillary reaction of an aptamer and a target and that the obtained K(d) values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.

Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection

Enzymatic farnesylation of oncogenic forms of Ras proteins is the initial step in a series of posttranslational modifications essential for Ras activity. The modification is catalyzed by the enzyme, protein farnesyltransferase (PFTase), which transfers a farnesyl moiety from farnesyl diphosphate to the protein. We employed capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection to develop a rapid and sensitive method for the determination of PFTase activity in vitro. The limited substrate specificity of PFTase allowed us to use a fluorescently labeled pentapeptide instead of a Ras protein as a substrate for the enzyme; the product of the enzymatic reaction was the farnesylated pentapeptide. The product was separated from the substrate by CE and quantified with LIF detection. Under optimal conditions, the separation was achieved within 10 min with a resolution of 86. The mass and concentration limits of detection for the farnesylated product were 10‐19 mol and 0.28 nM, respectively. By measuring the rate of accumulation of the farnesylated product, we were able to determine the kinetic parameters of the enzymatic reaction. For yeast PFTase as an enzyme and difluorocarboxyfluorescein‐labeled GCVIA peptide as a substrate, the values of kcat and KM were found to be (3.1 ± 0.3)×10‐3 s‐1 and (12.0 ± 1.2) νM, respectively. Our results suggest that CE‐LIF can be efficiently used for the determination of enzymatic activity of PFTase in vitro. After minor modifications, the developed method can be also applied to other reactions of enzymatic prenylation of proteins.

Kinetic methods in capillary electrophoresis and their applications

In recent years, capillary electrophoresis (CE) has been one of rapidly growing analytical techniques to study affinity interactions. Quick analysis, high efficiency, high resolving power, low sample consumption, and wide range of possible analytes make CE an indispensable tool for studies of biomolecules and, in particular, studies of their interactions. In the article, we discuss kinetic methods in CE. The spectrum of proven applications of kinetic CE methods includes: (i) measuring equilibrium and rate constants of protein-ligand interaction from a single experiment, (ii) quantitative affinity analyses of proteins, (iii) measuring temperature in CE, (iv) studying thermochemistry of affinity interactions, and (v) kinetic selection of ligands from combinatorial libraries. We demonstrate that new kinetic CE method can serve as a Swiss army knife in the development and utilization of oligonucleotide aptamers. Uniquely, they can facilitate selection of smart aptamers - aptamers with pre-defined binding parameters. We believe that further development of kinetic CE methods will provide a variety of methodological schemes for high-throughput screening of combinatorial libraries for affinity probes and drug candidates using CE as a universal instrumental platform.