Sergey N. Krylov

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.

Correlating Cell Cycle With Metabolism in Single Cells: Combination of Image and Metabolic Cytometry

BACKGROUND We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.

AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

ABSTRACT 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.

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.

Capillary Electrophoresis for Quantitative Studies of Biomolecular Interactions

■ CONTENTS Kinetic Capillary Electrophoresis 157 Applications 159 Aptamers 159 KCE for Quantitative Characterization of Aptamer-Target Binding 159 KCE-Based Aptamer Selection 160 Small Molecules 161 Cyclodextrins 162 Proteins 162 Others 164 Instrumentation and Methodology 164 Sample Preparation 164 Capillary Coatings 165 KCE-MS 165 Computational and Mathematical Approaches 166 Validation 167 Concluding Remarks 169 Author Information 169 Corresponding Author 169 Notes 169 Biographies 169 Acknowledgments 170 References 170

Single‐cell analysis using capillary electrophoresis: Influence of surface support properties on cell injection into the capillary

Capillary electrophoresis (CE) is an important tool of chemical cytometry. Whole‐cell analysis using CE starts with cell injection into the capillary by either siphoning or electroosmosis. However, strong adherence of the cell to the support surface can prevent efficient cell injection and lead to irreproducible analysis. Here we evaluated several surfaces as potential cell supports for HT29 cells (human colon adenocarcinoma). These cells strongly adhered to the surface of untreated glass or polystyrene. Hydrophobic coating with dimethyldichlorosilane (DMS) or Sigmacote® did not significantly reduce cell adhesion. In contrast, cell adhesion was reduced significantly when the surface was modified with hydrophilic polymers (hydrogels) such as poly(2‐hydrohyethyl methacrylate) (PHEMA) and polyvinyl alcohol (PVA). In addition to their pronounced antiadhesive properties, PHEMA and PVA coatings were the most biocompatible (had highest survival of cells in contact with surface). Hydrogel‐coated polystyrene plates were tested as a commercial alternative to hydrogel‐coated glass slides. The cell adhesive properties of such plates were similar to those of PHEMA and PVA. However, the biocompatibility of the plates was lower than that of the other surfaces tested. Moreover, in contrast to PHEMA‐ and PVA‐coated glass slides, the plates were sensitive to UV light and therefore should not be used when fluorescent image microscopy with UV excitation precedes CE. The analyses of the data obtained showed that PHEMA‐ and PVA‐coated glass slides were the most suitable cell supports for cell injection into the capillary.

Direct Quantitative Analysis of Multiple miRNAs (DQAMmiR)

MicroRNAs (miRNAs) are short RNA molecules (18–25 nucleotides) that were recently proven to play an important role in the regulation of cellular processes, and their abnormal expression is associated with pathologies such as cancer. A change in the cellular status is typically associated with a simultaneous change in the level of several miRNAs. For example, abnormal expression of two miRNAs was found to be indicative of colorectal cancer in humans. Therefore, both the study of the biological role of miRNA and the use of miRNA for informative disease diagnostics require accurate quantitative analysis of multiple miRNAs. Most methods of miRNA detection are indirect (e.g. PCR, microarrays, SPR, next generation sequencing, etc.), that is, they require chemical or enzymatic modifications of miRNA prior to the analysis. Not only do these modifications make the analysis more complex and timeconsuming but they also reduce the accuracy of the method owing to different efficiencies for modifications of different miRNAs. There are a few direct methods that do not require any modification of the target miRNA. Northern blotting does not require any modifications, however, the method can be tedious and although it can be quantitative its sensitivity is limited. Signal-amplifying ribozymes, in situ hybridization, bioluminescence detection, and two-probe single-molecule fluorescence are other direct miRNA detection methods however, the first two methods are only semi-quantitative while the latter two can hardly be used for multiple miRNAs. Cheng et al. used rolling-circle amplification (RCA), which does not require modification of the miRNA to detect low concentrations of miRNA and can be run in parallel; however, the process is tedious taking over 8 h and the amplification step can potentially lead to biases in quantitation. Thus, there is currently no method for direct quantitative analysis of multiple miRNAs. Herein we report the first direct quantitative analysis of multiple miRNAs (DQAMmiR). DQAMmiR uses miRNAs directly, without any modification, and accurately determines concentrations of multiple miRNAs without the need for calibration curves. This approach was achieved using a capillary-electrophoresisbased hybridization assay with an ideologically simple combination of two well-known separation-enhancement approaches: 1) drag tags on the DNA probes, and 2) single strand DNA binding protein (SSB) in the buffer. In this proof-of-principle work, we developed DQAMmiR for three miRNAs (mir21, 125b, 145) known to be deregulated in breast cancer. DQAMmiR opens the opportunity for simple, fast, and quantitative fingerprinting of up to several tens of miRNAs in basic research and clinical applications. The availability of suitable commercial instruments for DQAMmiR makes the method practical for a large community of researchers. We based DQAMmiR upon a classical hybridization approach, in which an excess of labeled DNA probes are bound to their complementary miRNA targets. Electrophoresis can be used to efficiently separate oligonucleotides, but simultaneously separating the hybrids from each other and from the unbound probes is challenging and so far has not been achieved. We solved the separation problem through a combination of two well-known mobility-shift approaches: 1) drag tags on the probes and 2) single strand DNA binding (SSB) protein in the buffer. This hypothetical approach is illustrated in Figure 1, in which the miRNAs and their complimentary ssDNA probes are shown as short lines of the same color, drag tags are shown as parachutes, fluorescent labels are shown as small green circles, and SSB is shown as a large black circle. In the hybridization step, an excess of the probes is mixed with the miRNAs, thus leading to all miRNAs being hybridized but with some probes left unbound to miRNA. A short plug of the hybridization mixture is introduced into a capillary prefilled with an SSBcontaining buffer. SSB binds all ssDNA probes but does not bind the double-stranded miRNA–DNA hybrid. When an electric field is applied, all SSB-bound probes move faster than all the hybrids (SSB works as a propellant). Different drag tags make different hybrids move with different velocities. SSB-bound probes, however, can move with similar velocities if the drag tags are small with respect to SSB. In such a case, a fluorescent detector at the end of the capillary generates separate signals for the hybrids and a cumulative signal (one peak or multiple peaks) for the excess of the probes. The amounts of the different miRNAs are finally determined from integrated signals (peak areas in the graph) by a simple mathematical approach. We reserve the term of direct quantitative analysis of multiple miRNAs and its abbreviation of DQAMmiR for the specific approach described above. To experimentally test the viability of our hypothetical DQAMmiR, we decided to use three miRNAs known to be deregulated in breast cancer: mir21 (5’-UAGCUUAUCAGA CUGAUGUUGA-3’), mir125b (5’-UCCCUGAGACCCUAACUU GUGA-3’), and mir145 (5’-GUCCAGUUUUCCCAGGAAUCCC U-3’). Three ssDNA probes were designed and all are labeled with Alexa 488 at the 5 end; the 3 end was reserved for drag tags. To separate the three hybrids we needed only two probes modified with drag [*] D. W. Wegman, Prof. S. N. Krylov Department of Chemistry, York University 4700 Keele Street, Toronto, Ontario M3J 1P3 (Canada) E-mail: skrylov@yorku.ca

Selection of smart small-molecule ligands: the proof of principle.

The development of drugs and diagnostics with desirable characteristics requires smart small-molecule ligandsligands with predefined binding parameters of interaction with the target. Here, we propose a general approach for selection of such ligands from highly diverse combinatorial libraries of small molecules by methods of kinetic capillary electrophoresis (KCE). We deduct three fundamental requirements for the combinatorial library to suit the KCE-based selection of smart ligands and suggest a universal design of the library for selecting smart small-molecule ligands: every small molecule in the library is tagged with DNA that encodes the structure of the molecule. Finally, we use several DNA-tagged small molecules, which represent a hypothetical library, to prove experimentally selection of smart small-molecule ligands by the proposed approach.

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.