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702–706  1996 Oxford University Press Nucleic Acids Research, 1996, Vol. 24, No. 4 Use of a high affinity DNA ligand in flow cytometry Kenneth A. Davis, Barnaby Abrams, Yun Lin1 and Sumedha D. Jayasena1,* Becton Dickinson Immunocytometry Systems, 2350 Qume Drive, San Jose, CA 95131, USA and 1NeXstar Pharmaceuticals Inc., 2860 Wilderness Place, Boulder, CO 80301, USA Received October 11, 1995; Revised and Accepted December 17, 1995 ABSTRACT To investigate the feasibility of using oligonucleotides in flow cytometry we describe a model system consisting of human neutrophil elastase (HNE) coated on 3.3 µm beads and a high affinity DNA ligand for HNE isolated by in vitro selection (SELEX). In this system the fluoresceinated DNA ligand was equally effective as an anti-HNE antibody in detecting HNE on beads. The location on and the chemistry of attachment of fluorescein to the DNA ligand is critical for the sensitivity of detection. DNA constructs in which fluorescein was conjugated via an ethylene glycol tether to either the 5′-end or near the 3′-end gave much higher signals than did probes with fluorescein directly conjugated to either end. Second-step staining with strepavidin-conjugated phycoerythrin was accomplished using a biotinylated DNA ligand in the initial staining of HNE beads. These data suggest that instead of, or in addition to, antibodies high affinity oligonucleotide probes can be useful in diagnostic applications based on flow cytometry. specific single-stranded oligonucleotide sequences with desired properties from large random sequence libraries (3,4). The SELEX process has permitted the isolation of specific and high affinity oligonucleotide ligands for a wide range of target molecules, including nucleic acid binding proteins, non-nucleic acid binding proteins and small organic molecules (reviewed in 5). In most cases SELEX-derived ligands have similar affinities and specificities to those of antibodies. In the present study, using a model system, we describe the behavior of a SELEX-derived high affinity oligonucleotide ligand in flow cytometry. In this model polystyrene beads coated with human neutrophil elastase (HNE) and a DNA sequence that binds HNE with high affinity (6) were used. The performance of the DNA ligand was compared with an anti-HNE antibody in detecting HNE on beads under flow cytometry. By synthesizing various DNA constructs containing one or two fluorescein molecules at different positions of the sequence we found that the site and chemistry of attachment of fluorescein are important for detection. This study demonstrates the feasibility of the use of high affinity oligonucleotide ligands in flow cytometry. MATERIALS AND METHODS INTRODUCTION Materials Antibodies have been commonly used in flow cytometry, a technique that is unique in its capability to perform simultaneous multiparameter analysis and to separate (or sort) unique cell types from heterogeneous mixtures. In flow cytometry particles or cells prestained with a target-specific ligand (typically an antibody) conjugated to a fluorophore are passed through a region where they intersect a laser beam. The characteristics of scattered light provide information on the size, shape and granularity of particles, whereas the emitted fluorescence indicates the binding properties of the ligand (1). Flow cytometers are capable of measuring these properties on thousands of cells in a few seconds. Flow cytometry, once used mainly as a research tool, has now made its way into clinical applications. A widely used application is the monitoring of HIV-infected patients to obtain their absolute CD4 counts (number of T-helper cells), a surrogate marker for the progression of the disease (2). In addition, the technique is also being used in immunophenotyping in leukemia and bone marrow screening in patients who undergo transplantation (2). Systematic Evolution of Ligands by EXponential enrichment (SELEX) is an in vitro selection/amplification technique to enrich A spacer phosphoramidite that introduces an 18 atom spacer arm (six ethylene glycol units) into oligonucleotides, fluorescein-ON phosphoramidite and symmetrical branching 3′–3′ linking CPG were purchased from Clontech (Palo Alto, CA). Mouse anti-HNE antibody was obtained from Dako Corp. (Carpinteria, CA). Rat anti-mouse IgG1, clone X56 (from Becton Dickinson) was labeled with fluorescein isothiocyanate (FITC) to contain approximately one fluorescein per molecule of antibody. Fluoricon Polystyrene Assay Particles (3.3 µm) were obtained from IDEXX Laboratories Inc. (Westbrook, ME). HNE was obtained as a salt-free lyophylized solid from Athens Research and Technology (Athens, GA). All other reagents were of analytical grade. Enzymes were purchased from commercial sources. * To whom correspondence should be addressed Oligonucleotides Oligonucleotides containing fluorescein were chemically synthesized by standard solid phase chemistry using cyanoethyl phosphosphoramidites. Symmetrical branching 3′–3′ linking CPG was used to synthesize the tail-to-tail dimer. After deprotection 703 Nucleic Acids Acids Research, Research,1994, 1996,Vol. Vol.22, 24,No. No.14 Nucleic 703 DNA sequences were purified by denaturing polyacrylamide gel electrophoresis to ensure size homogeneity. Preparation of HNE beads Polystyrene beads were successively washed with phosphatebuffered saline (PBS) containing 0.1% SDS, PBS containing 0.01% Tween 20 and then with acetate buffer (50 mM NaOAc, 0.15 M NaCl, pH 5.65). Beads (0.5% solids) in acetate buffer were coated with HNE (0.5 µg/cm2 bead surface area) for 30 min and then washed and suspended (∼5 × 105 beads/µl) in acetate buffer containing 2% bovine serum albumin (BSA). HNE-coated beads were stored at 4
C until use. Ligand binding to HNE in solution DNA ligands containing fluorescein either internally or near their 3′-ends were radiolabeled at their 5′-ends with [γ-32P]ATP and T4 polynucleotide kinase. DNAs carrying fluorescein at their 5′-ends were radiolabeled at their 3′-ends with [α-32P]ddATP and terminal deoxynucleotidyl transferase. Radiolabeled DNAs were purified by denaturing polyacrylamide gel electrophoresis. Gel-purified DNAs resuspended at a final concentration of 1 nM in the standard binding buffer (150 mM NaCl, 100 mM Tris–HCl, pH 7.0, 2 mM MgCl2 and 6 mM KCl) were heated to 70
C for 3 min and cooled to room temperature to facilitate secondary structure formation. Gel-purified radiolabeled DNA (