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
[mu]
m 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.
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 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.
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 IgG
1
, clone X56 (from Becton Dickinson) was labeled with fluorescein isothiocyanate
(FITC) to contain approximately one fluorescein per molecule of antibody. Fluoricontm Polystyrene Assay Particles (3.3 [mu]m) 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.
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 DNA sequences were purified by denaturing
polyacrylamide gel electrophoresis to ensure size homogeneity.
Polystyrene beads were successively washed with phosphate-buffered 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 [mu]g/cm
2
bead surface area) for 30 min and then washed and suspended (~5 * 10
5
beads/[mu]l) in acetate buffer containing 2% bovine serum albumin (BSA). HNE-coated beads were stored at 4oC until use.
DNA ligands containing fluorescein either internally or near their 3'-ends were radiolabeled at their 5'-ends with [[gamma]-
32
P]ATP and T4 polynucleotide kinase. DNAs carrying fluorescein at their 5'-ends were radiolabeled at their 3'-ends with [[alpha]-
32
P]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 MgCl
2
and 6 mM KCl) were heated to 70oC for 3 min and cooled to room temperature to facilitate secondary
structure formation.
Gel-purified radiolabeled DNA (<50 pM) was incubated with varying amounts of HNE in the binding buffer
containing 0.02% human serum albumin (HSA) for 10 min at 37oC. DNA/protein mixtures were filtered through pre-wet nitrocellulose filters (0.2 [mu]m) and the filters immediately washed with 5 ml binding buffer.
The radioactivity retained on the filters was counted. Retention of DNA on the
filters in the absence of HNE was determined and used for background
correction. Assuming equimolar binding of DNA to HNE, a non-linear least squares method was used to fit the data using the software
package Kaleidagraph (Synergy Software, Reading, PA) to obtain the equilibrium
dissociation constant
K
d
(
6
).
Staining of HNE-coated beads was achieved by incubating beads (~3 * 10
5
) with varying concentrations of fluoresceinated oligonucleotides in 50 [mu]l binding buffer (100 mM Tris-HCl, pH 7.0, 150 mM NaCl, 6 mM KCl, 2 mM MgCl
2
, 0.5% BSA) for 30 min at room temperature. The beads were then washed with 2 ml
binding buffer and suspended in 0.5 ml for analysis. Staining with mouse anti-HNE was accomplished by the same method except that bound antibody was
detected by a second-step staining with FITC-labeled rat anti-mouse antibody (X56-FITC). To obtain comparative measurements between the
antibody and DNA ligand X-56 was stoichiometrically labeled with FITC. Analysis was performed on a
FACScantm flow cytometer (Becton Dickinson). The sensitivity of the flow cytometer
was adjusted so that the autofluorescence of the unstained beads was at the
very low end of the scale (first decade).
For the determination of
K
d
values the observed fluorescence signal was plotted against the log
concentration of the fluoresceinated ligand.
The number of fluoresceins per oligonucleotide was determined
spectrophotometrically. An extinction coefficient of 7200 M
-1
cm
-1
at 494 nm was used for both free fluorescein in solution and fluorescein
tethered to DNA. DNA concentrations were based on the extinction coefficients
13800, 6500, 10500 and 7900 M
-1
cm
-1
at 260 nm for A, C, G and T respectively. The relative quantum yield for each
fluorescein-labeled oligonucleotide was obtained by measuring the fluorescence
(excitation at 494 nm and emission at 518 nm) in binding buffer relative to the fluorescence of a solution of FITC.
The concentration of each oligonucleotide was adjusted so that the absorbance
at 494 nm was approximately the same and small differences from the absorbance
of the reference FITC solution were normalized (fluorescence * gain
-1
* OD
494
-1
).
In the present study we show that the attachment of fluorescein at different
locations on a DNA ligand for HNE did not decrease its binding affinity for its
protein target. This result indicates that the two structural motifs of DNA
proposed to be involved in protein binding (
6
) were not grossly altered in these constructs. Although the different DNA
constructs used in the present study retained their high affinity binding to
HNE, fluorescence intensities were quite sensitive to the position and the way
in which the fluorescein molecule was attached. Certain sequences gave barely
detectable signals in flow cytometry, whereas others generated signals that
were comparable with those of an antibody.
Differences in signal intensity observed in flow cytometry of various constructs
do not correlate with their binding affinities, nor do they correlate with the
number or the position of fluoresceins in each construct. To investigate
whether these observations could be due to the intrinsic fluorescence of each
sequence we analyzed fluorescence characteristics such as the relative quantum
yield and the average number of fluoresceins per molecule (data not shown).
These values did not correlate with the observed signal intensities of these
ligands, suggesting that the fluorescence behavior of these molecules is
complex. The proposed secondary structure of the sequence predicts that the two
ends are close to each other and thus the local environment of the two ends is
expected to be the same (
6
). However, fluoresceins attached to either the 3'- or 5'-ends exhibit significantly different signal
intensities. This could be due to differences in the way in which the two ends
of the oligonucleotide interact with amino acid residues on the surface of HNE,
changing the properties of the local environment of the fluorophore and
affecting the quantum yield of fluorescence. In general, however, attachment of
fluorescein through an ethylene glycol linker improved signal intensity. The
generality of the observations on the position of fluorescein on this ligand
may not be applicable to a different ligand. However, It is not difficult to
try several oligonucleotide constructs for a given ligand to identify the most
suitable position for fluoresceination that gives a maximal signal upon binding
to the target. Similarly, it is also possible to identify a position for a
fluorophore in a ligand that gives maximal quenching upon interaction with a
target, a feature that may be useful in other diagnostic applications, such as
homogeneous detection.
SELEX-derived oligonucleotide ligands exhibit monovalent interaction with their
targets, whereas naturally occuring antibodies are bivalent or multivalent. The
dimers used in the study were designed to provide a bivalent target
interaction. The two dimers were synthesized with a flexible ethylene glycol
linker between the two monomeric units. Affinities of the two dimers were
higher than the monomeric forms, probably due to an avidity contribution to the
molecular interaction.
For certain flow cytometric applications, where amplification of the signal is
necessary, second-step staining of the primary probe is advantageous. Using biotinylated DNA
and SA-PE as reagents for primary and secondary staining respectively we
demonstrated the feasibility of using oligonucleotides in second-step staining. The affinity of the DNA-HNE interaction measured by second-step staining was lower than that measured directly (compare
circles in Fig.
3
a and b). The decrease in affinity could be due to the streptavidin-biotin interaction influencing binding between HNE and DNA. This effect
may be overcome by changing the presentation of biotin on the oligonucleotide.
In antibodies relatively small target recognition (antigen binding) sites are
localized in a defined region in a structurally large protein. This feature
allows the attachment of reporter molecules to sites away from those required
for target recognition. SELEX-derived ligands, on the other hand, are generally small and contain only
the necessary functional groups to interact with their targets and therefore
may require additional appendages to bear reporter molecules without loosing
their function. The ethylene glycol linkages used in this study appear to meet
this requirement. Although antibodies carrying multiple fluoresceins are
brighter than probes with a single fluorescein, fluorescence quenching limits
the brightness of probes containing a large number of fluoresceins. We have
stained cells with a SELEX-derived oligonucleotide containing a single fluorescein. The intensity of
oligonucleotide staining is ~5-fold less than that obtained with a commercially available antibody
containing three to five fluoresceins per molecule (unpublished data),
indicating that probes with more than one fluorescein molecule are generally
better. Oligonucleotide ligands are not resticted in the number of reporter
molecules that can be attached to them. Various approaches can be deviced to
incorporate more than one fluorescein per oligonucleotide molecule (e.g.
complexation of a biotinylated oligonucleotide ligand with streptavidin labeled
with several fluorosceins) to enhance the sensitivity of oligonucleotide-based probes in flow cytometry (unpublished results).
Nucleic acid-based probes in flow cytometry may offer certain benefits. (i) Unlike
antibodies, oligonucleotides derivatized with various molecular probes
(fluorophores) at defined positions can be obtained by direct chemical
synthesis. Functionalities such as amino and thiol groups that are useful to
derivatize oligonucleotides can also be placed in an oligonucleotide chain
during synthesis (
7
). (ii) Although most flow cytometric studies are based on cell surface
proteins, detection of intracellular proteins has also been demonstrated (
8
). The small size of SELEX-derived oligonucleotide probes (<20 kDa) may be advantageous over structurally large antibodies (~160 kDa) when intracellular proteins are targeted. Nuclease
degradation of oligonucleotide-based probes can be overcome by using chemically modified oligonucleotide
random libraries for selection (
9
,
10
). (iii) The presence of the Fc region on antibodies causes undesirable non-specific binding to Fc receptors on cells, creating difficulties in the
interpretation of flow cytometric data (
11
). The use of oligonucleotides may overcome this problem. (iv) Antibody-based reagents are generally stored at low temperature due to their
thermal sensitivity. Nucleic acid ligands are generally thermostable and,
further, their native structure can be restored after thermal denaturation,
allowing them to be shipped and store at room temperature.
Overall, the data presented here suggests that oligonucleotide-based high affinity ligands may be useful as probes in flow cytometric
applications. The development of monoclonal antibodies to specific cell surface
proteins has significantly contributed to the diversification of flow
cytometric applications in both research and clinical applications. The
feasibility of the use of SELEX-derived oligonucleotides in flow cytometry is expected to expand the
repertoire of available probes, making the technique even more versatile.
We thank Barry Polisky, Larry Gold and Dan Nieuwlandt for critical reading of
the manuscript.
REFERENCES
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