Nucleic Acids Research

Materials

The full-length extracellular domain of human CD4 (amino acids 1-370) produced in a mammalian expression system (Chinese hamster ovary cells) was purchased from Dupont NEN (Boston, MA) as a 1 mg/ml solution in a buffer consisting of 350 mM NaCl, 50 mM 2-[N-morpholino] ethane sulfonic acid (MES), pH 6.0, and stored in -70°C until use. Polymethylmethacrylate (PMMA) beads of 6.1 µm in diameter with amino functional groups on the surface were purchased from Bangs Laboratories (Carmel, IN). Polystyrene beads (3.2 µm in diameter) were from IDEXX Laboratories (Westbrook, ME). Streptavidin (SA), Fluorescein isothiocyanate (FITC), Iodoacetamido-LC-biotin, NHS-LC-biotin and 5-iodoacetamidofluorescein were purchased from Pierce Chemical company (Rockford, IL). Guanosine-5[prime]-O-(2-thiodiphosphate) (GDP-[beta]-S) was purchased from Calbiochem (La Jolla, CA). SA conjugated to phycoerythrin (PE) was from Becton Dickinson (San Jose, CA). Deoxyoligonucleotides were synthesized by standard cyanoethyl phosphoramidite chemistry and purified by electrophoresis on denaturing polyacrylamide gels.

CD4 attached to SA beads (SA-CD4 beads)

Polymethylmethacrylate amino beads were washed sequentially as follows: once with 10 vol of phosphate-buffered saline (PBS) containing 0.1% sodium azide, twice with 3 vol of PBS containing 0.1% sodium dodecyl sulfate, and twice with PBS alone. The washed beads were then suspended at 3% (w/v) in PBS containing 1.5 mM NHS-LC-biotin and incubated with gentle mixing for 1 h at room temperature. The beads were then washed twice with 3 vol of PBS, and suspended and stored in PBS containing 0.1% sodium azide and 2% non-fat dry milk.

The above beads were suspended in PBS at 2.5% (w/v), mixed with SA (0.4 mg/ml) for 2 h at room temperature, and then washed five times with 6 vol PBS containing 0.1% sodium azide and 0.5% BSA. The beads were finally stored as a 2.5% (w/v) suspension in this buffer at 4°C.

Biotinylation of CD4 was performed by incubating CD4 in PBS (pH 7.4) at 1 mg/ml with 0.1 mM NHS-LC-Biotin for 2 h at room temperature. Unreacted biotin was removed by passing the reaction mixture through a Sephadex G-50 column. The extent of biotinylation was assayed by binding biotinylated CD4 to polystyrene beads coated with an anti-CD4 antibody (Leu3a) and staining with FITC-labeled SA as compared to staining with an appropriate FITC-labeled second anti-CD4 antibody (e.g., FITC-L120).

CD4 on L200 beads (L200-CD4 beads)

In this approach, CD4 was bound to an anti-CD4 monoclonal antibody that had been adsorbed onto polystyrene beads. Polystyrene beads (3.3 µm in diameter) were washed with 50 mM MES buffer (pH 6.0) and suspended at 0.5% (w/v) in the same buffer. The anti-CD4 monoclonal antibody (L200) was added to 0.5 µg/cm² of bead surface area and incubated for 30 min at room temperature. The beads were then washed twice with 2 vol of PBS containing 0.5% BSA and stored in this buffer at a bead concentration of ~3 × 10⁸/ml.

Immediately prior to use, 0.6 µg of CD4 in 100 µl of PBS containing 0.1% sodium azide and 0.5% BSA was incubated with 10 µl of L200 beads (3.2 × 10⁶ beads) at room temperature for 1 h with gentle mixing. Unbound CD4 was removed by washing the beads twice with 200 µl of the same buffer.

Affinity selection

The starting random sequence 2[prime]-F-pyrimidine-containing RNA library [5[prime]-GGGAGACAAGAAUAAACGCUCAA-[N]₄₀-UUCGACAGGAGGCUCACAACAGGC-3[prime]] consisting of a 40 nt contiguous random region was obtained by in vitro transcription of the corresponding synthetic DNA template. One nanomole of gel-purified DNA template was subjected to four rounds of polymerase chain reaction (PCR) in a 1 ml reaction volume with 5[prime]-TAATACGACTCACTATA GGGAGACAA-3[prime] (5[prime]-primer) and 5[prime]-GCCTGTTGTGAGCCTCCTGTCGAA-3[prime] (3[prime]-primer). The resulting PCR products were transcribed in vitro by T7 RNA polymerase (1000 U) in a 2 ml reaction consisting of 3 mM each of 2[prime]-F-CTP and 2[prime]-F-UTP, 1 mM each of ATP and GTP, 50 µCi of [[alpha]-³²P]ATP, 40 mM Tris-HCl (pH 8.0), 12 mM MgCl₂, 1 mM Spermidine, 5 mM DTT, 0.002% Triton X-100 and 4% polyethelene glycol (w/v) for 10-12 h at room temperature. After transcription, the template DNA was digested with RNase-free DNAse I. The full-length transcription products were purified on 8% polyacrylamide gels ran under denaturing conditions. In subsequent rounds of selections, in vitro transcriptions were carried out in 500 µl reaction volume with 100 pmol template DNA and 100 U T7 RNA polymerase.

Affinity selection was initiated with 2 nmol of the 2[prime]-F-pyrimidine-containing RNA random sequence library. RNA suspended in 1 ml PBS containing 2 mM MgCl₂ (binding buffer) was heated to 80°C for 3 min and chilled on ice, before being transferred to room temperature. Once equilibrated at room temperature, 20 µl of SA-CD4 beads were added to the RNA suspension, incubated at room temperature for 15 min with gentle mixing. Beads were sedimented by centrifugation in a picofuge for 1 min and the supernatant was removed. RNA molecules that were trapped between beads were removed by a single wash with 100 µl of the binding buffer. The bead-retained RNA was recovered by extracting with 1:2 (v/v) mixture of neutralized phenol:fresh 7 M urea. Beads were then re-extracted with a fresh aliquot of phenol/urea mixture and the two aqueous phases combined. Extracted RNA was ethanol precipitated in the presence of 5 µg tRNA as a carrier. The recovered 2[prime]-F-pyrimidine-containing RNA was reverse transcribed by avian myeloblastosis virus reverse transcriptase in a 50 µl reaction volume consisting of 50 mM Tris-HCl (pH 8.3), 60 mM NaCl, 6 mM Mg(OAc)₂, 10 mM DTT, 0.4 mM dNTPs, 5 U of the reverse transcriptase and 50 pmol of the 3[prime]-primer at 48°C for 45 min. The resulting cDNA was amplified by PCR with the 5[prime]- and the 3[prime]-primers, and the resulting PCR products were used as the template for in vitro transcription to obtain RNA for the next round of selection.

In subsequent rounds, the enriching 2[prime]-F-pyrimidine RNA libraries were pre-exposed to 50 µl of the same beads lacking CD4 (control beads) to remove sequences that bind to sites other than CD4. In addition, during the course of selection, wash volumes (50-1000 µl) and washing times (10-60 min) were gradually increased to effectively remove low affinity molecules and also to enrich for molecules with slow off-rates.

Selections were started with SA-CD4 beads (1-6 rounds) and then switched to L200-CD4 beads to eliminate the enrichment of undesirable RNA molecules that bind to components other than CD4. To further increase the stringency of the selection of high affinity binding aptamers to CD4, selection rounds 12-15 were carried out with low density L200 beads on which the number of CD4 molecules per bead was decreased by ~10-fold.

The cDNA derived from the 15th round of selection was amplified by PCR with primers (5[prime]-CAGAAGCTTAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3[prime] and 5[prime]-GACTGGATCCGCCTGTTGTGAGCCTCCTGTCGAA-3[prime]) that introduce BamHI and EcoRI restriction sites at the termini of the resulting duplex DNA. The duplex DNA was gel purified under native conditions, digested with BamHI and EcoRI and cloned into plasmid pUC18 vector that has been previously digested with the same enzymes. Clones were sequenced by standard dideoxy sequencing technique.

Preparation of biotinylated and fluoresceinated RNA aptamers

The strategy used to derivatize RNA with either biotin or fluorescein is summarized in Figure 1. To prepare RNA derivatized with biotin (or fluorescein; see below), in vitro transcriptions were carried out in a reaction mixture containing a 10-fold excess of GDP-[beta]-S over GTP. Under these conditions GDP-[beta]-S is incorporated at the 5[prime]-end of the transcript (20). Approximately 2 nmol of gel purified RNA transcripts were suspended in 100 µl of a buffer consisting of 50 mM ammonium bicarbonate (pH 8.0), 2 mM EDTA and 4 mM DTT. After a 15 min incubation at room temperature, 300 µl of 4 mM iodoacetyl-LC-biotin in N,N-dimethyl formamide was added, mixed and the pH of the reaction mixture was adjusted to 7.5-8.0 with 2 N NaOH. The reaction was allowed to proceed at room temperature for 8-10 h. Unreacted biotin was removed by passing the RNA suspension through a 30 kDa molecular weight cutoff spin filter (Schleicher & Schull). The filter-retained RNA was washed three times with 400 µl TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0), recovered and precipitated with ethanol.

Figure 1. Schematic representation of strategies used to derivatize in vitro-transcribed RNA. (a) Derivatization of RNA with biotin for the preparation of multivalent aptamer complexes containing different fluorophores. (Single aptamer molecules labeled with fluorescein were obtained by treating RNA carrying terminal [beta]-thiophosphate with 5-iodoacetamidofluorescein instead of iodoacetyl-LC-biotin that was used for biotinylation of aptamers.) (b) Making multivalent RNA ligands labeled with PE or fluorescein. In this approach, the biotinylated RNA was incubated with SA conjugated to PE or to fluorescein. The reaction mixture was then fractionated on Superose 6[trade] columns to remove unreacted RNA and SA-fluorophore.

a    b

To derivatize the in vitro-transcribed RNA with fluorescein, DTT-treated RNA (1-2 nmol) suspended in 50 µl of a buffer consisting of 50 mM ammonium bicarbonate (pH 8.0), 2 mM EDTA and 10 mM DTT, was incubated with 50 µl of 200 mM 5-iodoacetamidofluorescein in N,N-dimethyl formamide for 2-3 h at room temperature. Fluoresceinated RNA was purified as described above for biotinylated RNA.

Preparation of SA-PE:RNA conjugates

RNA, bearing biotin at the 5[prime] end, was mixed with PE conjugated to SA (SA-PE) at a 3:1 molar ratio of RNA:SA-PE in PBS buffer (pH 7.4) and incubated for 16 h at 4°C. The mixture was subjected to size exclusion chromatography on a Superose6[trade] HR 10/30 column using an elution buffer consisting of 50 mM sodium phosphate, 150 mM NaCl, 2 M urea and 0.1% NaN₃ (pH 7.2). Fractions (0.35 ml) were collected at a flow rate of 0.2 ml/min, and those containing SA-PE:RNA conjugates were pooled. Urea was removed by passing the SA-PE:RNA complexes through Sephadex G-50 spin columns equilibrated with the binding buffer. The stoichiometry of RNA and PE in complexes were calculated by measuring UV absorption of the complex at 260 nm (for RNA) and 565 nm (for PE).

Flow cytometric analysis

All flow cytometric analyses were performed on a FACScan[trade] model flow cytometer (Becton Dickinson Immunocytometry systems, San Jose, CA). Binding of RNA or antibodies to CD4 presented on beads was analyzed by incubating beads(~9 × 10⁴ SA-CD4 beads or ~2.5 × 10⁵ L200-CD4 beads) in 50 µl volume of PBS buffer containing 2 mM MgCl₂ and 1 mg/ml BSA for 20 min at room temperature in the dark. Beads were then washed with 2 ml of the same buffer, suspended in 0.5 ml of the buffer and subjected to flow cytometric analysis within 30 min.

Binding of RNA aptamers (or antibodies) to cell surface CD4 was determined using the same procedure described for beads, except that ~10⁵ cells for B6 and BW5147, and 5 × 10⁴ CD4 positive T-helper lymphocytes/50 µl of peripheral blood mononuclear cells (PBMCs) were used for a given experiment.

Binding between aptamers complexed to SA and CD4 on beads may be multivalent in nature. However, in calculating equilibrium dissociation constants (K_d values) we used a simple monovalent binding model to avoid the complexity contributed by avidity and polyvalency. The K_d values were calculated by using non-linear least squares method as described (7). The amounts of CD4 on L200 and SA beads were estimated to be 130 000 and 300 000 molecules/beads, respectively. This was based upon a comparison of staining intensities of beads with those of lymphocytes containing ~100 000 CD4 molecules/cell. Hence, the total amount of bead-bound CD4 used in a single staining experiment was estimated to be <0.1 pmol.

Aptamer enrichment

A collection of aptamers that bind human CD4 with high affinity was enriched from an initial random sequence library of 2[prime]-F-pyrimidine-containing RNA with ~10¹⁴ molecular complexity. Enrichment was achieved by iterative affinity selection using CD4 presented on beads. Two types of affinity matrices, biotinylated CD4 captured on SA beads (SA-CD4 beads) and CD4 captured onto beads coated with an anti-CD4 antibody (L200-CD4 beads), were used for selection. Enrichment of sequences that recognize molecular components of the affinity matrices other than CD4, for example, SA or anti-CD4 antibody, was curbed by switching between the two types of affinity matrices during selection.

Figure 2. Binding analysis of the affinity-enriched RNA library (15th round) and the unselected random sequence library to L200 (0.01% w/v) and SA (0.025% w/v) beads with and without CD4. Both RNA libraries were directly labeled with fluorescein at the 5[prime] ends. Closed circles: binding of the enriched RNA library to CD4 on L200 beads; closed squares: binding of the enriched RNA library to CD4 on SA beads; open circles: binding of the enriched RNA library to L200 beads; open squares: binding of the enriched RNA library to SA beads; triangles: binding of the unselected random sequence RNA library to SA beads.

Binding of aptamers to CD4 was investigated by flow cytometric analysis. This required the attachment of a fluorophore to the aptamers. The strategy used to derivatize in vitro transcribed RNA aptamers with fluorophores is shown in Figure 1. This strategy utilizes the introduction of reactive [beta]-thiophosphate group at the 5[prime] position of in vitro transcribed RNA (20).

Figure 2 shows the binding characteristics of the enriched aptamer library obtained after 15 rounds of affinity selection on CD4 beads. Compared to the unselected starting library (closed triangles), the enriched library after 15 rounds of selections showed significant improvement of binding to SA-CD4 beads (closed squares) as well as to L200-CD4 beads (closed circles). There was no detectable binding to control beads lacking CD4 (open symbols), indicating that affinity-enriched RNA library bound to CD4 on beads.

Recognition of cell surface CD4 by affinity-enriched aptamer library

The observation that the affinity-enriched RNA library binds to recombinant CD4 on beads prompted us to investigate its binding to CD4 expressed on cell surfaces. This was initiated with a mouse T cell line transfected with human CD4 (B6 cells). The parent mouse cell line (BW5147), lacking human CD4, was used as a control. Figure 3 compares the results of cellular staining using the affinity-enriched aptamer library (from the 15th round) with those of an anti-CD4 antibody (Leu3a). In this experiment both probes were labeled with fluorescein. Figure 3a-1 and b-1 show the auto fluorescence of the two cell types. Fluoresceinated anti-CD4 antibody binds strongly to B6 cells (b-2), but not to BW4157 (a-2). Analogous to anti-CD4 antibody, the fluoresceinated aptamers also show exclusive binding to B6 cells (b-3) and not to the control BW4157 cells lacking human CD4 (a-3). The fact that the enriched aptamer library does not bind to BW4157 cells (the parent mouse T cell line) containing mouse CD4 with 55% sequence identity to the human protein indicates that these aptamers are specific for human CD4.

Figure 3. Staining of mouse T-cell lines with and without human CD4 on surface with a monoclonal antibody (Leu3a-FITC) and the affinity-enriched RNA library directly labeled with fluorescein. Mouse T-cell line (BW5147) (a-1-a-3) and the same cell line transfected with human CD4 (B6) (b-1-b-3) were incubated at room temperature for 20 min (50 000 cells in 50 µl final volume) in PBS, 2 mM MgCl₂ and 0.1% BSA. Cells were washed with 2 ml of buffer and suspended in 0.5 ml of buffer for analysis. In each plot the ordinate represents the frequency of events (or number of cells), whereas the abscissa indicates the fluorescence intensity. (a-1 and b-1) Autofluorescence; (a-2 and b-2) staining with 60 nM Leu 3a-FITC that stains human CD4; (a-3 and b-3) staining with 200 nM fluoresceinated affinity-enriched RNA aptamer library (round 15).

Aptamer recognition site on CD4

As indicated in Figure 2, the enriched aptamer library bound similarly to CD4 on both types of beads, indicating that the aptamer binding site(s) on CD4 is accessible when CD4 was presented on both types of beads. The presentation of the CD4 molecule on the two types of beads is different. L200 antibody recognizes the V1 domain of the CD4 molecule and thus, this domain is at least partially sterically hindered on L200 CD4 beads. On the other hand, staining of SA-CD4 beads with a panel of anti-CD4 antibodies indicated that the two termini of the CD4 molecule (i.e., V1 and V4 domains) are equally accessible (data not shown), suggesting that the biotinylation site on CD4 is predominantly in the middle of the molecule.

To determine the approximate binding site(s) on CD4 of the enriched aptamer library, we performed an antibody blocking experiment with a panel of monoclonal antibodies whose binding epitopes on CD4 are known (Fig. 4a) (21). In this experiment, CD4, on either SA beads or L200 beads, was preincubated with a given unlabeled monoclonal antibody before being exposed to the enriched aptamer library labeled with fluorescein. The results of this experiment are summarized in Figure 4b. Antibodies L113, L121 and L120 that bind to the V4 region completely blocked aptamer binding, whereas those binding elsewhere on CD4 exerted varying degrees (~10-50%) of blockage. Overall, these data suggest that the majority of aptamers in the enriched library interact with the V4 region of CD4. The ability of some antibodies that bind to the V1 region and one antibody that binds to the V2 region to partially block the RNA binding may suggest the existence of aptamers that recognize these two domains of CD4 as well. Alternatively, the latter observation could be due either to long range steric hindrance or to the conformational changes in CD4 caused by antibody binding.

Figure 4. (a) Schematic view of binding sites on CD4 of different monoclonal antibodies that were used to probe the aptamer binding site(s) on CD4. (b) Interference (or blocking) of the binding of RNA aptamer library (round 15) to CD4 on beads by different CD4-specific antibodies. Beads were preincubated with 1 µg of an unlabeled antibody for 10 min at room temperature prior to the addition of fluoresceinated aptamer library to a final concentration of 100 nM. CD4 on L200 beads was used for experiments with L113, L117 and L121, whereas CD4 on SA beads was used for the rest.

Individual aptamers with high-affinity binding to CD4

Since further selections beyond round 15 did not significantly improve the affinity of RNA to CD4 on beads, the sequence complexity of the 15th round library was analyzed by molecular cloning and sequencing. As shown in Figure 5a, sequences could be grouped into four classes based on sequence similarity. Approximately 60% of the identified sequences (total of 65) fell into classes I, II and III. The remaining sequences were grouped under class IV. Members of classes I, II and III, show a high degree of sequence similarity within each class. On the other hand, class IV represents a group of sequences that are highly rich in pyrimidines and have little or no similarity to one another. Moreover, class IV sequences do not appear to contain any noticeable secondary structural motif. A close examination of the sequence sets in classes I, II and III revealed the occurrence of self complementary regions (indicated by arrowheads), suggesting that these sequences have the potential to exist in stem-loop structures (Fig. 5b). In all three cases helical segments of stem-loop structures constitute nucleotides from the variable region (shown in blue, purple and red) and from the 3[prime]-fixed region (indicated by lower case green letters). Stem-loop structures predicted for aptamers in classes I, II and III are different with respect to both helical and loop regions. Hence, the secondary structure that appears to be important for high-affinity recognition seems to tolerate some degree of sequence variations. Further studies are required to understand exactly how the predicted secondary structures participate in target recognition. Nucleotides from the 3[prime]-fixed region form the base of the stems in secondary structures predicted for aptamers in classes I, II and III. However, the requirement of the 3[prime] fixed region for the function of aptamers could only be discerned by determination of the minimal sequence required for high-affinity binding to CD4.

Figure 5. (a) 2[prime]-F-pyrimidine-containing RNA aptamer sequences derived from affinity selection on recombinant human CD4. Only the 40 nt random region is shown. Certain sequences contained 39 nt instead of 40. Sequences were grouped into four classes. Only a few examples of class IV sequences are shown. Nucleotide stretches shown in blue, purple and red are self complementary, as indicated by arrowheads. Nucleotides in green have a complementary region in the 3[prime]-fixed region. (b) Predicted secondary structures for three aptamers representing classes I (Aptamer 2), II (Aptamer 54) and III (Aptamer 7). For each aptamer, only the secondary structural motif is illustrated. Nucleotides shown in lower case letters are from the 3[prime]-fixed region.

a    b

To determine binding affinities of individual aptamers, complexes of aptamers with SA-PE were prepared for several members from each class. Fluorescence intensities of CD4 beads (L200 beads) stained with aptamer:SA-PE complexes were measured by flow cytometry. The mean fluorescence of the beads was plotted against the concentration of aptamer:SA-PE complex. The equilibrium dissociation constant (K_d) was calculated as described in Materials and Methods. Figure 6 shows binding curves for four representative aptamers, one from each class. Representative aptamers from classes I, II and III (clones 9, 12 and 7) showed high-affinity binding to CD4 beads. In contrast, the RNA sequence from class IV (clone 31) showed no binding above background levels to CD4 beads. A similar analysis of several other clones showed that only aptamers belonging to classes I, II and III exhibited a high-affinity interaction with CD4, but not sequences from class IV.

Figure 6. Binding analysis of a representative aptamer from each of the four classes. Biotinylated RNA aptamer sequences complexed to SA-PE were incubated with CD4 presented on L200 beads and then analyzed by flow cytometry. The mean fluoresence of beads was plotted against the concentration of Aptamer:SA-PE complex. The ratios of aptamer to SA-PE are listed inTable 1.

Aptamers in aptamer:SA-PE complexes used to evaluate their binding interactions with CD4 were multimeric in nature; i.e., more than one molecule of an aptamer was complexed to SA attached to PE. Contributions from avidity must be considered under such conditions where polyvalent interactions may exist. As a consequence, it is expected that the apparent K_d values of aptamers summarized in Table 1 could be lower than the `true' solution K_d values measured under conditions of monovalent interactions. We were unable to measure solution K_d values of aptamer interaction with CD4 using the nitrocellulose filter binding technique due to poor retention of the protein on nitrocellulose filters.

Overall, the K_d values in the subnanomolar range indicate a tight binding of RNA aptamers to CD4. The affinity of the enriched library was slightly lower than those of individual aptamers from classes I, II and III. This may be due to the presence of RNA sequences classified in class IV in the enriched library (~40%) that do not bind to CD4.

Antibody blocking experiments with several representative aptamers from classes I, II and III were carried out to investigate the binding sites of individual aptamers on CD4. The results were qualitatively similar to those observed for the enriched library (Fig. 4a). Antibodies that bind to the V4 domain of CD4 effectively blocked individual aptamer binding (data not shown). This indicates that affinity-selected aptamers recognize the V4 domain of CD4.

Staining of human lymphocytes by individual aptamers

The positive staining observed with the enriched aptamer library on mouse T cells expressing human CD4 (Fig. 3) indicated that oligonucleotide aptamers selected against a recombinant protein would recognize the same protein naturally expressed on a cell surface. To investigate the specific binding of an RNA aptamer to CD4⁺ cells in a heterogeneous mixture of cells, we chose human PBMCs. In this experiment, an antibody to CD3 was used to identify the T cell subset of PBMCs (Fig. 7). The abscissas of Figure 7 represent the fluorescence intensity of CD3(Leu4)-Cy5/PE. Gate (or box) R1 of Figure 7a shows cells that stained with CD3(Leu4)-Cy5/PE alone and represents the total T cell population in this sample. In Figure 7b, CD4⁺ T cells (T-helper cells) are stained with a PE-labeled antibody to CD4 (Leu 3a), as well as an antibody to CD3. The population of cells positive for both antibodies, colored in red (gate R2), represents 61% of the total T cells. Figure 7c shows the results when an aptamer to CD4 (aptamer-9:SA-PE) was used in place of the antibody to CD4 in a double staining experiment. A similar result was obtained with the aptamer and, once again, the double positive CD4⁺ cell population, shown in red, represents 61% of the total T cells. Although there is a difference in intensity of staining between the two classes of probes, both stained a similar subset of T cells, and overall, the performance characteristics of the RNA aptamer are similar to those of the antibody in this flow cytometric assay.

Simultaneous staining of cell surface CD4 with an antibody to CD4 and an aptamer to CD4

Antibody blocking experiments revealed that an antibody recognizing the V1 domain of CD4 (i.e., Leu3a) did not noticeably interfere with the binding of an aptamer (i.e., aptamer-12). Thus, the antibody Leu3a and RNA aptamer-12 should bind CD4 simultaneously. To test whether this can be achieved, PBMCs were simultaneously stained with Leu3a and aptamer-12 and analyzed by flow cytometry (Fig. 8). In this experiment, PBMCs were stained with three different probes, each labeled with a distinct fluorophore: aptamer-12 labeled with SA-PE (Aptamer-12:SA-PE); an antibody to CD4 labeled with FITC (Leu3a-FITC); and an antibody to CD14 labeled with PerCP (LeuM3-PerCP). Staining patterns of PBMCs produced by LeuM3-PerCP and Leu3a-FITC show two different CD4⁺ cell populations (Fig. 8). T helper cells (CD4⁺ T cells), with a high density of CD4, stained brightly with the anti-CD4 antibody (Fig. 8a; red). On the other hand, monocytes, with a low density of CD4 (22), were dimly stained (Fig. 8a; green). This assignment is confirmed with CD14 (LeuM3)-PerCP, a monocyte marker, which only stained the cell population with a low density of CD4 (green). The staining pattern in Figure 8b, obtained with Aptamer-12:SA-PE, is very similar to that obtained with the anti-CD4 antibody (Fig. 8a). Figure 8c displays cells stained simultaneously with the two classes of probes to CD4. Cells simultaneously stained by the two CD4 probes lie on a diagonal, thus both populations of cells, T cells and monocytes, that were stained by the antibody Leu3a were also stained by the RNA aptamer. This suggests that these two probes are binding a common target, CD4 on cell surface.

Figure 7. Staining of T lymphocytes in a human PBMC preparation with either a monoclonal antibody to CD4 (Leu 3a-PE) or an RNA aptamer selected for CD4 binding (Aptamer-9:SA-PE). PBMCs were incubated in a buffer consisting of PBS, 2 mM MgCl₂ and 0.1% BSA either with two antibodies: CD3-Cy5-PE and CD4(Leu 3a)-PE, or with an antibody and RNA complex: CD3-Cy5-PE and Aptamer-9:SA-PE for 15 min at room temperature. Cells were washed with 2 ml and suspended in 0.5 ml of the same buffer for analysis. In all three panels abscissas indicate the staining intensity of CD3-CY5-PE that binds to T cells. (a) Staining with 14 nM CD3-Cy5-PE alone; (b) double staining with 14 nM CD3-Cy5-PE (abscissa) and 5 nM CD4(Leu 3a)-PE (ordinate); and (c) double staining with 14 nM CD3-Cy5-PE (abscissa) and 150 nM Aptamer-9:SA-PE (ordinate). Gate R1 (in green) includes CD3⁺ T cells, whereas gate R2 (in red) includes T cells that are stained with either CD4(Leu 3a)-PE or Aptamer-9:SA-PE and represent CD4⁺ T cells. The observed percentage of CD4⁺ T cells in each panel is also shown. This figure only shows cells selected by gating on lymphocytes by scatter.

Figure 8. Simultaneous staining of human PBMCs with an affinity-selected RNA aptamer and an antibody to CD4. PBMCs (~1.2 × 10⁵) in 50 µl of PBS containing 2 mM MgCl₂ and 0.1% BSA were incubated with 25 nM Aptamer-12:SA-PE, 2.5 nM CD4 (Leu3a)-FITC and 15 nM CD14 (LeuM3)-PerCP at room temperature for 30 min. Cells were washed with 2 ml of buffer, suspended in 0.5 ml of buffer, analyzed and cell staining with these three reagents compared. (a) Staining pattern observed with CD4 (Leu3a)-FITC [staining CD4⁺ T cells (red) and monocytes (green)] and CD14 (LeuM3)-PerCP [staining monocytes only (green)]; (b) staining pattern observed with (Aptamer-12:SA-PE) [staining CD4⁺ T cells (red) and monocytes (green)] and CD14 (LeuM3)-PerCP [staining monocytes only (green)]; (c) staining pattern observed with (Aptamer-12:SA-PE) and CD4 (Leu3a)-FITC [staining CD4⁺ T cells (red) and monocytes (green)].

Table 1. Equilibrium dissociation constants (K_ds) of several individual aptamers and the 15th round affinity-enriched RNA library in their SA-PE complex forms for interaction with CD4 presented on L200 beads

Aptamer	Stoichiometrya	Kd (complex)b
7	3.2	0.5 ± 0.05
9	3.3	0.9 ± 0.09
12	1.9	0.5 ± 0.07
21	2.9	0.4 ± 0.02
Enriched library (15th round)	3.2	1.5 ± 0.08

Stoichiometry was obtained by absorption spectroscopy at 260 nm (for RNA aptamer) and 565 nm (for PE) for each Aptamer:SA-PE complex.
^aAptamer molecules per SA-PE conjugate.
^bApparent K_d values in nM.

DISCUSSION

Previous studies utilizing aptamers labeled with fluorescein indicated that oligonucleotides can be used in flow cytometry (5,10). In this report, we have further expanded the utility of aptamers in flow cytometry by demonstrating the attachment of the fluorescent protein PE and a direct comparison with antibodies. This study was facilitated by the identification of nuclease-resistant 2[prime]-F-pyrimidine-containing RNA aptamers with high-affinity and specificity for human CD4.

The starting random sequence library of 2[prime]-F-pyrimidine-containing RNA showed little or no detectable binding to CD4. However, after 15 rounds of affinity selection, the enriched library showed marked enhancement of binding to CD4 on beads. In spite of exhaustive counter selection, binding analysis of individual sequences revealed that ~40% of the enriched library contained sequences that did not interact with CD4. These sequences, grouped in class IV, were rich in pyrimidines and showed no sequence similarity among themselves. The remaining 60% of the sequences, grouped in classes I, II and III, were found to be aptamers that bound CD4 with high affinity and specificity. These aptamers, with high affinity binding to CD4, have predicted stem-loop structures.

Flow cytometric analysis requires that probes are conjugated to fluorescent reporter molecules. Previously, we described the use of a fluorescein-conjugated DNA aptamer in flow cytometry (5). We found that the signal intensity of fluorescein was dependent upon the site and the way of its attachment to the DNA sequence. In the case of chemically synthesized aptamers, the optimal site of fluorophore attachment such that they retain the functions of both the fluorophore and the aptamer can easily be determined by analyzing several different constructs. On the other hand, sites for facile conjugation to an RNA molecule produced by in vitro transcription are rather limited. In this study, we devised a strategy to conjugate fluorophores at the 5[prime]-end of an RNA aptamer sequence through the introduction of reactive [beta]-thiophosphate moiety. Conjugation to the 5[prime] end of RNA with either a small fluorophore like fluorescein or a structurally complex and a large fluorophore like PE preserved both the affinity characteristics of aptamers and the fluorescent characteristics of fluorophores, allowing us to use such conjugates effectively in flow cytometry. Here we demonstrated that aptamers complexed with SA-PE retain their binding capacity to their cognate target, CD4. It is worth noting that the aptamer which mediates binding to CD4 is relatively small (~25 kDa) compared to the rest of the complex consisting of 60 kDa SA and 240 kDa PE. A logical extension of this result is the possible use of aptamers conjugated to drug molecules for targeted drug delivery.

The apparent K_d values, as obtained by flow cytometric analysis, of individual aptamers for their interactions with recombinant CD4 on beads were in the 0.4-1 nM range. These affinity measurements were obtained with aptamer:SA-PE conjugates consisting of more than one aptamer molecule per complex and CD4 presented on a solid support. Under these conditions of polyvalency, avidity is expected to play a role in molecular interactions. Hence, it is possible that the `true' solution K_d values of aptamers are actually higher than those calculated in this study. On the other hand, the aptamer:SA-PE complexes studied contained 2-3 aptamers per complex molecule, a valency which is not very different from the bivalency of most antibodies. Thus, the affinities of aptamer:SA-PE complexes can be compared to those of antibodies. Bi-specific antibodies that combine the specificities of two different antibodies have been engineered for various applications, including immunodiagnostics and therapy. The generation of such bi-specific antibodies is a tedious process requiring the manipulation of genes, the expression of correct constructs in cell lines and a final purification of antibodies (23). However, unlike antibodies, aptamers are produced by chemical synthesis, a controlled and reproducible process. Hence, the production of bi-specific (or even multi-specific) aptamer complexes is relatively easier than the production of bi-specific antibodies. Dimeric aptamers with specific linkers can be synthesized directly by solid phase synthesis (5,24). Alternatively, biotinylated monomers with different specificities can be complexed with SA to obtain multimeric complexes of different specificities.

Flow cytometry allows for the measurements on cells of simultaneous interactions of multiple probes labeled with distinct fluorophores. Here we demonstrated the simultaneous binding of an aptamer and an antibody to CD4. This observation suggests that such an aptamer/antibody pair could be used in a sandwich ELISA, similar to an assay previously described for vascular endothelial growth factor (6). These results suggest that aptamers, or aptamers in conjunction with antibodies, may be useful in the development of novel diagnostic assays.

Overall, the results of the present study demonstrate that aptamers with high affinity and specificity can be used in flow cytometry. There are several attractive features inherent to aptamers that are not common to antibodies. Aptamers are relatively small in size, low in complexity and lack Fc receptors. They are produced by controlled, reproducible and accurate chemical synthesis with little or no lot-to-lot variation. Aptamers are also stable upon storage and can be shipped and stored at room temperature. Once denatured aptamers can be renatured fairly easily and quickly. No animals or cell lines are necessary for the selection or production of aptamers and hence, it is possible to raise aptamers to target molecules that are difficult to use in vivo, such as toxins and molecules with poor immunogenicity. Aptamers can be readily conjugated to a variety of reporter molecules and the site of conjugation can be controlled as well. Unlike antibodies, the active structures of aptamers can be established by changes in temperature, metal ions, salt concentration and pH (8,10,24-26). As a result, aptamers can be used as sensors or molecular switches that respond to those stimuli, making them versatile and attractive for different diagnostic platforms.

Several decades ago, the discovery of monoclonal antibodies made a great impact upon the expansion of technologies based on molecular recognition. Today, a different class of molecules, aptamers, is emerging as being capable of meeting the requirement for molecular recognition. Consequently, aptamers are expected to play a role in therapeutic and diagnostic applications. Accumulating evidence indicates that aptamers can work together with antibodies in a variety of diagnostic assay formats, further expanding their utility.

ACKNOWLEDGEMENTS

We thank Barry Polisky and Larry Gold of NeXstar Pharmaceuticals Inc. for critical reading of the manuscript. We also acknowledge helpful discussions with Vernan Maino of Beckton Dickinson Immunocytometry Systems.

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*To whom correspondence should be addressed. Tel: +1 303 546 7629; Fax: +1 303 444 0672; Email: sumedha@nexstar.com

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