A ssDNA Aptamer That Blocks the Function of the Anti-FLAG M2 Antibody

Abstract

Using SELEX (systematic evolution of ligands by exponential enrichment), we serendipitously discovered a ssDNA aptamer that binds selectively to the anti-FLAG M2 antibody. The aptamer consisted of two motifs (CCTTA and TGTCTWCC) separated by 2-3 bases, and the elimination of one or the other motif abrogated binding. The DNA aptamer and FLAG peptide competed for binding to the antigen-binding pocket of the M2 antibody. In addition, the aptamer eluted FLAG-tagged proteins from the antibody, suggesting a commercial application in protein purification. These findings demonstrate the feasibility of using SELEX to develop ssDNA aptamers that block the function of a specific antibody, a capability that could lead to the development of novel therapeutic modalities for patients with systemic lupus erythematosus, rheumatoid arthritis, and other autoimmune diseases.

Figure 1

SELEX with or without the Flag-Stn1 protein yields the same bipartite consensus. A randomized pool of ssDNA molecules was incubated with an in vitro translation system programmed with either a Flag-Stn1 plasmid (Flag-Stn1 present) or water (Flag-Stn1 absent), after which protein/DNA complexes were recovered by immunoprecipitation using the anti-Flag M2 antibody. After six successive rounds of selection, the remaining oligonucleotides were cloned and sequenced. (a) Oligonucleotides selected in the presence of the Flag-Stn1 protein. The selected ssDNA molecules share a common bipartite consensus, which consists of a CCTTA (pink) and TGTCTWCC (green) motifs. Nonnucleotide letters denote ambiguous sequencing reads (R = A/G, Y = C/T, S = C/G, W = A/T, K = T/G, M = A/C). (b) Oligonucleotides selected in the absence of the Flag-Stn1 protein. The same bipartite consensus is shared among the ssDNA molecules selected by the mock-programmed in vitro translation system.

Figure 2

The bipartite ssDNA consensus binds directly to the anti-Flag M2 antibody. (a) Sequence and graphical representation of the probes used. All probes were labeled at the 5′-end with [³²P]. Probes were designed to carry none, one, or both of the identified motifs. Motif A = CCTTA (pink). Motif B = TGTCTWCC (green). (b) The anti-Flag M2 antibody binds selectively to probe ABA. The indicated probes (80,000 cpm) were incubated with the listed antibodies (1 μg), after which protein/DNA complexes were resolved by electrophoresis in a native polyacrylamide gel. (c) Titration of the M2 antibody. A constant amount of probe ABA was incubated with an increasing concentration of M2 antibody, and the protein/DNA complexes were resolved by native electrophoresis. At the higher antibody concentrations, a larger protein/DNA complex is observed (top arrow), which may represent the product of antibody oligomerization. (d) Binding isotherm of probe ABA interacting with the anti-Flag M2 antibody. The scatter plot shows the amount of ABA bound (amount present in both protein/DNA complexes) as a function of antibody concentration. Dotted line shows fitting of the data to a “one site” saturation binding curve. Nonlinear regression of the data allowed for the determination of the dissociation constant (K_D = 80 ± 7 nM). Error on the value of the K_D represents the 95% confidence interval of the best-fit curve.

Figure 3

The 3XFLAG peptide blocks the binding of ABA to the M2 antibody. (a) The 3XFLAG peptide prevents formation of the ABA/M2 complex. The M2 antibody was incubated with an increasing concentration of 3XFLAG peptide after which the [³²P]-ABA probe was added. Protein/DNA complexes were resolved by electrophoresis in a native polyacrylamide gel. (b) The 3XFLAG peptide elutes the ABA probe already bound to the M2 antibody. The [³²P]-ABA was first captured by magnetic beads coated with the M2 antibody. The beads were then incubated with an increasing concentration of 3XFLAG peptide and the amount of [³²P]-ABA released and remaining on the beads was counted by scintillation (n = 1).

Figure 4

3XABA oligonucleotide blocks the interaction of Flag-tagged proteins with the M2 antibody. (a)-(b) The 3XABA oligo blocks the binding of Flag-TRF2^ΔB to M2-coated beads. Magnetic beads coated with the M2 antibody were incubated in the absence (No Comp) or presence of the indicated competitor (3XABA, 3XCTR, 3XFLAG). In vitro translated [³⁵S]-labeled Flag-TRF2^ΔB was then added and the amount captured by the beads was determined by SDS-PAGE electrophoresis and exposure to a PhophorImager cassette (a). In a second experiment done in triplicate, the amount of [³⁵S]-labeled protein captured was counted by scintillation (b). The amount of [³⁵S]-labeled protein captured in the absence of competitor (No Comp) was arbitrarily set to 100%. In both experiments, beads coated with normal mouse IgG were included as negative control for the capture (IgG). Data represent the mean ± S.D. (n = 3). (c)-(d) The 3XABA oligo elutes the Flag-TRF2^ΔB proteins already bound to M2-coated beads. The [³⁵S]-Flag-TRF2^ΔB protein was first captured by magnetic beads coated with the M2 antibody. The beads were then incubated in the absence (No comp) or presence of the indicated competitor (3XABA, 3XCTR, 3XFLAG). The amount of [³⁵S]-Flag-TRF2^ΔB released was determined by SDS-PAGE electrophoresis and exposure to a PhophorImager cassette (c). In a second experiment done in triplicate, the amount of [³⁵S]-labeled protein released was counted by scintillation (d). The amount of [³⁵S]-labeled protein released by the boiling (total) was arbitrarily set to 100%. In both experiments, beads boiled to release to all of the captured [³⁵S]-labeled protein were included as positive control for the elution (Total). Data represent the mean ± S.D. (n = 3).