Ligand-Guided Selection of Target-Specific Aptamers: A Screening Technology for Identifying Specific Aptamers Against Cell-Surface Proteins

Abstract

We report on a new strategy for identifying highly specific aptamers against a predetermined epitope of a target. Termed "ligand-guided selection" (LIGS), this method uniquely exploits the selection step, the core of SELEX (Systematic Evolution Exponential enrichment). LIGS uses a naturally occurring stronger and highly specific bivalent binder, an antibody (Ab) interacting with its cognate antigen to outcompete specific aptamers from a partially enriched SELEX pool, as a strategy. We demonstrate the hypothesis of LIGS by utilizing an Ab binding to membrane-bound Immunoglobulin M (mIgM) to selectively elute aptamers that are specific for mIgM from a SELEX pool that is partially enriched toward mIgM expressing Ramos cells. The selected aptamers show specificity toward Ramos cells. We identified three aptamer candidates utilizing LIGS that could be outcompeted by mIgM Ab, demonstrating that LIGS can be successfully applied to select aptamers from a partially evolved cell-SELEX library, against predetermined receptor proteins using a cognate ligand. This proof-of-concept study introduces a new biochemical-screening platform that exploits the binding of a secondary stronger molecular entity to its target as a partition step, to identify highly specific artificial nucleic acid ligands.

FIG. 1.

Outline of ligand-guided selection. Color images available online at www.liebertpub.com/nat

FIG. 2.

Flow cytometric analysis of evolved library and LIGS. Fluorescence intensity on x-axis is indicative of binding of fluorescently labeled R10 that evolved round 13 pool or anti-IgM. (a) Analysis of binding of evolved pool from round 13 against Ramos cells. At the 13th round, an increase in fluorescence intensity was observed, indicating that the library was partially evolved with Ramos-specific DNA aptamer sequences. (b) Pool from round 13 during LIGS, introduction of anti-IgM Ab to round 13 bound Ramos cells, resulted in decreased fluorescence intensity. (c) Binding of anti-IgM mAb during LIGS showing Ab binding to Ramos cells (blue line). LIGS, ligand-guided selection. Color images available online at www.liebertpub.com/nat

FIG. 3.

Sequence alignment of the major family of LIGS using ClustalX2. Sequences repeated in the competitively eluted library were synthesized and screened against Ramos cells. Also, sequences from the main library share common motifs with competitively eluted sequences that are synthesized and screened against Ramos cells. MainLib, sequences obtained from SELEX-13 round; ComLib, sequences obtained from competitively eluted SELEX-13 round. Three specific aptamer candidates R1, R10, and R15 were evaluated and their Bmax/2 was calculated. Color images available online at www.liebertpub.com/nat

FIG. 4.

Analysis of specificity of R1, R10, and R15. Specificity of aptamers was analyzed against mIgM-expressing Ramos cells and mIgM-negative Jurkat.E6. Color images available online at www.liebertpub.com/nat

FIG. 5.

Investigation of epitope identity. Flow cytometric competitive binding analysis of R1, R10, and R15 in the presence of IgM (a) and competitive blocking of anti-IgM binding by R 1.1 (b). Each FITC-labeled library (0.5 μM for R10 and 0.4 μM for R1, and R15) was incubated for 60 min on ice with 1 × 10⁵ Ramos cells that were preincubated with either anti-IgM or anti-CD20 followed by washing with 3 mL of wash buffer; they were subsequently analyzed by flow cytometry. Aptamer fluorescence intensity on the x-axis is indicative of binding of each aptamer. Thus, an increment of fluorescence intensity directly translates into aptamer binding to pretreated Ramos cells. When the cells are preincubated with anti-CD20, all three aptamers show an increase in fluorescence intensity (blue line). Aptamer fluorescence intensity on the x-axis shifts to a lower value in the presence of the anti-IgM antibody (red line), indicating that the anti-IgM substantially blocks R1, R10 and effects R15 binding to its target. No difference in fluorescence intensity was observed for the random control (gray–black), when Ramos cells were preincubated with anti-IgM antibody or anti-CD20, compared with an un-evolved pool from round 0 (black line). Binding of the corresponding antibody indicated in the lower panel. (b) 10 × 10⁴ Ramos cells were incubated with either the random control or R1.1 for 45 min on ice and added to a serially diluted anti-IgM solution. The competitive blocking was allowed for an additional 35 min. followed by wash and were analyzed by flow cytometry. FITC, fluorescein isothiocyanate. Color images available online at www.liebertpub.com/nat