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.

Ligand-guided selection of aptamers against T-cell Receptor-cluster of differentiation 3 (TCR-CD3) expressed on Jurkat.E6 cells

“…A two-step polymerase chain reaction (PCR) was employed to expand the evolved library as reported elsewhere. 10 A single-stranded DNA was made using avidin agarose beads (Pierce) and desalted using NAP-10 columns (GE) as described by Sefah et al 11 For subsequent SELEX, we followed Zumrut et al 8 .…”

“…Ligand Competition: As introduced in Zumrut et al, LIGS was used to selectively elute aptamers against TCR complex 8 . Briefly, the enriched 16 th library of ss-DNA cell-SELEX was folded by heating and subsequent cooling for 20 minutes.…”

“…Following incubation, the eluted 16 th fraction obtained through competition and found in the supernatant was collected and amplified by PCR. A two-step PCR was performed as described in Zumrut et al 8 . First, the whole fraction resulting from LIGS was amplified using 10-PCR cycles.…”

“…To address this, we recently developed a method called Ligand-guided Selection, (LIGS), which selects aptamers that specifically bind a predetermined epitope expressed on the target cell surface 8 . LIGS takes advantage of the partition step in cell-based SELEX and introduces a secondary, pre-existing high-affinity ligand, in effect a monoclonal antibody (mAb), to outcompete and elute specific aptamers binding to the receptor target of the antibody, not the cell.…”

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines

“…To this end, we introduced a variant of SELEX called “LIgand-Guided Selection” (LIGS) that allows the identification of specific aptamers against known (i.e., SELEX) cell-surface proteins. 12,13 In particular, LIGS identifies aptamers specific for a predetermined epitope expressed on the cell surface at its native environment. In terms of protocol, LIGS interrupts the selection process of SELEX and introduces a strong, high-affinity bivalent antibody (Ab), which interacts with its cognate epitope to outcompete and replace specific aptamers from an enriched SELEX pool.…”