AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

Abstract

The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization.

Delivery of Cell-Specific Aptamers to the Arterial Wall with an Occlusion Perfusion Catheter

“…Aptamer tissue concentration was determined using both aptamer fluorescence binding and internalization (AFBI) assay 22 and qRT-PCR. At 1 h post-delivery, Apt01 showed higher arterial retention as compared to Apt14 and NSC (AFBI post-extraction, 1.880 ± 0.841 nM versus 1.409 ± 0.425 nM versus 1.165 ± 0.634 nM, p = 0.0277).…”

A modified SELEX approach to identify DNA aptamers with binding specificity to the major histocompatibility complex presenting ovalbumin model antigen

“…Additionally, the use of synthetic peptide–MHC molecules tagged with magnetic beads in protein-SELEX simplifies the process compared to the time-consuming retrieval of aptamers from cells in cell-SELEX. 53,54 Moreover, aptamers that are incubated with cells during positive selection may be internalized, 55 posing further challenges for aptamer purification. On the other hand, cell-SELEX provides the advantage of preserving the natural conformation and appropriate post-translational modifications of proteins exhibited on the cell membrane, enabling the identification of biologically relevant aptamers that can be directly applicable to in vivo settings.…”

A CRISPR View of Biological Mechanisms

“…This process is labor intensive and requires experience to prevent cell disruption 78 . The use and development of mRNA aptamers have also been limited by time and expense constraints 79,80 . CRISPR provides a simpler method of RNA targeting without the need for extensive protein libraries or genetic manipulations.…”