A comparative analysis of cell surface targeting aptamers

Abstract

Aptamers represent a potentially important class of ligands for the development of diagnostics and therapeutics. However, it is often difficult to compare the function and specificity of many of these molecules as assay formats and conditions vary greatly. Here, with an interest in developing aptamer targeted therapeutics that could effectively deliver cargoes to cells, we chemically synthesize 15 aptamers that have been reported to target cell surface receptors or cells. Using standardized assay conditions, we assess each aptamer’s binding properties on a panel of 11 different cancer cell lines, correlate aptamer binding to antibody controls and use siRNA transfection to validate each aptamer’s binding to reported target receptors. Using a subset of these molecules known to be expressed on prostate cancers, we use near-infrared in vivo imaging to assess the tumor localization following intravenous injection. Our data demonstrate some surprising differences in the reported specificity and function for many of these molecules and raise concerns regarding their cell targeting capabilities. They also identify an anti-human transferrin aptamer, Waz, as a robust candidate for targeting prostate cancers and for future development of aptamer-based therapeutics.

Aptamers used for molecular imaging and theranostics - recent developments

“…In a recent systematic analysis of a panel of commonly used aptamers and cell lines, Kelly et al . established a number of important findings that further highlight the need for standardized assay conditions and test protocols to distinguish “real” binding sequences from non-specifically binding sequences 207 . Notably, only 4 out of 11 tested aptamers demonstrated specific binding under normalized assay conditions.…”

Aptamers for SARS‐CoV‐2: Isolation, Characterization, and Diagnostic and Therapeutic Developments

“…However, due to the differences in measurement methods and the use of different targets (S1 protein, trimeric spike protein or pseudoviruses), the reported K d values may not accurately reflect their true affinity ranking. As such, the reported K d value for an aptamer to a target does not necessarily translate to its ability to function in vivo , necessitating the requisite for standardized assay conditions [65] . A head‐to‐head comparison of these aptamers using the same target and same experimental methods would be needed to allow proper affinity ranking.…”

“…As such, the reported K d value for an aptamer to a target does not necessarily translate to its ability to function in vivo, necessitating the requisite for standardized assay conditions. [65] A head-to-head comparison of these aptamers using the same target and same experimental methods would be needed to allow proper affinity ranking.…”

Identification of novel aptamers targeting cathepsin B-overexpressing prostate cancer cells

“…Fitting to Langmuir isotherm, the calculations were determined using the model Hill 1 equation However, it is well known that the selected aptamers may not recognize the same protein in conditions different from those selected or in their native conformation in a cell. 58 Therefore, we investigate whether Apt01 and Apt08rc possess the ability to recognize CatB using a prostate cell line (PC-3), which is described in the literature as a cell line with secretion of CatB at the cellular membrane. [59][60][61] PC-3 cells were incubated with increasing concentrations of FAM-labelled Apt01 or Apt08rc for 1 h at room temperature.…”