Probing interactions between human lung adenocarcinoma A549 cell and its aptamers at single‐molecule resolution

Abstract

Because cell-specific aptamers have high potential for biomedical applications, investigation of the interaction between cell and its aptamers may be of key importance for an improved understanding of biochemical processes. Herein, the interaction between human lung adenocarcinoma A549 cell and its four aptamers was explored using single-molecule force spectroscopy (SMFS). The values of the unbinding force varied from 117.1 to 171.0 pN at the loading rate of 1.8 × 10(5) pN/s. Based on the dependence of singe molecule force on the atomic force microscopy loading rate, the corresponding kinetic parameters were obtained. The results revealed two activation barriers and two transient states in the unbinding process of aptamer/cell interaction. More importantly, the binding sites on A549 cells with its four aptamers were defined to be different using SMFS and flow cytometry. This work demonstrated that SMFS can be used as a powerful tool for exploring the aptamer/cell binding behavior at the single-molecule level, and may provide valuable information for the design and application of aptamer probes.

Elucidation of the effect of aptamer immobilization strategies on the interaction between cell and its aptamer using atomic force spectroscopy

“…According to our previous work (Wang et al , ), all cells were cultured in RPMI 1640 medium containing 15% fetal bovine serum and then were maintained in an incubator at 37 °C with 5% CO 2 . Next, the cells were seeded on 35 mm dish at a final cell density of 5 × 10 4 cells per milliliter for 24–36 h. Before experiments, the subconfluent cells (about 80–90%) were rinsed four times using 2 ml 0.01 M phosphate‐buffered saline (PBS) buffer (pH 7.4), and then 2.5 ml RPMI 1640 medium without fetal bovine serum was added.…”

“…That is, the biophysical differences of an aptamer immobilized by different strategies can be observed using SMFS (Zhang and Yadavalli, ). Furthermore, SMFS could not only obtain the biomolecular interaction with nanometer positional accuracy and piconewton force sensitivity (Jin et al ., ; Yang et al ., ; Steffens et al , ) but also be suitable for the investigation of living cell under physiological conditions (Formosa et al , ; Wang and Yadavalli, 2014; Wang et al , ). Therefore, SMFS has the potential to become a powerful tool to evaluate aptamer immobilization strategies at the living cell level.…”

Investigation of the interactions between aptamer and misfolded proteins: From monomer and oligomer to fibril by single‐molecule force spectroscopy

“…The aptamer was attached to AFM tip (Si 3 N 4 , spring constants of 0.06 N·m −1 , Benyuan Nano Instrument Co, China) by covalent cross‐linking according to the method from references . The tip was firstly immersed in ethanol, alkaline solution (NH 4 OH/H 2 O 2 /H 2 O, 1:1:5 v /v) for 30 minutes, respectively.…”

Evaluation of Medicine Effects on the Interaction of Myoglobin and Its Aptamer or Antibody Using Atomic Force Microscopy

“…AFM silicon nitride (Si 3 N 4 ) tips (Benyuan Nano Instrument Co., China) were covalently attached by the aptamer (or antibody) according to previously reported procedures. − In detail, the tips were first cleaned and hydroxized through the treatment with chloroform, HF acid, alkaline solution (NH 4 OH/H 2 O 2 /H 2 O, 1:1:5 v/v), and piranha solution (98% H 2 SO 4 /H 2 O 2 , 7:3 v/v), respectively, to generate Si–OH groups. (CAUTION: “Piranha” solution reacts violently with organic materials; it must be handled with extreme care.)…”