Following aptamer–ricin specific binding by single molecule recognition and force spectroscopy measurements

Phys. Chem. Chem. Phys.

Lou

Park

et al. 2015

We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL(-1)). The off-rate values of the ricin-aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10(-4) s(-1) and (1.82 ± 0.067) × 10(-2) s(-1), respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.

“…5 The same tip was used to measure the DFS force-distance curves. In the first method (M1), the thiol-modified aptamer was directly immobilized on the gold substrate.…”

Section: Apparatusmentioning

confidence: 99%

Section: The Affinity Of the Aptamer For Ricin Measured By Afm-dfsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Surface conformations of an anti-ricin aptamer and its affinity for ricin determined by atomic force microscopy and surface plasmon resonance

Phys. Chem. Chem. Phys.

Lou

Park

et al. 2015

“…A polyethylene glycol (PEG) chain with molecular weight of 2000, PEG2000, was used as linker and spacer to connect aptamer to the gold coated AFM tip surface. 25 The PEG2000 was treated as an elastic polymer molecule in solvent, and its physical behaviors follow WLC model. 26 The aptamer formed a stable folding structure in PBS buffer, and the applied force will distort and break the connection between aptamer and ricin during the unbinding process.…”

mentioning

confidence: 99%

Determining the elastic properties of aptamer-ricin single molecule multiple pathway interactions

Applied Physics Letters

Park

Kwon

et al. 2014

Self Cite

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

J of Molecular Recognition

Zhou

Yang

et al. 2014

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.