Characterization of the specific interaction between the DNA aptamer sgc8c and protein tyrosine kinase-7 receptors at the surface of T-cells by biosensing AFM

Abstract

We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (k_off = 5.16 s^-1) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per μm². Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s^-1 of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per μm² in the cell membrane.

Keywords: DNA aptamer; Energy landscape; Molecular recognition; PTK7; Recognition imaging; Single molecule force spectroscopy; T-cell.

Graphical Abstract

The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s⁻¹ of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per μm² in the cell membrane

Fig. 1

(A) DNA sequence of the used sgc8c aptamer. (B) Tip chemistry. (1) Inert silicon nitride cantilever are amino-functionalized using APTES gas phase silanization. The heterobifunctional crosslinker NHS-PEG-PDP (2a) or NHS-PEG-Acetal (2b) is coupled allowing binding NH₂-terminated (4) DNA aptamers [after deprotection of the acetal group (3b)] or SH-terminated sgc8c aptamers (3a) respectively

Fig. 2

SMFS experiments: (A) Optical Image of T-cells on Cell-Tak coated glass slide. The bio-functionalized cantilever (upper middle) is placed above a single cell before approaching. (B) Typical force distance cycle using a sgc8c functionalized tip on a T-cell. In the retraction part (black line) a downwards bending is observable as a result of specific interaction. In contrast, in the presence of free sgc8c this interaction is blocked (inset). (C) Typical probability density function before (black) and after (red) addition of free ligand. The red curve is normalized to the relative binding probability. The significance of the proof experiment is shown in the inset. (D) Probability density functions of sgc8c–T-cell interactions at different pulling velocities. The most probable unbinding force increases at higher pulling velocities. The fit of the resulting loading rate dependence of the rupture force is shown in the inset

Fig. 3

TREC experiments: (A) Schematic of TREC setup. The upper part of the oscillation is used to gain the recognition image, whereas the lower part is influenced by the sample topography. Topography (B1) and simultaneously acquired recognition (B2) image on a T-cell membrane using sgc8c functionalized tips. A superposition of topography and recognition is shown at image (B3). After addition of free aptamers the topography (C1) remains unchanged, whereas the recognition spots (C2) is completely abolished as a result of blocked PTK7 receptors (illustrated in C3). Scale bar for all AFM images is 500 nm