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 (koff = 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 AbstractThe 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

DNA Aptamers in the Detection of Leukemia Cells by the Thickness Shear Mode Acoustics Method

“…Our experiments showed that the binding probability was highest for MOLT‐4 cells (48.6±3.9 %) and lowest for U266 cells (3.3±1.5). The binding probability for Jurkat cells were 37.5±5.0 % and this is in good agreement with work by Leitner at al . Figure SI‐3 in Supporting information summarizes all experiments on the cells including U266 cells that do not contain PTK7 receptors.…”

“…The principle of this measurement is based on the direct relation between voltage of photodiode and the bending of the cantilever, which can be considered as a spring. Using the Hooke's law it is possible to transform this bending into the force . For each AFM tip we have measured 5 retraction velocities in the range from 300 to 7500 nm/s.…”

“…at room temperature and washed again in PBS for three times. Afterwards the cells were left in PBS and used immediately for force measurement or stored in the fridge for a maximum 2 days …”

“…SMFS method allowed study of molecular interactions between aptamers and PTK7 receptors. This approach has been applied in our recent work for study the interactions of sgc8c aptamers with Jurkat and U266 cells . However, interaction of aptamers with MOLT‐4 cells was not studied so far by SMFS and not compared with acoustics method.…”

<p>Nucleic Acid Aptamer: A Novel Potential Diagnostic and Therapeutic Tool for Leukemia</p>

“…Thus, rapid, selective and specific methods are urgently in demand. Based on the specific interaction of DNA Sgc8c aptamer and PTK-7 on ALL cells,85 a growing number of sophisticated diagnostic methods have been created. In one report, biotin-modified semiconductor QDs were labeled with the Sgc8 aptamer via streptavidin, then a stronger fluorescent signal would be generated by biotin amplification interactions when Sgc8 aptamer identified CCRF-CEM cells in T-ALL,67 In another report, dual-aptamer (Sgc8c and ATP aptamers)-functionalized graphene oxide (DAFGO) complex, consisting of bispecific aptamers (Sgc8c and ATP aptamers) and graphene oxide (GO), was designed for internalization into Molt-4 T-ALL cells,86 Sgc8c aptamer was selected and characterized to target to PTK-7.…”