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

Michael Leitner,Andreas Ebner,Alexandra Poturnayova,Ivana Karpisova,Tibor Hianik,Maja Snejdarkova,Constanze Lamprecht,Sabine Weich

doi:10.1007/s00216-017-0238-5

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 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

Highlights

Cancer is a major societal challenge and its detection and identification at the earliest stage are crucial for efficient and successful treatment
We performed atomic force microscopy (AFM) recognition imaging to probe the distribution of protein tyrosine kinase-7 (PTK7) receptors for the first time on the single receptor level on lymphoma cells
The DNA aptamer sgc8c (Fig. 1A) has been designed to recognize the receptor PTK7 in the plasma membrane. It is an important player in extracellular signaling and highly expressed on the surface of leukemia cells and especially T-Acute lymphoblastic leukemia (ALL) cells

Summary

Introduction

Cancer is a major societal challenge and its detection and identification at the earliest stage are crucial for efficient and successful treatment. Aptamers are synthetic short single-stranded DNA or RNA oligonucleotides that fold into unique threedimensional shapes These structures enable highly selective and specific targeting of molecules with high affinities comparable to those of antibodies. The aptamer was linked to the tip of the AFM cantilever and brought into contact with the plasma membrane of HeLa cells In their proof of principle experiment rupture forces of 46 ± 26 pN between sgc8c and PTK7 on the cell surface were measured only at one given force load and showed that the binding strength of aptamer and antibody to cancer cells was about equal under these setting. We expand on this work, and include dynamic aspects of the molecular recognition between sgc8c and PTK7 on Jurkat T-cells by conducting single molecule force spectroscopy (SMFS) under variation of the force load. We performed AFM recognition imaging to probe the distribution of PTK7 receptors for the first time on the single receptor level on lymphoma cells

Materials and methods

Results and discussion

Conclusions

Compliance with ethical standards