Selection and Characterization of an α6β4 Integrin blocking DNA Aptamer

Abstract

The heterodimeric laminin receptor α6β4 integrin plays a central role in the promotion of tumor cell growth, invasion, and organotropic metastasis. As an overproduction of the integrin is often linked to a poor prognosis, the inhibition of integrin α6β4 binding to laminin is of high therapeutical interest. Here, we report on the combination of a cell-systematic evolution of ligands by exponential enrichment and a bead-based selection resulting in the first aptamer inhibiting the interaction between α6β4 integrin and laminin-332. This Integrin α6β4-specific DNA Aptamer (IDA) inhibits the adhesion of prostate cancer cells (PC-3) to laminin-332 with an IC50 value of 149 nmol/l. The Kd value concerning the aptamer's interaction with PC-3 cells amounts to 137 nmol/l. Further characterization showed specificity to α6 integrins and a half-life in murine blood plasma of 6 hours. Two truncated versions of the aptamer retained their binding capacity, but lost their ability to inhibit the interaction between laminin-332 and PC-3 cells. Confocal laser scanning microscope studies revealed that the aptamer was internalized into PC-3-cells. Therefore, in addition to the adhesion-blocking function of this aptamer, IDA could also be applied for the delivery of siRNA, microRNA or toxins to cancer cells presenting the integrin α6β4.

Figure 1

Flow cytometric analyses. (a) An enrichment of binding nucleic acids in higher rounds could be detected via flow cytometry. Green: library round 12, blue: library round 10, violett: library round 6, red: starting library, grey: untreated cells. (b) FACS analysis of cloned nucleic acids. Green: nc28, blue: nc36, pink: nc41, orange: nc 47, grey: nc50, violet: nc25, red: starting library, grey: untreated cells. The nucleic acid nc28 showed the highest affinity to PC-3 cells.

Figure 2

Determination of the K_d value for the interaction of IDA with PC-3 cells (blue); control DNA (green). A dilution series from 500 to 15.6 nmol/l was incubated with a constant amount of cells and analyzed by flow cytometry. Bound DNA was normalized to 500 nmol/l as 100%.

Figure 3

IDA inhibits the interaction between PC-3 cells and laminin-332. (a) PC-3 cells were incubated with IDA (violet), nc36 (blue) or without DNA (green) in a laminin-coated plate. Only IDA showed a reduced binding of PC-3 cells. LnCap cells not presenting the integrin showed no adhesion (red). (b) Dilution series of IDA (1.25 µmol/l–39 nmol/l) to determine the IC₅₀ value and the K_i.

Figure 4

Determination of the specificity of IDA. (a) Flow cytometry analysis. IDA binds to PC-3 cells (blue) with a higher affinity than to β4 integrin knockdown cells (green). The auto fluorescence of knockdown cells is shown in dark grey, the fluorescence of PC-3 cells in light grey. (b) Electrophoretic mobility shift assay. The aptamer binds to murine (green) and humane (violet) recombinant α6β4 integrin, as well as humane α6β1 (blue), but not to recombinant α4β1 (red).

Figure 5

Confocal laser scanning microscopy analyses. (a) IDA is internalized by PC-3 cells. (b) The nonbinding DNA showed no fluorescence signal.

Figure 6

Stability assay of IDA in murine plasma. The half-life turned out to be around 6 hours.