Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate

A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA t o immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trime thyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-m ica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capa ble of binding of DNA molecules such that, after UV cross-linking, they wit hstand a thorough rinsing with SDS. Observations with atomic force microsco py showed that derivatized surfaces remain smooth, so DNA molecules are eas ily visualized. Linear and circular DNA molecules were photochemically immo bilized on the surface. The molecules are distributed over the surface unif ormly, indicating rather even modification of AP-mica with trioxalen. Gener ally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces re main quite similar. This suggests that UV cross-linking does not induce for mation of a noticeable number of single-stranded breaks in DNA molecules.