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 to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl 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-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules.
Shlyakhtenko
L S
LS
Department of Microbiology, Arizona State University, Tempe, Arizona 85287-2701, USA.
Gall
A A
AA
Weimer
J J
JJ
Hawn
D D
DD
Lyubchenko
Y L
YL
D000538:Aluminum Silicates / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D010777:Photochemistry; D010957:Plasmids / Q000648:ultrastructure; D013057:Spectrum Analysis; D014307:Trioxsalen / Q000031:analogs & derivatives*; D014466:Ultraviolet Rays
D000538:Aluminum Silicates; D003432:Cross-Linking Reagents; D004247:DNA; C011934:mica; D014307:Trioxsalen