AFM imaging in solution of protein-DNA complexes formed on DNA anchored to a gold surface.
A chemical procedure for anchoring DNA molecules to gold surfaces was used to facilitate the imaging of DNA and DNA-protein complexes in buffer solution by tapping mode atomic force microscopy (TMAFM). For preparing flat gold surfaces, a novel approach was employed by evaporating small amounts of gold onto freshly cleaved mica to give flat films that were stable under aqueous buffer conditions. The thickness of the investigated films ranged from 1 to 10 nm. For typical films of 4-6 nm, which were stable under aqueous buffer conditions, the root mean square (RMS) roughness ranged between 0.25 and 0.5 nm, as measured by atomic force microscopy (AFM). This roughness is comparable to that of obtained by the template stripped gold (TSG) technique, which is widely used in scanning probe microscopy but involves more preparation steps. In order to visualize DNA and DNA-protein complexes by TMAFM, the DNA was chemisorbed to the gold surface through a linker carrying a terminal thiol group at the 5'-end of each of the DNA strands. The modified DNA fragments were bound to the gold films and imaged in buffer solution, while unmodified DNA could not be visualized. Since the DNA was not dried during the process, it can be assumed that its native conformation was retained. This mode of anchoring did not prevent interaction with proteins, as confirmed by the observation that the topology of a complex formed by adding the protein to a surface-anchored DNA was the same as that obtained by anchoring a pre-formed complex to the gold surface. We attribute this observation to the fact that the DNA is anchored to the gold surfaces only through its ends, therefore the DNA-support interaction is minimized but imaging is still possible.
Medalia
O
O
Department of Chemistry, The Weizmann Institute of Science, Rehovot, Israel. omedalia@biochem.mpg.de
Englander
J
J
Guckenberger
R
R
Sperling
J
J
D001426:Bacterial Proteins / Q000648:ultrastructure; D004247:DNA / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000648:ultrastructure; D006046:Gold / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D011506:Proteins / Q000648:ultrastructure*; D012808:Sigma Factor; D013171:Spores, Bacterial / Q000648:ultrastructure; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties; D014157:Transcription Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D012808:Sigma Factor; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D006046:Gold; D004247:DNA