Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials.
The procedure of surface functionalization based on the use of 1-(3-Aminopropyl)silatrane (APS) instead of our early procedure utilizing aminopropyl triethoxy silane (APTES) is described. Unlike APTES, APS is less reactive and extremely resistant to hydrolysis and polymerization at neutral pH. The kinetics of DNA adsorption to APS-mica was studied. The results are consistent with a diffusion controlled mechanism suggesting that DNA molecules bind irreversibly with the surface upon immobilization. This conclusion is supported by the data on imaging of supercoiled DNA, the labile conformations of which are very sensitive to the conditions at the surface-liquid interface. In addition, we demonstrated directly that the segments of DNA molecules could move along the surface if the sample is imaged in aqueous solution without drying of the sample. Using the time-lapse mode of AFM imaging we visualized the transition of purine-pyrimidine sequence in supercoiled DNA from intramolecular triple-helical conformation (H-form) into the B-helix upon the change of pH from acidic (pH 5) to neutral. The mechanisms of the H-to-B transitions and the correlation of the local structural transitions with a global DNA conformation are discussed.
Shlyakhtenko
Luda S
LS
Department of Microbiology, Arizona State University, Tempe, AZ 85287, USA.
Gall
Alexander A
AA
Filonov
Alexander
A
Cerovac
Zoran
Z
Lushnikov
Alexander
A
Lyubchenko
Yuri L
YL
D000327:Adsorption; D000538:Aluminum Silicates; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000138:chemical synthesis / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods; D017646:Organosilicon Compounds / Q000138:chemical synthesis / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties
D000538:Aluminum Silicates; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004278:DNA, Superhelical; D017646:Organosilicon Compounds; C015503:silatrane; D004247:DNA; D019809:Streptavidin; C011934:mica