| Popis: |
Cellular functions emerge from biochemical reactions that occur within rather crowded cellular compartments while, on the contrary, standard in vitro techniques do not allow to study how biomolecules work at such high concentrations.Nanotechnology allows confinement of biomolecules into packed systems where the level of crowding is closer to the one in cells, and provides analytical methods to investigate the effect of crowding on biomolecular functions.In our experimental work we studied the mechanism by which restriction enzymes work inside brushes of short double stranded (ds)DNA molecules, confined on ultra-flat gold surfaces by using nanografting, an atomic force microscopy (AFM) based nanolithography method. dsDNA molecules have a restriction site at half height, and therefore, successful restriction reactions lead to a 50% decrease of the brush height with respect to the surrounding surface, that we measure by AFM.We address the effect of confinement on these reactions by varying the dsDNA density, and we unequivocally show that the confinement has a quantifiable effect on the mechanisms of enzyme diffusion and reaction.1) We show that enzyme molecules do not access the dsDNA brush directly from the solution, but 2D-diffuse inside the brush, with access exclusively from the sides. Moreover, the access is arrested when the DNA density reaches a certain critical threshold.2) We show that when the dsDNA density is sufficiently high, the restriction reaction can successfully occur on a dsDNA site having only partial consensus for the enzyme.Our findings demonstrate that, in crowded systems, enzymes may work very differently than in solutions. These findings may have broad implications on the development of in vitro approaches to investigate the effect of crowding on biochemical reactions, and can improve our understanding of molecular mechanisms in cells. |
