科研之友
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摘要
The expression of genes in a cell in response to external signals or internal programs occurs within an environment that is compartmentalized and dense. Reconstituting gene expression in man-made systems is relevant for the basic understanding of gene regulation, as well as for the development of applications in bio- and nanotechnology. DNA polymer brushes assembled on a surface emulate a dense cellular environment. In a regime of significant chain overlap, the highly charged nature of DNA, its entropic degrees of freedom, and its interaction with transcription/translation machinery lead to emergent collective biophysical and biochemical properties, which are summarized in this Account. First, we describe a single-step photolithographic biochip on which biomolecules can be immobilized. Then, we present the assembly of localized DNA brushes, a few kilo-base pairs long, with spatially varying density, reaching a DNA concentration of similar to 10(7) base pairs/mu m(3), which is comparable to the value in E. coli. We then summarize the response of brush height to changes in density and mono- and divalent ionic strength. The balance between entropic elasticity and swelling forces leads to a rich phase behavior. At no added salt, polymers are completely stretched due to the osmotic pressure of ions, and at high salt they assume a relaxed coil conformation. Midrange, the brush height scales with ratio of density and ionic strength to the third power, in agreement with the general theory of polyelectrolyte brushes. In response to trivalent cations, DNA brushes collapse into macroscopic dendritic condensates with hysteresis, coexistence, and a hierarchy of condensation with brush density. We next present an investigation of RNA transcription in the DNA brush. In general, the brush density entropically excludes macromolecules, depleting RNA polymerase concentration in the brush compared to the bulk, therefore reducing transcription rate. The orientation of transcription promoters with respect to the surface also affects the rate with a lower value for outward compared to inward transcription, likely due to local changes of RNA polymerase concentrations. We hypothesize that equalizing the macromolecular osmotic pressure between bulk and brush with the addition of inert macromolecules would overcome the entropic exclusion of DNA associated proteins, and lead to enhanced biochemical activity. Finally, we present protein synthesis cascades in DNA brushes patterned at close proximity, as a step toward biochemical signaling between brushes. Examining the synthesis of proteins polymerizing into crystalline tubes suggests that on-chip molecular traps serve as nucleation sites for protein assembly, thereby opening possibilities for reconstituting nanoscale protein assembly pathways.
- 出版日期2014-6
