Synthetic gene brushes: a structure–function relationship

Abstract

We present the assembly of gene brushes by means of a photolithographic approach that allows us to control the density of end-immobilized linear double-stranded DNA polymers coding for entire genes. For 2 kbp DNAs, the mean distance varies from 300 nm, where DNAs are dilute and assume relaxed conformations, down to 30 nm, where steric repulsion at dense packing forces stretching out. We investigated the gene-to-protein relationship of firefly luciferase under the T7/E.Coli-extract expression system, as well as transcription-only reactions with T7 RNA polymerase, and found both systems to be highly sensitive to brush density, conformation, and orientation. A 'structure-function' picture emerges in which extension of genes induced by moderate packing exposes coding sequences and improves their interaction with the transcription/translation machinery. However, tighter packing impairs the penetration of the machinery into the brush. The response of expression to two-dimensional gene crowding at the nanoscale identifies gene brushes as basic controllable units en route to multicomponent synthetic systems. In turn, these brushes could deepen our understanding of biochemical reactions taking place under confinement and molecular crowding in living cells.

Compartmentalization by directional gene expression

“…For brush assembly we used glass slides, 18 × 18 mm 2 , with nine separate reaction chambers enclosed in a sample holder (6)(7)(8). The density of DNA brushes on the same slide and duplicate slides could be fixed to within 5%.…”

“…We propose an in vitro methodology to emulate gene expression in a boundary-free compartment. The approach is based on a photolithographic biochip for fabricating dense phases of surface-bound DNA, thus forming DNA brushes (6)(7)(8). In general, brushes are formed when end-attached linear polymers collectively stretch because of excluded volume interaction when packed to distances at which there is significant overlap between neighboring molecules (9).…”

RNA Polymerase Activity Assay on Biochips: Correlation between Template DNA Density and RNA Synthesis

“…[8][9][10][11] The biosynthetic reaction on surface of microchips was first realized by Buxboim et al and they developed and utilized the microchips assembled by photolithographic approach on silicon dioxide (SiO2) for controlling the cell-free expression. 7,8,12 This approach allows us to control the immobilized DNA and regulate the biosynthetic reactions in a more controlled manner. 7 Recently, it has been reported that there exists attenuation of protein synthesis in a translation coupled with a transcription system on DNA-grafted biochips, which is likely due to molecular crowding caused by the translational machinery.…”

“…[6][7][8] As a new research tool, cell-free gene expression has been applied to DNA-based biochips, which showed possibilities of developing synthetic biological systems and biochemical materials at nanoscale. [8][9][10][11] The biosynthetic reaction on surface of microchips was first realized by Buxboim et al and they developed and utilized the microchips assembled by photolithographic approach on silicon dioxide (SiO2) for controlling the cell-free expression.…”

“…7,8,12 This approach allows us to control the immobilized DNA and regulate the biosynthetic reactions in a more controlled manner. 7 Recently, it has been reported that there exists attenuation of protein synthesis in a translation coupled with a transcription system on DNA-grafted biochips, which is likely due to molecular crowding caused by the translational machinery. 7 Here, we investigated T7 RNAP enzyme activity of RNA synthesis on self assembled monolayers (SAMs) on gold surface which presents template DNA at various surface densities ranging from 5% to 50%.…”