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.

Figure 1

(A) Daisy biochip platform. Daisy molecules form a photolithographic monolayer (yellow) on silicon dioxide (light blue) in a single step. Following UV light photo-deprotection through an image mask, we bind biotin (blue circles) to free amines via NHS (Supplementary information). Streptavidin-conjugated dsDNA (SA-DNA, brown) are then immobilized with the mask grayscale translated into surface density of dsDNA imaged by dual fluorescent tags, FITC (green) covalently attached to the SA, and cy5 (red) covalently attached to the DNA at its surface-distal end. Scale bar is 50 μm. (B) Assembly of uniform linear dsDNA brushes. The packing of linear dsDNA polymer brushes following uniform deprotection of daisy-coated chips was quantified by radioactive labeling (Supplementary information) of 420 (green), 2160 (red), and 2900 (blue) bp DNA. Each radioactive spot in a 3 × 3 array contained a different amount of DNA (shown above the graph) as imaged by phosphorimaging densitometry (Methods and materials). The signal from the adsorbed DNA was converted to number of molecules using a calibration curve deduced from spots of known amounts of radioactive DNA on the surface (left). The equilibrium mean distance between dsDNAs was dictated by their solution concentration (main plot).

Figure 2

(A) Protein biosynthesis on a chip and in solution. Transcription/translation of luciferase using the T7/E. Coli-extract system in solution and on the chip with identical number of genes and reaction volume; the arrows indicate the position and direction of the T7 promoter within the 2160-bp-long dsDNA. The total amount of luciferase synthesized after 3 h of incubation is plotted. (B) Expression from top- and bottom-oriented genes. Chips with varying amounts of DNA, and hence density, were prepared with genes oriented either with the promoter directed into the surface (top) or toward the solution (bottom) sides. The amount of luciferase synthesized as a function of gene number is plotted (Material and methods). (C) Effect of brush structure on expression. Transcription/translation on a chip from variable ‘gene-only' (left bottom scheme) and fixed ‘gene/dummy' density brushes (right bottom scheme). The ‘gene/dummy' brushes were maximally packed at the highest density with varying ratio of gene-to-dummy DNA. The amount of expressed luciferase produced in a 10 μl reaction volume placed on top of a 7 mm² surface covered with DNA is plotted as a function of gene number. (D) Effect of brush structure on expression utility. Transcription/translation on a chip from variable ‘gene-only' and fixed ‘gene/dummy' density brushes. The expressed luciferase per gene is plotted as a function of mean intergene distance. (E) Transcription-only configuration in a buffer on a chip as a function of gene number for ‘top' and ‘bottom' orientations. (F) Transcription-only configuration in a buffer on a chip from variable ‘gene-only' and fixed ‘gene/dummy' density brushes.

Figure 3

mRNA and luciferase synthesis spatial profiles. The mRNA spatial profile (A) away from the brush (at z=0) relative to synthesis in solution (chip, red; solution, dashed black) was computed based on rate equations (Supplementary information) and the measured transcription and translation rates. (B) The luciferase profile does not reach steady state and is shown for t=30, 55, and 80 min, relative to solution (chip, red; solution, black). (C) Replotting Figure 2A after rescaling gene and protein numbers by brush and diffuse layer heights, h_brush=500 nm and h_luc=140 μm.