Synthetic gene brushes: a structure-function relationship.
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.
Buxboim
Amnon
A
Department of Materials and Interfaces, The Weizmann Institute of Science, Rehovot, Israel.
Daube
Shirley S
SS
Bar-Ziv
Roy
R
D001709:Biotechnology / Q000379:methods; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D008954:Models, Biological; D036103:Nanotechnology / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D011108:Polymers / Q000737:chemistry; D014176:Protein Biosynthesis; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry
D011108:Polymers; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases