Cell-free gene expression in localized DNA brushes on a biochip has been shown to depend on gene density and orientation, suggesting that brushes form compartments with partitioned conditions. At high density, the interplay of DNA entropic elasticity, electrostatics, and excluded volume interactions leads to collective conformations that affect the function of DNA-associated proteins. Hence, measuring the collective interactions in dense DNA, free of proteins, is essential for understanding crowded cellular environments and for the design of cell-free synthetic biochips. Here, we assembled dense DNA polymer brushes on a biochip along a density gradient and directly measured the collective extension of DNA using evanescent fluorescence. DNA of 1 kbp in a brush undergoes major conformational changes, from a relaxed random coil to a stretched configuration, following a universal function of density to ionic strength ratio with scaling exponent of 1/3. DNA extends because of the swelling force induced by the osmotic pressure of ions, which are trapped in the brush to maintain local charge neutrality, in competition with the restoring force of DNA entropic elasticity. The measurements reveal in DNA crossover between regimes of osmotic, salted, mushroom, and quasineutral brush. It is surprising to note that, at physiological ionic strength, DNA density does not induce collective stretch despite significant chain overlap, which implies that excluded volume interactions in DNA are weak.DNA biophysics | synthetic biology D ouble-helix DNA polymers exhibit relaxed random-walk configurations at lengths beyond the persistence scale l p = 50 nm, occupying volume to maximize their entropy. Unfolding DNA entropic degrees of freedom to full contour-length stretch requires large forces of 500 k B T/l p (50 pN) using a force-extension apparatus (1, 2). However, the transition of DNA into an ordered stretched state can also result from an entropy increase in a coupled system when chains experience significant overlap. Such is the case of polymer brushes where individual polymers stretch to minimize the free energy of the brush (3). For charged polymers, such as DNA, the collective extension also increases the mixing entropy of the ions that are trapped within the brush to maintain local charge neutrality (4-7).In the past two decades, DNA brushes have become useful in a range of applications such as next-generation sequencing (8, 9), hybridization arrays (10-14), protein biosynthesis compartments (15-18), and coated particle assembly (19,20). The utility of the diverse DNA-based reactions carried out in such brushes requires an in-depth understanding of their basic materials properties. To date, the focus has been on short DNA brushes (∼100 bp) (21, 22) having negligible polymer degrees of freedom. The compression of a few kilobase-pair DNA brushes on beads, as deduced from optical trapping force measurement and Brownian motion analysis (23, 24), was shown to behave as a power of −1/3 with ionic strength. For flexible polyelectrol...