Entropy-driven collective interactions in DNA brushes on a biochip

Abstract

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...

Heterogeneous Morphology and Dynamics of Polyelectrolyte Brush Condensates in Trivalent Counterion Solution

“…It was also observed that for C Spd > C * Spd , the collapsed domains were smaller in a denser brush of shorter chains, which is consistent with the prediction from our scaling argument R c ∼ N 0.4 σ −0.2 . The scaling relationship of R c ∼ N 0.4 σ −0.2 further explains the various size of octopus-like micelle domain depicted in (2), (4), (6) in Fig. 7.…”

“…Double-stranded DNA brushes on a biochip [4] at a cell-like density (∼ 10 4 kb/µm 3 ) have been developed as a platform to study cell-free gene expression [5]. Due to the negative charges along the backbone, this synthetic system behaves like a well-defined strong polyelectrolyte brush [5], the height of which can be varied by modulating the ionic strength of monovalent salt (NaCl) solution and the grafting density [6]. Recently, it has been reported that trivalent counterions, spermidine 3+ (Spd 3+ ), can induce a collapse transition of DNA brush into fractal-like dendritic macroscopic domains [7].…”

Dendritic and Nanowire Assemblies of Condensed DNA Polymer Brushes

“…In the osmotic regime, a precondensed 3.5 kb brush extends to a height of ∼1 μm, which is well beyond the evanescent excitation length 31 and is therefore not measurable by TIRF. Condensation was triggered by adding a single aliquot of concentrated Spd solution ( Figure 1G and Movie S2).…”

“…29 The brush was immersed in a low-salt solution (3 mM NaCl), an osmotic brush regime in which the osmotic pressure due to the confined ions induces maximal swelling of the brush up to the 1.2 μm chain contour length. 31 The Spd concentration (C Spd ) was incrementally increased by adding minute volumes of concentrated Spd solution to the reservoir. Passing 60 μM Spd initiated a major change in the surface fluorescence, which began with the emergence of a single bright spot surrounded by a ∼1 μm thick perimeter with reduced fluorescence ( Figure 1A and Movie S1 in the Supporting Information).…”

Transcription Driven Phase Separation in Chromatin Brush

“…6,7,16,20 Although the distribution of DNA chain segments in DNA brushes has not been fully resolved, 16,17 we here use the Alexander approximation, which assumes that the local concentration of Kuhn segments is uniform in the brush region. 21 This treatment highlights the roles played by transcription for chromatin structures, rather than the details of brush models.…”