Modeling DNA beacons at the mesoscopic scale

We report model calculations on DNA single strands which describe the equilibrium dynamics and kinetics of hairpin formation and melting. Modeling is at the level of single bases. Strand rigidity is described in terms of simple polymer models; alternative calculations performed using the freely rotating chain and the discrete Kratky-Porod models are reported. Stem formation is modeled according to the Peyrard-Bishop-Dauxois Hamiltonian. The kinetics of opening and closing is described in terms of a diffusion-controlled motion in an effective free energy landscape. Melting profiles, dependence of melting temperature on loop length, and kinetic time scales are in semiquantitative agreement with experimental data obtained from fluorescent DNA beacons forming poly(T) loops. Variation in strand rigidity is not sufficient to account for the large activation enthalpy of closing and the strong loop length dependence observed in hairpins forming poly(A) loops. Implications for modeling single strands of DNA or RNA are discussed.Comment: 15 pages, 17 figures, submitted to Eur. J. Phys.

Photoinduced gel–liquid transition of DNA fluid generates remote-controlled nonequilibrium microflow

We demonstrate remote-controlled microflow using photoresponsive DNA fluid. The DNA fluid was fabricated through the self-assembly of branched DNA motifs, nanostructures of base-paired single-stranded DNAs (ssDNAs): Three ssDNAs hybridized in the stem region to form a branched motif possessing sticky ends (SEs); via the hybridization of SEs, the branched DNA motifs self-assembled into DNA fluid, which was micrometer-scale condensates. The motifs were equipped with a photoresponsive capability by introducing azobenzene (Azo), well-studied photoisomerizable compound44, in the SEs. The photoswitchable Azo isomerization enabled the reversible association/dissociation between branched DNA motifs, leading to photoreversible fluidity regulation via gel–liquid–dispersed state transition. To design an energy-transducing system, we exploited the following sequence-specific programmability of DNA. By cross-linking the photoresponsive DNA motif with a branched DNA motif possessing nonphotoresponsive orthogonal SEs, we achieved photocontrollable “transporter” DNA and “cargo” DNA fluids; the photoresponsive DNA fluid hydrodynamically performed mechanical actions upon the nonphotoresponsive DNA fluid by transducing light energy. Notably, we discovered multiple modes in the generated fluid’s mechanical action as a function of the applied temperature and the Azo insertion site in the SE. Each flow mode was characterized as a single-peak-shaped profile in the temperature-dependent flow mobilities. The highest and lowest mobilities were obtainable in moderate and lower and higher temperature ranges, respectively. This anomalous profile was analogous to the reentrant phase behavior of DNA microstructures41,50,51, which behave as solid-like only in a moderate temperature range and as liquid-like in the lower and higher temperature ranges. Following thermodynamic arguments on the observed reentrancy, we describe the observed flow-mobility behavior as a manifestation of a transient non-equilibrium state in the SEs binding stability. We also explain the mode-changeability of the mechanical actions with experimental support by explaining the role of the Azo insertion site in the SE in determining the degree of the nonequilibrium in the binding stability

Liquid DNA coacervates form porous capsular hydrogels via viscoelastic phase separation on microdroplet interface

Liquid-liquid phase separation (LLPS) droplets of biopolymers are known as functional microdroplets in living cells and have recently been used to construct protocells and artificial cells. The formation of DNA coacervates (also referred to as DNA droplets) from branched DNA nanostructures and the control of their physical properties via DNA nanostructure design were demonstrated previously. For the construction of artificial cells or protocells, however, even though physical effects such as surface tension, wetting, and viscoelasticity are more important in a tiny (micrometer-sized), confined environment than in a bulk solution environment, they have not been explored yet. This study shows that a tiny, confined environment using a water-in-oil (W/O) microdroplet interface modulates the phase separation dynamics of DNA coacervates, leading to micrometer-sized porous capsular structures. The porous structures were produced via two types of viscoelastic phase separation (VPS) processes in DNA coacervates: (i) simple VPS and (ii) cluster-cluster aggregation after VPS. Finally, it was shown that environmental chemical stimulation can manipulate porous capsular DNA hydrogels extracted from W/O microdroplets. These results provide an approach for designing and fabricating artificial cells or protocells with complex structures and physicochemical properties

Tunable synthetic phenotypic diversification on Waddington’s landscape through autonomous signaling

Phenotypic diversification of cells is crucial for developmental and regenerative processes in multicellular organisms. The diversification concept is described as the motion of marbles rolling down Waddington’s landscape, in which the number of stable states changes as development proceeds. In contrast to this simple concept, the complexity of natural biomolecular processes prevents comprehension of their design principles. We have constructed, in Escherichia coli, a synthetic circuit with just four genes, which programs cells to autonomously diversify as the motion on the landscape through cell–cell communication. The circuit design was based on the combination of a bistable toggle switch with an intercellular signaling system. The cells with the circuit diversified into two distinct cell states, “high” and “low,” in vivo and in silico, when all of the cells started from the low state. The synthetic diversification was affected by not only the shape of the landscape determined by the circuit design, which includes the synthesis rate of the signaling molecule, but also the number of cells in the experiments. This cell-number dependency is reminiscent of the “community effect”: The fates of developing cells are determined by their number. Our synthetic circuit could be a model system for studying diversification and differentiation in higher organisms. Prospectively, further integrations of our circuit with different cellular functions will provide unique tools for directing cell fates on the population level in tissue engineering