Microscopic agents programmed by DNA circuits.

Information stored in synthetic nucleic acids sequences can be used in vitro to create complex reaction networks with precisely programmed chemical dynamics. Here, we scale up this approach to program networks of microscopic particles (agents) dispersed in an enzymatic solution. Agents may possess multiple stable states, thus maintaining a memory and communicate by emitting various orthogonal chemical signals, while also sensing the behaviour of neighbouring agents. Using this approach, we can produce collective behaviours involving thousands of agents, for example retrieving information over long distances or creating spatial patterns. Our systems recapitulate some fundamental mechanisms of distributed decision making and morphogenesis among living organisms and could find applications in cases where many individual clues need to be combined to reach a decision, for example in molecular diagnostics.

Pursuit-and-Evasion Reaction-Diffusion Waves in Microreactors with Tailored Geometry.

Out-of-equilibrium chemical systems may self-organize into structures displaying spatiotemporal order, such as traveling waves and Turing patterns. Because of its predictable chemistry, DNA has recently appeared as an interesting candidate to engineer these spatiotemporal structures. However, in addition to the intrinsic chemical parameters, initial and boundary conditions have a major impact on the final structure. Here we take advantage of microfluidics to design controlled reactors and investigate pursuit-and-evasion chemical waves generated by a DNA-based reaction network with Predator-Prey dynamics. We first propose two complementary microfabrication strategies to either control the initial condition or the two-dimensional geometry of the reactor where the waves develop. We subsequently use them to investigate the effect of curvature in wave propagation. We finally show that DNA-based waves can compute the optimal path within a maze. We thus suggest that coupling configurable microfluidics to programmable DNA-based dissipative reaction networks is a powerful route to investigate spatiotemporal order formation in chemistry.

Fourier analysis to measure diffusion coefficients and resolve mixtures on a continuous electrophoresis chip.

We report a method to measure diffusion coefficients of fluorescent solutes in the 10(2)-10(6) Da molecular mass range in a glass-PDMS chip. Upon applying a permanent electric field, the solute is introduced through a narrow channel into a wide analysis chamber where it migrates along the injection axis and diffuses in two dimensions. The diffusion coefficient is extracted after 1D Fourier transform of the resulting stationary concentration pattern. Analysis is straightforward, requiring no numerical integration or velocity field simulation. The diffusion coefficients measured for fluorescein, rhodamine green-labeled oligonucleotides, and YOYO-1-stained dsDNA fragments agree with the literature values and with our own fluorescence correlation spectroscopy measurements. As shown for 151 and 1257 base pair dsDNA mixtures, the present method allows us to rely on diffusion to quantitatively characterize the nature and the composition of binary mixtures. In particular, we implement a DNA hybridization assay to illustrate the efficiency of the proposed protocol for library screening.

Quadruplex-based molecular beacons as tunable DNA probes.

Molecular beacons (MBs) are fluorescent nucleic acid probes with a hairpin-shaped structure in which the 5' and 3' ends are self-complementary. Due to a change in their emissive properties upon recognition with complementary sequences, MBs allow the diagnosis of single-stranded DNA or RNA with high mismatch discrimination, in vitro and in vivo. Whereas the stems of MB hairpins usually rely on the formation of a Watson-Crick duplex, we demonstrate in this report that the preceding structure can be replaced by a G-quadruplex motif (G4). Intramolecular quadruplexes may still be formed with a central loop composed of 12 to 21 bases, therefore extending the sequence repertoire of quadruplex formation. G4-MB can efficiently be used for oligonucleotide discrimination: in the presence of a complementary sequence, the central loop hybridizes and forms a duplex that causes opening of the quadruplex stem. The corresponding G4-MB unfolding can be detected by a change in its fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using G4-MB instead of traditional MB. In particular, the intrinsic feature of the quadruplex motif facilitates the design of functional molecular beacons by independently varying the concentration of monovalent or divalent cations in the medium.