Topological digestion drives time-varying rheology of entangled DNA fluids

Abstract

Understanding and controlling the rheology of polymeric complex fluids that are pushed out-of-equilibrium is a fundamental problem in both industry and biology. For example, to package, repair, and replicate DNA, cells use enzymes to constantly manipulate DNA topology, length, and structure. Inspired by this feat, here we engineer and study DNA-based complex fluids that undergo enzymatically-driven topological and architectural alterations via restriction endonuclease (RE) reactions. We show that these systems display time-dependent rheological properties that depend on the concentrations and properties of the comprising DNA and REs. Through time-resolved microrheology experiments and Brownian Dynamics simulations, we show that conversion of supercoiled to linear DNA topology leads to a monotonic increase in viscosity. On the other hand, the viscosity of entangled linear DNA undergoing fragmentation displays a universal decrease that we rationalise using living polymer theory. Finally, to showcase the tunability of these behaviours, we design a DNA fluid that exhibits a time-dependent increase, followed by a temporally-gated decrease, of its viscosity. Our results present a class of polymeric fluids that leverage naturally occurring enzymes to drive diverse time-varying rheology by performing architectural alterations to the constituents.

Cooperative Rheological State‐Switching of Enzymatically‐Driven Composites of Circular DNA And Dextran

“…This digestion co be seen using time-resolved gel electrophoresis as shown in Figure 1C and Figure 5 and described below. [21] Sample Preparation: Aqueous composite solutions comprising varying volume fractions of 11c* solutions of DNA (ϕ DNA ) and dextran (ϕ dex = 1 − ϕ DNA ) (Figure 1A) were prepared at a total volume of 200 μL that included 20 μL of 10× CutSmart Buffer (0.5 M Potassium Acetate, 0.2 M Tris-acetate, 0.1 M Magnesium Acetate; New England BioLabs) and DNA volume fractions of ϕ DNA = 0, 0.25, 0.5, 0.75 or 1. The buffer conditions and temperature (20 °C) provided good solvent conditions for the DNA.…”

“…The buffer conditions and temperature (20 °C) provided good solvent conditions for the DNA. [21,40,[41][42][43] Prior to each measurement, the sample was mixed and equilibrated on a rotator at 4 °C for 12-24 h. This mixing method, developed in Ref. [25], was shown to be sufficient and necessary to homogenize the DNA and dextran phases, resulting in no visible signs of phase separation or large-scale aggregation, assessed via bulk rheology (Figure S4, Supporting Information) and fluorescence imaging of single DNA molecules in composites (Figure S5, Supporting Information).…”

“…To estimate c e for the fully digested composites, we recall that ApoI digestion results in fragments with minimum, maximum, and average lengths of l f,min ≃ 64 bp, l f,max ≃ 3160 bp, and 〈l f 〉 ≃ 854 bp, respectively. [39] Combining the relations c * ≈ lR −3 G and R G ≈ l 𝜈 , where 𝜈 ≃ 0.588 is the Flory exponent for flexible polymers in good solvent conditions (which we have here), [21,35,40,[41][42][43] yields c * L (l) ≈ l 1−3𝜈 , from which we can derive an entanglement concentration expression c e (l f )…”

“…Conversely, the same study showed that solutions of entangled linear DNA gradually become less viscous over time under the action of restriction enzymes that cut the long chains into many short fragments. [ 21 ] This effect was rationalized as arising from a decreasing number of entanglements per chain as the lengths of the chain fragments l f become comparable to or lower than the nominal length between entanglements l e . In these studies, the enzymes act as catalysts to break the sugar‐phosphate DNA backbone at specific sites, thereby irreversibly altering the DNA, which, in turn, pushes the systems out‐of‐equilibrium, driving them to new thermodynamic equilibria.…”

Enzymatic cleaving of entangled DNA rings drives scale-dependent rheological trajectories

“…However, only recently has the action of restriction enzymes, that alter the topology and/or length of biopolymers such as DNA, been appreciated as a potential driver of changes to viscoelastic properties. [12][13][14][15][16][17][18] Yet, this process is ubiquitous in diverse processes including nuclear division, DNA repair, transcription and signaling. 1,3,19,20 For example, DNA naturally occurs in supercoiled circular, relaxed circular (ring), and linear topologies with lengths that scale many decades.…”