Transient self-organisation of DNA coated colloids directed by enzymatic reactions

Abstract

Dynamic self-organisation far from equilibrium is a key concept towards building autonomously acting materials. Here, we report the coupling of an antagonistic enzymatic reaction of RNA polymerisation and degradation to the aggregation of micron sized DNA coated colloids into fractal structures. A transient colloidal aggregation process is controlled by competing reactions of RNA synthesis of linker strands by a RNA polymerase and their degradation by a ribonuclease. By limiting the energy supply (NTP) of the enzymatic reactions, colloidal clusters form and subsequently disintegrate without the need of external stimuli. Here, the autonomous colloidal aggregation and disintegration can be modulated in terms of lifetime and cluster size. By restricting the enzyme activity locally, a directed spatial propagation of a colloidal aggregation and disintegration front is realised.

Figure 1

Colloidal structure formation coupled to enzymatic reactions. 1 μm sized polystyrene colloids are functionalized with two sorts of ssDNA docking strands and can be distinguished via fluorescence. The structure formation is induced by complementary RNA-linker strands which link the two different species of colloids. The production of the RNA-linker is realised using a T7 Polymerase under use of free dsDNA templates and NTPs in solution. The disintegration process of the so formed colloidal clusters is achieved by the activity of the ribonuclease RNaseH, which degrades the RNA-linkers once bound to DNA.

Figure 2

Colloidal aggregation induced by RNA production. (a) The RNA production was measured over time. Here, the T7 polymerase and the DNA template concentrations were kept constant while the fuel NTP was varied. A linear production rate in the nanomolar range can be observed over three hours of polymerisation. The dashed lines represent linear fits, which obey a linear dependency. The RNA concentration is visualised by the DNA/RNA intercalator SyGr II and can be converted into the effective RNA concentration (S2). The decrease of the signal in the beginning is due to the heating of the chamber, because the dye is more fluorescent at room temperature. (b) The NTP dependent RNA production is transferred to the colloids and monitored using bright field microscopy. The structure formation is depicted after one hour, showing that the enzymatic reaction of the RNA linker strands indeed induces the colloidal aggregation. The average cluster size was determined in a.U. using image analysis and rises with the NTP concentration (26, 53 and 112 a.U., respectively).

Figure 3

Optical properties of the colloidal solution. (a) Colloids are illuminated by a green 1 mW laser beam. The sample containing monodispersed colloids absorbs the laser beam, which can be seen by the outline of the sample holder (red dotted line) on the detection wall. The colloidal volume fraction is too high and causes absorption and scattering of the light beam. (b) In contrast, the colloidal gelation leads to the transmission of the laser beam. Here, huge volume fractions arise, where no colloids are present.

Figure 4

Colloidal disintegration. The colloidal aggregation is induced by the addition of 100 nM RNA-linker strands. The complete disintegration of the colloidal structures was realised using a RNaseH concentration of 0.1 U/μl. The cluster size decreased from 103 a.U. to 24 a.U. within 30 min.

Figure 5

Transient RNA production. (a) The RNA production and degradation are linear reactions. In the absence of degradation, a linear production is achieved (blue curve). Adding a degradation term, results in a steady state were production and degradation equilibrates. The dashed lines represent the calculations obtained from eq. 1. (b) To realise a transient behaviour with linear equations, fuel (NTP) consumption is necessary to reduce the production rate over time. The blue line shows the RNA production with a limited reservoir of NTP. Limited production and an additional degradation can be seen by the red lines. Here, transient behaviour can be observed, which can be controlled by varying the RNaseH concentration. The corresponding dashed lines represent the calculations obtained from eq. 2, which takes the fuel consumption into account. The black dashed line depicts the pure production without fuel consumption (eq. 1) and fits well with the production curve in the first 10 minutes.

Figure 6

Transient colloidal structure formation. Colloidal structure formation is induced using the enzymatic setup of Fig. 5b (0.19 U/μl RNaseH). The structure formation starts after several minutes and reaches its peak after one hour of polymerisation. The aggregation is followed by a disintegration, which is completed in approximately two hours.

Figure 7

Controlling the colloidal structure formation. (a) The transient structure formation is controlled in terms of lifetime and cluster size by varying the enzymatic reactions. Here, the RNA production was kept constant, while the degradation was increased. (b) The maximal cluster size decreases with increasing RNaseH concentration. (c) The time at which the maximal cluster size occurs shortens with increasing RNaseH concentration. (d,e) Bright field images of the maximal cluster size for the shortest and the longest pulse, respectively.

Figure 8

Controlling transient colloidal structure formation by hierarchical hybridisation reactions. (a) The transient RNA production (blue line) has a fast initial phase and is followed by a relative slow degradation. Adding a complementary RNA to the system (here 50 nM) reduces the unbound RNA, which is potentially able to link colloids (green line) (b). Comparison of the transient structure formation with and without complementary RNA. The complementary RNA-linker acts like an aggregation threshold and delays the aggregation and accelerates the disintegration.

Figure 9

Propagation of aggregation front. (a) The reservoir of the diffusion chamber is functionalised with DNA Templates. By that, the RNA production is restricted locally, while the degradation takes place globally. The aggregation was analysed at different positions (distance 400 μm) in the diffusion channel. (b) RNA is produced locally at the reservoir and diffuses into the channel, resulting in an aggregation front, which propagates into the channel. (c) Using the enzymatic conditions of a transient RNA pulse leads to an aggregation, which starts almost simultaneously for both positions. Here, the disintegration starts at position 1 and propagates along the channel.