Transient co-assemblies of micron-scale colloids regulated by ATP-fueled reaction networks

Self-assembly of colloidal particles offers an attractive bottom-up approach to functional materials. Current design strategies for colloidal assemblies are mostly based on thermodynamically controlled principles and lack autonomous behavior. The next advance in the properties of colloidal assemblies will come from coupling these structures to out-of-equilibrium chemical reaction networks furnishing them with autonomous and dynamic behavior. This, however, constitutes a major challenge of carefully modulating the interparticle potentials on a temporal circuit program and avoiding kinetic trapping and irreversible aggregation. Herein, we report the coupling of a fuel-driven DNA-based enzymatic reaction network (ERN) to micron-sized colloidal particles to achieve their transient co-assembly. The ERN operating on the molecular level transiently releases an Output strand which links two DNA functionalized microgel particles together into co-assemblies with a programmable assembly lifetime. The system generates minimal waste and recovers all components of the ERN after the consumption of the ATP fuel. The system can be reactivated by addition of new fuel as shown for up to three cycles. The design can be applied to organize other building blocks into hierarchical structures and materials with advanced biomimetic properties.

All oligonucleotides (except amine-modified oligonucleotides) were purchased from Integrated DNA Technologies Inc. (IDT) and Biomers GmbH (as listed below in Table S1).The oligonucleotides as received were dissolved in 1X TE buffer, pH = 8.0 (Thermo Fischer).Amine-modified oligonucleotides were synthesized following a well-defined procedure (as described in section 3.4).

General Characterization Methods and Instruments
DLS measurements were performed on the LS Instruments NanoLab 3D at 25 ᵒC operating with a red laser (λ = 685 nm) and a scattering angle of ϴ = 90 ᵒ using standard disposable PS cuvettes (BRAND GmbH & Co. KG).The distributions of the hydrodynamic radii were obtained by a CONTIN mode analysis.
The temperature-controlled fluorescence measurements were performed on a TECAN (SPARK control v3.1) microplate plate reader using Corning® 384-Well black polystyrene plate with non-binding surface.Excitation and emission wavelengths for Atto488 are 485 nm and 535 nm and for Atto647N are 620 nm and 679 nm respectively.p values were calculated by performing a t-Test (Two-Sample Assuming Equal Variances) in Microsoft excel using a built-in Data analysis tool pack.
The amine-modified oligonucleotides were synthesized on a H-8 custom LNA, DNA/RNA automatic synthesizer from K&A Laborgeräte.The synthesized oligonucleotides were purified by High Performance Liquid Chromatography (HPLC) on a Dionex Ultimate 3000 (Thermo Fischer Scientific).

Synthesis of surfactant-free, poly(2,2,2-trifluoroethyl methacrylate)-Rhodamine labeled core particles.
The synthesis is analogous to a previous report.The stirring rate was increased to 600 rpm and reaction was allowed to run for 4 hours.The resulting core/shell MG particles (amount of AA moieties assuming full conversion = 1250 µmol per g of MG) were filtered while hot and purified by dialysis (MWCO 8000 Da) against deionized water.The resulting core-shell MGs were further purified via centrifugation (5 × 25 min, 11000 rpm, 15 ᵒC, replacement of the supernatant with Milli Q water per centrifugation step).

Synthesis and purification of amine-modified oligonucleotide sequences.
The oligonucleotides were synthesized at 10 μmol scale employing the standard solid phase β-cyanoethylphosphoramidite chemistry in trityl-on mode.The DNA phosphoramidites (DMT (dimethoxytrityl)-dT, DMT-dA(bz), DMT-dG(dmf) and DMT-dC(ac)) were diluted to 50 mM with dry acetonitrile and synthesis occurred from the 3' towards the 5' end of the oligonucleotides on packed solid phase columns.
Cleavage of the oligonucleotides (DMT-on) from the solid support and base deprotection was achieved in one step to ensure optimal yields.The 34 µmol/g controlled pore glass (CPG) solid support was treated with 10 mL of ice-cold ammonia solution (30-32 % NH 3 ) overnight at room temperature to detach the DNA from the CPG support.The cleaved DNA (DMT-on) in ammonia was diluted with 10 mL of disodium phosphate buffer (75 mM containing 1 mM EDTA, pH = 8.3) and the crude product was obtained upon freeze drying.The obtained DNA was redispersed in MilliQ water and purified by preparative reverse phase-HPLC (RP-HPLC) followed by freeze drying to remove the solvent.
The DMT group was cleaved from the purified product by making a 2 wt % solution of the dry DNA in NaOAc/HOAc buffer (200 mM, pH 4.0, 200 mM NaCl) and heating the mixture to 50 °C for one hour.After neutralizing the reaction mixture with disodium phosphate buffer (750 mM, 10 mM EDTA), the synthesized DNA was precipitated into a 5-fold excess of isopropanol to remove contaminants and to exchange the counterions to sodium.The precipitate was dissolved in MiliQ water and freeze dried.The synthesized and purified strands were stored at -20 °C until further use.
The purity of the obtained oligonucleotides was confirmed with analytical HPLC.

DNA annealing
All DNA strands were used as received.All the sequences are provided in Supplementary Table S1 and Table S2.
The DNA strands received from IDT and Biomers were dissolved in TE buffer (10 mM Tris-HCl, pH = 8.0) to prepare a stock solution of 1 mM and stored at -20 ᵒC for further use.The complementary DNA strands intended for double stranded complexes, i.e., Complex 1 and Substrate 1 were dissolved in annealing buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl 2 , pH = 8.0) with the same stoichiometry at -20 ᵒC overnight to prepare a stock solution of 0.125 mM.

CLSM image treatment and analysis
All CLSM images were processed using ImageJ (Fiji).All images except in Fig. 3a were acquired as a z-stack.
Atto488, Atto647 and Rhodamine B channels were merged together to form a composite image and then compiled as a z-projection for representation purposes.The size of the co-assemblies was determined by thresholding the compiled images.The masks obtained upon thresholding were analyzed in terms of size and total amount. 3

Lifetime calculation
The lifetime of the transient state is defined as the time that a transient profile (either obtained from timedependent fluorescence measurements or assembly size analysis) takes to decrease to half of the initial and maximum value.5 Supplementary Tables Table S1.DNA sequences for the oligonucleotides synthesized following a well-defined procedure (section 3.4) with their abbreviations and modifications.

1
Definition of activity units of both enzymesDefinition of the Weiss Unit to describe the activity of T4 DNA ligase: 0.01 Weiss Unit [WU] of T4 DNA Ligase is the amount of enzyme required to catalyze the ligation of greater than 95 % of 1 μg of λ/HindIII fragments at 16 °C in 20 minutes.Unit definition to describe the activity of BsaI: One Unit [U] is defined as the amount of enzyme required to completely digest 1 μg of pXba DNA in one hour at 37 °C in 50 μl assay buffer containing acetylated BSA added to Fig. 3-4 5' Amino Modifier C6 dT NH2-z* (z*-T 20 ) GATAGAGATCGTGTGTTAC TTTTTTTTTTTTTTTTTTTT Fig. 3-4 3' Amino Modifier C6 Fig. S1 Free energy change for the interaction between (a) Output and long strand of Substrate in the presence of A1 and A2 in the State A of the system, (b) Output and A1 and A2 in the absence of long strand of Substrate 1 in the State B of the system calculated with NUPACK simulations setting the temperature at 37 ᵒC and salt concentrations at 50 mM NaCl, 10 mM MgCl 2 .Output preferably remains bound within Substrate 1 and does not show any interaction with A1 or A2 in State A of the system.Only in State B of the system in the absence of long strand of Substrate, Output forms Assembly Complex with A1 and A2.

Fig. S2 (
Fig. S2 (a) Equilibrium concentrations of all the components obtained from NUPACK 4 simulations upon mixing 5 µM each of Output, A1 and A2 setting the temperature at 37 ᵒC and salt concentrations at 50 mM NaCl, 10 mM MgCl 2 .The desired Assembly Complex between Output, A1 and A2 is formed in 54.8 % yield.(b), (c) Experimental verification of the equilibrium concentration of desired Assembly Complex between Output, A1-Atto488 and A2-ABMNQ535.(b) Time-dependent fluorescence intensity measurements of A1-Atto488 (red), A2-ABMNQ535 (black), and a 1:1 mixture of A1-Atto488, A2-ABMNQ535 upon introduction of Output in the system (blue).The remaining fluorescence (blue) corresponds to the uncomplexed or free A1-Atto488.(c) With the help of calibration curve between fluorescence Intensity and concentration of free A1-Atto488, the amount of uncomplexed A1-Atto488 in the equivalent mixture of A1-Atto488, A2-ABMNQ535, and Output can be calculated as 2.4 µM.Assuming a 100% quenching efficiency between Atto488 and BMNQ535 and zero fluorescence contribution from BMNQ535, the equilibrium concentration of Assembly complex can be calculated as 2.6 µM accounting for 52% yield.This means the experimental yield shows only 5% deviation from simulated yield (NUPACK, 54.8%).Experimental conditions: all species present at 5 µM concentration in 1X NEB CutSmart buffer at 37 ᵒC.

Fig. S3
Fig. S3 (a) Schematics for experimental verification of percentage yield of Assembly Complex in Static and Transient systems.(b) Time-dependent fluorescence intensity measurements of A1-Atto488 and a 1:1 mixture of A1-Atto488, A2-ABMNQ535 upon introduction of Output in the system.The percentage of fluorescence decrease (grey, dotted line) provides a Reference point which indicates the minimum fluorescence that can be observed in the system.Experimental conditions: all species present at 5 µM concentration in 1X NEB CutSmart buffer at 37 ᵒC.(c) Time-dependent fluorescence intensity changes demonstrating static and transient complexation of A1-Atto488 and A2-BMNQ535 upon ATP-fueled ligation induced release of Output.For the Static system, in the absence of BsaI, fluorescence intensity decreases approximately to the Reference point.This clearly indicates that Output must have released with 100% efficiency.But, in case of transient system, restriction can set in already at the hemi-ligated intermediate (e.g., Substrate 1, Complex 1 and only one of the Inputs) without completing to the fully ligated state which is the condition for expulsion of Output.Because of this, fluorescence intensity decreases by only 25% which indicates that Output is released with 44.6% efficiency with respect to Reference point.Since only 52% of Output released can form Assembly Complex (Fig.S2b), a final yield of 23% can be attributed to Assembly Complex.Experimental conditions: A1-Atto488 and A2-BMNQ535 at an equimolar concentration of 5 µM are dissolved in 1X NEB CutSmart buffer containing 20 µM Complex 1, 5 µM Substrate 1, 10 µM Input 1 and Input 2 at 37 ᵒC.For transient system (green curve), 0.8 WU µL -1 of T4 DNA ligase and 0.8 U µL -1 of BsaI was used and system was fueled by 40 µM ATP; static system (blue curve) was carried out only with 0.8 WU µL -1 of T4 DNA ligase and fueled with 40 µM ATP.

Fig. S6
Fig. S6 Measurement of DNA grafting density on Particle2.(a) The binding capacity of z-A2-Atto647 onto Particle2 via z/z* hybridization is measured as a proxy for the DNA grafting density.The mean fluorescence intensity per µm 2 over the particle (F total , includes the contribution from both z-A2-Atto647 bound on Particle2 and free z-A2-Atto647 in the suspension) and in the background (F unbound , includes only free z-A2-Atto647 in the suspension) is measured for increasing amounts of z-A2-Atto647 via CLSM.Experimental conditions: Particle1 is incubated with increasing concentrations of z-A2-Atto647 (0.2-20 µM) in TE buffer (pH = 8.0) at 15 ᵒC at a final MG concentration of 0.05 wt %.F total and F unbound represent average fluorescence intensity measured from 10 different regions.(b) The data is fitted with linear equation where the slope provides fluorescence contribution from z-A2-Atto647 on the particle (F bound ).(c) With the help of calibration curve between mean fluorescence intensity per µm 2 (F, measured via CLSM) and concentration of free z-A2-Atto647, a corresponding DNA concentration for F bound is calculated to be 4.5  0.1 µM accounting for 2.7 × 10 3 strands/MG.Experimental conditions: Increasing concentrations of z-A2-Atto647 (4-40 µM) dispersed in TE buffer.F represents an average fluorescence intensity from 5 different regions.A laser with λ = 638 nm and power of 30 mW was used at 2% intensity throughout the experiment.Scale bars: (a), (c) 2 µm.

Fig. S7
Fig. S7Assembly size analysis on the particles from the CLSM images (Fig.3b) obtained during sequential ATP fueling and BsaI restriction (i.e., static system) The co-assemblies achieve a maximum average size of 9 µm 2 after 2h of ATP addition.The formed structures disassemble again after 15h of BsaI addition.The box represents 25-75 % of data, whiskers represent 5-95 % of data, a solid circle represents the mean, and horizontal bar the median of the assembly size distribution in box charts.

Fig. S10
Fig. S10 Control for transient co-assemblies of MGs regulated by upstream ATP-fueled ERN without ATP addition.(a) Time dependent ex situ CLSM imaging shows absence of any co-assembly between Particle1-A1-Atto488 and Particle2-A2-Atto647 without ATP addition.All CLSM images are represented as merged composite compiled as a z-projection.Experimental conditions: Particle1-A1-Atto488 and Particle2-A2-Atto647 suspended as an equimolar mixture in 1X NEB CutSmart buffer at a final MG concentration of 0.05 wt %, 20 µM Complex 1, 5 µM Substrate 1, 10 µM Input 1 and Input 2 at 37 ᵒC.(b) Assembly size analysis on the particles obtained from two different z-projections at each time interval.The average assembly size increased to 2.5 times of its initial value indicating formation of small clusters only after 24 h of monitoring the system.This might result because of saltinduced non-specific aggregation of MGs. 5 The box represents 25-75 % of data, whiskers represent 5-95 % of data, a solid circle represents the mean, and horizontal bar the median of the assembly size distribution in box charts.Scale bars: 10 µm.