Network topology-directed design of molecular CPU for cell-like dynamic information processing

Abstract

Natural cells (NCs) can automatically and continuously respond to fluctuant external information and distinguish meaningful stimuli from weak noise depending on their powerful genetic and protein networks. We herein report a network topology-directed design of dynamic molecular processing system (DMPS) as a molecular central processing unit that powers an artificial cell (AC) able to process fluctuant information in its immediate environment similar to NCs. By constructing a mixed cell community, ACs and NCs have synchronous response to fluctuant extracellular stimuli under physiological condition and in a blood vessel-mimic circulation system. We also show that fluctuant bioinformation released by NCs can be received and processed by ACs. The molecular design of DMPS-powered AC is expected to allow a profound understanding of biological systems, advance the construction of intelligent molecular systems, and promote more elegant bioengineering applications.

Fig. 1.. Network topology–directed design of DRN-based fluctuation filter.

(A) Flow diagram of a threshold gate simplified from stimulus threshold of NCs. (B) Static threshold gate design with a consumptive and irreversible threshold gate (BC). (C) NCs that can sense and response fluctuant stimulus. (D) The incoherent FFL used in this design with two pathways connecting input and output having the different signs. (E) Flow diagram of fluctuation filter integrating incoherent FFLs and threshold gates. Black dashed line denotes inhibition. (F) Flow diagram of network topology–directed fluctuation filter that can process a continuous and ever-changing input wave. Thr denotes threshold.

Fig. 2.. Blueprint of DMPS-powered AC that has dynamic information processing ability.

DMPS, as the mCPU of AC, contained three modules: a signal convertor for biosignal reception, a fluctuation filter for signal process, and an intensity accumulator for signal output and storage. The extracellular fluctuation of stimuli will, in turn, cause intracellular fluctuation of stimuli through influx or efflux driven by osmotic pressure. The fluctuant stimuli inside the AC are transduced into a fluctuant DNA analog via a signal convertor. The fluctuation filter can determine whether the fluctuant DNA analog signal exceeds or falls below a cutoff threshold value and only outputs the overflowing signal. This filtration process is continuous and autonomous, regardless of whether fluctuation goes up or down. The filtered signal can be recorded through the intensity accumulator and can be lastly reported by fluorescence output.

Fig. 3.. Construction and operation of DMPS.

(A) PAGE analysis of K⁺-specific AI treated with different concentrations of K⁺. G4, G-quadruplex. (B) PAGE analysis of K⁺-specific AI (in the presence of initial 40 mM K⁺) treated with different concentrations of CE. (C) PAGE analysis of fluctuation filter–controlled reaction pathways. Lanes 1 to 5: I, B, C, D, and E; lanes 6 to 9: BC, DE, IC, and IE; lanes 10: BC + DE + 1 equivalent. I (initial I, below cutoff value); and lane 11: BC + DE + 2 equivalent. I (including overflowing I). (D) PAGE analysis of fluctuation filter–controlled reaction pathways. Lanes 1 to 5: AI, IE, IC, DE, and BC; lane 6: IC, IE, B, and D incubated with 1 equivalent. A (initial A); and lane 7: IC, IE, B, and D incubated with 2 equivalent. A (including overflowing A). (E) A total of 200 nM quenched duplex BC and 200 nM quenched duplex DE with 400 nM AI were alternately incubated with 40 mM K⁺ and 40 mM CE to test the fluorescence kinetics. a.u., arbitrary units. (F) PAGE analysis of CHA driven by strand D. Lanes 1 to 5: DE, D, Y1, Y1 + Y2, and D + Y1 + Y2; lane 6: DE + Y1 + Y2 + Y3; lane 7: Y1 + Y2 + Y3; and lane 8: D + Y1 + Y2 + Y3. (G) PAGE analysis of braking CHA reaction via strand E. Lane 1: 2-min CHA reaction (Y1 + Y2 + Y3 + D) and then incubated with 1 equivalent. E for 2 min; lane 2: 2-min CHA reaction; lane 3: Y1 + Y2 + Y3 + D + E simultaneously; and lane 4: CHA reaction for 10 min. (H) PAGE analysis of Van-specific AI treated with different concentrations of Van. L denotes 20–base pair (bp) DNA ladder.

Fig. 4.. Construction of DNA NP–anchored AC.

(A) AGE analysis of stepwise assembly of DNA NP. Lane L, 100-bp DNA ladder; lanes 1 to 6, self-assembly of corresponding numbers of ssDNA building blocks. (B) Statistical analysis of size distribution from more than 200 ACs in microscopic imaging. (C) Fluorescence imaging of quenched dsDNA AI-embedded (K⁺-responsive AI assembled from FAM-A with BHQ1-I; Van-responsive AI assembled from Cy3-A with BHQ2-I) ACs with or without Cy5-NP after 40 mM K⁺ or 100 μM Van was added. Scale bars, 20 μm. (D) Scheme illustrating DMPS could be regarded as mCPU to power AC. (E) Scheme illustrating AC-mediated protection of inner DNA molecules from nuclease-mediated degradation owing to the limited size of DNA NP. (F) SBR analysis when DMPS-AC and free DMPS were treated with 40 mM K⁺ at room temperature for 1 hour of kinetic scanning after incubation with 10% FBS or buffer at 37°C for different times, respectively. All data are means ± SD, n = 3. ****P < 0.0001; ns means no significant difference [two-way analysis of variance (ANOVA)]. (G) Fluorescence kinetics of DMPS-AC incubated with 40 mM K⁺ in buffer. (H) Fluorescence kinetics of free DMPS incubated with 40 mM K⁺ in buffer.

Fig. 5.. Dynamic information processing of DMPS-AC responded to fluctuant concentrations of K⁺ or Van.

(A) Scheme illustrating information processing of DMPS-AC responded to dynamic extracellular stimuli. (B) Fluorescence imaging kinetics of DMPS-AC incubated with alternating concentrations of 10 or 5 mM K⁺. (C) FI analysis and K⁺ treatment of (B). (D) Fluorescence imaging kinetics of DMPS-AC incubated with alternating concentrations of 6 or 5 mM K⁺. (E) FI analysis and K⁺ treatment of (D). (F) Fluorescence imaging kinetics of DMPS-AC incubated with alternating concentrations of 30 or 14 μM Van. (G) FI analysis and Van treatment of (F). (H) Fluorescence imaging kinetics of DMPS-AC incubated with alternating concentrations of 20 or 14 μM Van. (I) FI analysis and Van treatment of (H). All data are means ± SD (n = 3) in (C), (G), (E), and (I). Scale bars, 20 μm. All concentrations are more than cutoff value from black time points to red time points, and all concentrations are less than cutoff value from red time points to black time points in (B), (D), (F), and (H). BF, bright field.

Fig. 6.. mCPU-powered AC responded to extracellular fluctuant stimuli under live-cell environments.

(A) Scheme illustrating the fluorescence response of DMPS-AC and NC to fluctuant stimuli (K⁺ concentrations). (B) The mixture of DMPS-ACs and NCs (1:20) was incubated with fluctuant concentrations of 6 or 5 mM K⁺. FAM channel indicates signal intensity of DMPS-AC. pHrodo indicates the pH value of NCs; stronger red signal denotes bigger pH value. (C) Flow cytometry analysis of NCs in (B). (D) Statistical analysis of (C). (E) Scheme illustrating the fluorescence response of DMPS-AC and NC to fluctuant stimuli (Van concentrations). (F) The mixture of DMPS-ACs and NCs (1:50) was incubated with fluctuant concentrations of 20 or 10 μM Van. FAM channel indicates signal intensity of DMPS-ACs. Blue caspase 3/7 channel indicates the apoptosis level of NCs; stronger blue signal denotes higher apoptosis. (G) Flow cytometry analysis of NCs in (F). (H) Statistical analysis of (G). Rows denote DMPS-ACs of all control groups (0 hour) in (B) and (F). All data are means ± SD (n = 3) in (B), (D), (F), and (H). Scale bars, 50 μm.

Fig. 7.. Response of mCPU-powered AC with NC and in blood vessel–mimic circulation system.

(A) Scheme illustrating stimulated NC released K⁺ to DMPS-AC. (B) Representative fluorescence kinetic imaging of DMPS-AC and NC in RPMI 1640 medium by adding trigger, 5 μM amphotericin B, and 10 μM ouabain. (C) Quantification analysis of FAM intensity and proposed K⁺ concentration’s tendency of the group treated with trigger. Red arrow denotes adding trigger, and yellow arrow denotes adding 1 mM CE to reduce the concentration of K⁺ in (B). (D) Construction of circulation system by mimicking blood vessels and controlling the concentrations of K⁺ for diagnosis and readout of recorded results of NC and DMPS-AC. (E) Fluorescence kinetic imaging of the mixture of NCs and DMPS-ACs pipetted from (D). (F) Statistical analysis of (E). All data are means ± SD (n = 3) in (C), (E), and (F). Scale bars, 50 μm.