Unlocking Autonomy: Feedback Driven (Dis)Assembly Cycles in Minimal System

The Overarching Theme

Consider that every day we rely on food to nourish and sustain us. This food acts as a source of energy that fuels all living materials. At the heart of this energy exchange are mitochondria, which produce ATP (adenosine triphosphate)- the molecular currency that powers countless bodily functions, from muscle movement to brain activity. Take a deep dive into the production and destruction of ATP and you will find that the minimal building block of life, the cell, is the epicentre that regulates all critical functions through numerous catabolic-anabolic reactions. Powered by ATP and biocatalysts, such metabolic reaction networks help to provide critical components to maintain the living characteristics of the whole system. Therefore, the periodic changes of chemicals throughout the reaction networks depend on the energy inputs under non-equilibrium conditions that help maintain the catalytic reaction networks in the biochemical cell. Notably, such periodic changes in chemical components are not only limited to the metabolic network¹. From the microscopic to the systemic level, homeostasis controls mitosis, metabolism, signal transduction, circadian rhythms and many other important biological processes². Inspired by this complexity of natural systems, we were interested in synthetically realising systems that can exhibit periodic changes of one or more of their components in an autonomous manner with spatiotemporal control of properties. To install this capability, the synthetic system must be integrated into the feedback loop as seen in modern biochemical processes, from cell division to circadian rhythm.

So, what if we could build a complex autonomous system using minimal chemical building blocks? What if the resulting complex state could use energy to grow and feedback to repair, or even communicate via feedback loops, just like a biological system? These are the interesting questions that motivate us and from which we started our research journey.

The Blueprint and the Challenges

Energy driven (dis)assembly of cytoskeletal protein assemblies is one of the fascinating examples of homeostasis at the microscopic scale, achieved through self-regulation, feedback loops and oscillations³. Towards our goal of accessing a minimal chemical-based system capable of demonstrating multiple cycles of (dis)assembly, a two-component system has been designed that incorporates negative and positive feedback with appropriate time lags in the kinetics under non-equilibrium conditions. First, if the assembly is catalytic and can promote its own degradation, then the negative feedback could be installed (Fig. 1a). For this purpose, the building block A’BC/ABC was designed to have a tendency to self-assemble and, in the assembled state, the catalytic unit histidine could catalyse the ester (BC) and thus induce negative feedback. Furthermore, if one of the products of the catalytic degradation or the building block can facilitate the regeneration of the assembly, time-delayed positive feedback will be installed (Figure 1b).  With this in mind, building block A’BC/ABC was rationally designed with a free aldehyde group (Fig. 1d). The dynamic imine linkage was chosen because this linkage is known for its acute responsiveness to pH switches and one of the products is acid, which lowers the pH of the medium used to generate the second feedback (positive feedback). This coupling of negative and positive feedback loops could lead to autonomous (dis)assembly for several cycles in a closed system (Fig. 1c).

Now, to obtain multiple cycles in practice, the main challenges were the particular selection of the concentrations of the building blocks to induce both feedback (positive and negative) in the system and its consequence effect on maintaining the required time delay to keep the feedback orthogonal that help to observe the oscillation. During the first cycle the assembled state should show negative feedback (hydrolysis) to return to its initial state and the produced acid should be sufficient to induce the positive feedback to regain the assembled state and continue.

The Turning Point

We started with high throughput analysis to find periodic changes in (dis)assembly, and initially we found great difficulty in hitting the particular concentrations responsible for multiple (dis)assembly cycles. So, after many brainstorming discussions and a huge number of trials, we finally found a system that showed (dis)assembly cycle up to 2 cycles. However, at this point we were interested in why not more than 2 (dis)assembly cycles? And during the work on this project, it became clear that the intrinsic property of the building block also played an important role in the introduction of the feedback at the optimal time, in addition to the concentrations of the building blocks. The (dis)assembly observed in the initial (histidine) system for only two cycles can be attributed to the insufficient amount of ester remaining for reassembly given the higher critical aggregation concentration of the system. Therefore, we speculated that if additional hydrogen bonding capabilities could be installed in the system, the CAC would be lowered, providing the possibility of (dis)assembly for multiple cycles. In this regard, a new building block (a dipeptide of histidine and beta-alanine with an amide for additional hydrogen bonding) was used along with ester. Remarkably, we found multiple cycles in a closed system, which was an eye-opener for us. We were excited to see how a minimal synthetic network can exhibit periodic behaviour in a closed system, which is important in the absence of advanced biocatalytic systems. Furthermore, through mathematical modelling, the Gadgil group showed the theoretical possibility that the described set of reactions and regulatory interactions could result in autonomous (dis)assembly cycles.

The Future Perspective

Oscillation plays a critical role in the origin of life, driving the necessary chemical reactions and shaping the environmental conditions over time that are essential for life to emerge⁴. Energy cycles, such as day-night rhythms and tidal forces, provide the dynamic energy needed to synthesise complex organic molecules from simpler ones. In the future we would like to focus on developing a sustained oscillatory system and trying to understand how oscillations further influence molecular properties such as stability and chirality of molecules that are critical for life.

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3. Hess, H. & Ross, J. L.  Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots. Chem.  Soc. Rev. 46, 5570-5587 (2017).
4. Dunlap, J. C. Molecular bases for circadian clocks, Cell 96, 271–290 (1999).