Computing Arithmetic Functions Using Immobilised Enzymatic Reaction Networks

Abstract

Living systems use enzymatic reaction networks to process biochemical information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for molecular information processing using enzymes immobilised in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addition, subtraction and multiplication, using various concentrations of substrates or inhibitors as inputs and the production of a fluorescent molecule as the readout.

Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility–Mass Spectrometry

“…We also included G6PDH from the pentose phosphate pathway to introduce substrate competition between GPI and G6PDH and to mediate the concentration of NADH from which Pyr can be reduced to lactate (Lac). To compartmentalize the ERN in a flow reactor, we individually immobilized each enzyme on hydrogel beads 10,18 via the coupling between lysine residues and NHS-activated carboxylic acids on the hydrogel beads (see Supporting Information�Sections S2 and S3). Selected volumes (Tables S5−S9) of functionalized beads were pipetted into a CSTR (equipped with a filter on both the inlet and outlet to prevent the escape of the beads), to which we continuously flow the reaction inputs using programmable syringe pumps.…”

“…Living systems exploit complex cascades of enzymatic reactions to achieve key functions such as energy metabolism or maintaining homeostasis in changing environments. , Taking inspiration from biological systems, significant progress has recently been made in the forward design of in vitro enzymatic reaction networks (ERNs) with specific functionalities, ranging from the synthesis of added-value chemicals to the recycling of cofactors or processing of molecular inputs according to logic-gate responses. − The design of increasingly sizable and complex ERNs introduces crosstalk such as substrate competition, allostery, or inhibition. Achieving control and optimization toward desired outcomes, such as minimizing side products or limiting cofactor consumption, necessitates a thorough understanding of the kinetic parameters and interactions within ERNs, including those that are not readily identifiable. − …”

Exploring Emergent Properties in Enzymatic Reaction Networks: Design and Control of Dynamic Functional Systems

“…Rather than recreating digital infrastructure directly, recent work on small ERNs has aimed to establish modules and motifs capable of analogue computation. One example of this shows the development of small ERNs that are capable of arithmetic operations . The key insight here is that specific enzymatic interactions, such as cascading pathways, inhibitors, and parallel product conversion, can be interpreted as arithmetic functions if operated in the right parameter regime.…”

“…One example of this shows the development of small ERNs that are capable of arithmetic operations. 236 The key insight here is that specific enzymatic interactions, such as cascading pathways, inhibitors, and parallel product conversion, can be interpreted as arithmetic functions if operated in the right parameter regime. Similarly, specific forms of substrate competition between 238 They employed computational retrosynthesis tools to design enzymatic models that can act as transducers (converting different incoming metabolite signals into one substrate) and nonlinear actuators (to achieve sigmoidal responses) and when combined together can act as analogue adders (to perform a weighted sum of incoming signals, e.g., a perceptron).…”

Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy

“…Biocomputing, an ambitious field using biological tools for information processing, displays great potential in sensing systems as well as intelligent machines, which also provides a powerful tool for biomedical implementation including bio-imaging and precision medicine 1 , 2 , 3 , 4 . Recent studies on molecular diagnostics and gene expression profiling have demonstrated remarkable heterogeneity within cancer 5 , 6 .…”