Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA‐Based Recruitment Domains

Abstract

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Protein‐Templated Reactions Using DNA‐Antibody Conjugates

“…The increase observed on product formation at larger protein excesses might occur due to protein aggregation which leads to a non-specific colocalization of reactants, in accordance with previous observations. [42] Small 2023, 19, 2200971 A series of control experiments further supports that the reaction is templated by HSA (Figure S3, Supporting Information). A specificity test shows that the system is solely responsive to HSA, as no product is generated in the presence of other proteins (i.e., bovine serum albumin, BSA) (Figure S3, lane 3, Supporting Information).…”

“…The increase observed on product formation at larger protein excesses might occur due to protein aggregation which leads to a non‐specific colocalization of reactants, in accordance with previous observations. [ 42 ]…”

DNA‐Mediated Control of Protein Function in Semi‐Synthetic Systems

“…Recently, de Greef et al. have shown that DNA‐mediated dimerization can lead to the activation of caspase‐9, an enzyme involved in apoptosis [5c,14] . These studies have shed fundamental insight into proximity‐driven enzyme regulation and its possible role in programmed cell death.…”

Protein–Protein Communication Mediated by an Antibody‐Responsive DNA Nanodevice**

“…The complex, tightly regulated networks [1, 2] through which DNA, RNA and proteins interact underly the functioning of living systems [3–5] . One of the aims of synthetic biology is to create artificial pathways in which DNA, RNA and proteins interact with each other via analogously “programmed” reaction patterns to create new tools for sensing, drug‐delivery, cell imaging [6–14] . A widely used approach to this end is the rational design of synthetic DNA/protein communication that takes advantage of the many naturally occurring proteins that recognize and bind specific oligonucleotide sequences to, for example, regulate transcription or translation [15–21] .…”