iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition

Abstract

Linker engineering is rapidly gaining prominence as protein engineers and synthetic biologists construct increasingly sophisticated protein assemblies capable of executing complex molecular functions in the context of biosensing, biocatalysis, or biotherapeutics. Depending on the application, the structural and functional requirements imposed on the underlying linkers can differ vastly. At the same time, there is a distinct lack of methods to effectively code linkers at the level of DNA and tailor them to the functional requirements of different fusion proteins. Addressing these limitations, a scalable framework is presented to compose co-polymeric linkers of variable lengths and amino acid composition based on a limited number of linker fragments stored in sequence-verified entry plasmids. The assembly process is exemplified for Pro-rich linkers in the context of a Zn²⁺-responsive dual-readout BRET/FRET sensor while examining how linker composition impacts key functional properties such as ligand affinity, dynamic range, and their ability to separate structurally distinct domains.