DC Science Series #2

My project within the SYNSENSO network broadly aims to use split intein proteins to build novel logic gates that could improve biosensing in cell-free systems. Split inteins are proteins capable of ligating two proteins at the peptide level while simultaneously excising themselves from the fused proteins in a reaction called protein trans-splicing (PTS; details of the reaction in the middle panel of Fig. 1)¹. Based on this ligation reaction, split intein proteins have been applied as logic gates for biotechnological applications such as biosensing and biocomputation².

Logic gates are concepts borrowed from electronic circuits to describe biological systems. A logic gate generally refers to a system that receives inputs and triggers output signals. In a biological system, this logic can be interpreted through the following sequential steps: (i) the input signal refers to a target molecule being detected; (ii) next, an operation occurs that corresponds to an enzymatic reaction; and (iii) finally, this enzymatic reaction gives rise to an output signal. Split inteins are ideal triggers for releasing an output signal due to their PTS reaction. Therefore, split inteins are strong candidates for creating novel logic gate designs, especially considering their advantages: fast-reacting, modular, and particularly, their logic functionality¹,².

The main idea and novelty of the project lie in designing and characterising a chimeric protein containing split inteins to perform logic operations (Fig. 1). A chimeric protein is the fusion of proteins or their domains to either enhance a function or introduce a novel one. In theory, our chimeric protein design will detect nucleic acids via recognition domains and, consequently, release an output signal through the action of the split inteins. We are currently designing and screening such a chimeric construct by implementing a directed evolution pipeline. Ultimately, our project thus aims to create a foundational biosensing technology using the chimeric protein design and to demonstrate its biocomputational potential in cell-free biosensing for nucleic acid targets, mainly RNA.