Engineering Protein Switches for Rapid Diagnostic Tests

Abstract

Biological signaling pathways are underpinned by protein switches that sense and respond to molecular inputs. Inspired by nature, engineered protein switches have been designed to directly transduce analyte binding into a quantitative signal in a simple, wash-free, homogeneous assay format. As such, they offer great potential to underpin point-of-need diagnostics that are needed across broad sectors to improve access, costs, and speed compared to laboratory assays. Despite this, protein switch assays are not yet in routine diagnostic use, and a number of barriers to uptake must be overcome to realize this potential. Here, we review the opportunities and challenges in engineering protein switches for rapid diagnostic tests. We evaluate how their design, comprising a recognition element, reporter, and switching mechanism, relates to performance and identify areas for improvement to guide further optimization. Recent modular switches that enable new analytes to be targeted without redesign are crucial to ensure robust and efficient development processes. The importance of translational steps toward practical implementation, including integration into a user-friendly device and thorough assay validation, is also discussed.

Paving the way towards continuous biosensing by implementing affinity-based nanoswitches on state-dependent readout platforms

“…The amount of targets for which naturally occurring switches exist is however limited, and therefore, strategies have been described to develop synthetic switches from affinity binders, which we subdivide here in the following categories. 18–20…”

“…21 For monovalent targets however, either two affinity binders that can bind to distinct epitopes of the target molecule, or one affinity binder recognizing the target molecule and a second one binding to the target-receptor-complex, are required. 18,20,22 This strategy is highly modular and thus allows for straightforward design of affinity-based nanoswitches. 21 However, as small ligands cannot easily be detected using clamping due to their limited recognition domains, affinity-based nanoswitches have also been engineered based on competition, which relies on the attachment of a competitor to the bioreceptor (Fig.…”

“…However, by using a competitor, the overall affinity of the target towards the switch will be lower than its affinity for the native bioreceptor. 18,20,23–26 Lastly, synthetic switches can be developed by introducing an alternative folding, as depicted in Fig. 1C and 2C.…”

Context-Aware Diagnostic Specificity (CADS)

“…Rapid detection of proteins is critical in a vast array of diagnostic or monitoring applications [ 1 , 2 , 3 ]. Development of point of need (PON) devices is expanding rapidly, facilitated by technological improvements in miniaturization and enhanced access to scalable manufacturing equipment [ 4 ].…”

Engineering A Fluorescent Protein Color Switch Using Entropy-driven Beta Strand Exchange

“…Protein switches are versatile tools with applications in synthetic biology, molecular diagnostics and cellular biosensing [1][2][3][4] . Rapid advancements in the field of protein switch engineering using random, rational, semi-rational, and de novo approaches testify to the ongoing search for improved designs [5][6][7][8] .…”