Engineering Generalized Protein-Based Biosensors for Molecular Detection and Clinical Applications

Abstract

Protein-based conformational switches serve as powerful tools for the construction of biosensors and for the control of cellular processes. These proteins feature a binding domain that recognizes a specific analyte and is coupled to an output domain in such a way that the binding event causes the output domain to provide an observable signal. These signals can either be turn-on of fluorescence, luminescence, or enzymatic activity or consist of the sensor changing its color. A challenge in constructing these protein switches is finding binding domains capable of relaying a ligand binding event to the conformational change of an output domain. Generalized binding domains can address these challenges by providing a scaffold that can easily be modified to detect a different ligand. These generalized binding domains are small proteins with modifiable residues that can be selected to bind a ligand of choice, usually through phage display and similar selection techniques. Here, we present two approaches to make generalized protein switches. In the first approach, antibody mimetics nanobodies and monobodies are inserted in fluorescent proteins such that binding of their ligand causes an increase in fluorescence. This technique, named adaptable turn-on maturation (ATOM), was used to develop biosensors for WD-40 repeat protein 5 (WDR5), c-Abl src homology 2 (SH2) domain, hRas, postsynaptic density protein 95 (PSD95), gephyrin, HOMER1, and mCherry for use in mammalian cells. ATOM is, therefore, compatible with a variety of ligands due to its input domain being a generalized binding domain. Additionally, the ATOM mechanism can be used to convert many fluorescent proteins into biosensors. For demonstration, we made biosensors from Clover, mTurqoise, mTagRFP-t, mStayGold, mBaoJin, and GCaMP6s. In the second approach, we develop a luminescent protein switch from the enzyme nanoluciferase (nLucAFF) that switches color from green to blue upon DNA binding. We show that DNA-based devices can then be used to detect various ligands and relay that event to nLucAFF, which provides an output easily quantifiable by a cell phone. The nLucAFF protein was used to detect DNA sequences amplified from cytomegalovirus (CMV), dengue, and nCoV. Additionally, aptamers binding to serotonin and aptamers were used to detect these molecules by directing the nLucAFF color change. The initial version of nLucAFF was slow, dim, and had low sensitivity. These drawbacks were resolved in the next version, nLucAFF2, to achieve turn-on within 5 minutes and detect ligands down to 40 pM with a cell phone camera. The last chapter combines two ligand-binding domains to activate a small cytotoxic RNase, barnase, and paves the way for the development of multi-input protein switches that can potentially be generalized ligand-binding domains.