Design and application of genetically encoded biosensors

Abstract

In the past 5-10 years, the power of the green fluorescent protein (GFP) and its numerous derivatives has been harnessed toward the development of genetically encoded fluorescent biosensors. These sensors are incorporated into cells or organisms as plasmid DNA, which leads the transcriptional and translational machinery of the cell to express a functional sensor. To date, over 100 different genetically encoded biosensors have been developed for targets as diverse as ions, molecules and enzymes. Such sensors are instrumental in providing a window into the real-time biochemistry of living cells and whole organisms, and are providing unprecedented insight into the inner workings of a cell.

Figure 1

Schematics of design platforms for fluorescent sensors. (a) A translocation-based probe for detecting the plasma membrane PtdIns(4,5)P2 concentration [48]. The PH domain of PLC-δ1, which can selectively recognize PtdIns(4,5)P2, is fused with GFP. When PtdIns(4,5)P2 in the plasma membrane is decreased, the sensor translocates from the plasma membrane to the cytosol, which increases cytosolic fluorescence. Examples include sensors for phosphoinositides. (b) An intensity-based single FP probe GCaMP2 [49,50]. GCaMP2 consists of the M13 fragment from myosin light chain kinase (shown in purple), a circularly permutated EGFP (shown in green) and calmodulin (CaM, shown in red). Ca²⁺ binding promotes the binding of M13 to CaM, which alters the protonation state of the chromophore, leading to an increase in fluorescence intensity. Classic examples include sensors for Ca²⁺, Cl⁻ and H⁺. (c) A ratiometric single-FP-based redox sensor, roGFP [51]. The relative fluorescence intensity of the two excitation maxima of roGFP1 shifts depending on the redox state: reduction causes a decrease in the excitation at 400 nm and an increase in the excitation at 480 nm (arrows). Classic examples include the ratiometric H⁺ sensor pHlorin, the ratiometric Ca²⁺ sensor pericam, and sensors for cGMP and membrane potential. (d) A FRET sensor, ZapCY, activated by conformational change [52]. Conformational change of the Zn²⁺-binding domain (zinc fingers 1 and 2 of transcription factor Zap1) in the presence of Zn²⁺ leads to an increase in FRET between CFP and YFP. Examples of this sensing platform include cameleon Ca²⁺ sensors, as well as sensors for sugars, glutamate, Zn²⁺, cAMP, cGMP, NO and membrane potential. (e) The FRET-based sensor Phocus for kinase activity [53]. On phosphorylation of the substrate domain (shown in pink) by protein kinase, the adjacent phosphorylation recognition domain (shown in purple) binds to the phosphorylated substrate domain, which causes a change in FRET between CFP and YFP. Classic examples include sensors for kinases and GTPase activity (e.g. Raichu probes). (f) Schematic of a protease-activated FRET biosensor for caspase [54]. During apoptosis, activated caspase cleaves the DEVD amino acid sequence, which reduces the FRET between GFP and BFP. Examples of this sensor platform include those for caspases and matrix metalloproteases.

Figure 2

Examples of sensors that use bioluminescence. (a) Aequorin senses Ca²⁺ [55]. First, coelenterazine is added to apo-aequorin to form the intermediate activated aequorin. Subsequently, on binding to Ca²⁺, the coelenterazine is oxidized, leading to emission of blue light at 480 nm. (b) Schematic of a BRET sensor CAMYEL used to measure cAMP concentrations [6]. The inactive cytosolic mutant form of the cAMP-binding protein human Epac-1 (shown in green) was flanked by citrine (in yellow) and Renilla luciferase (in grey). On oxidation of its cell-permeable substrate coelenterazine h, the luciferase emits light at 480 nm. Changes in cellular cAMP levels alter BRET between luciferase and citrine (excitation at 513 nm, emission at 530 nm) such that increases in cAMP cause BRET to increase.

Figure 3

Historical evolution of the cameleon family of Ca²⁺ sensors. These sensors use Ca²⁺ binding to a specific domain to induce a conformational change and to alter the energy transfer between two FPs. The original Ca²⁺-binding domain is derived from Xenopus calmodulin and the 26-residue M13 calmodulin-binding peptide from skeletal muscle myosin light chain kinase. The E104Q mutation in the yellow cameleon (i.e. YC family) decreases Ca²⁺ affinity.

Figure I

Steps involved in engineering a genetically encoded sensor.