Genetically encodable materials for non-invasive biological imaging

Abstract

Many questions in basic biology and medicine require the ability to visualize the function of specific cells and molecules inside living organisms. In this context, technologies such as ultrasound, optoacoustics and magnetic resonance provide non-invasive imaging access to deep-tissue regions, as used in many laboratories and clinics to visualize anatomy and physiology. In addition, recent work has enabled these technologies to image the location and function of specific cells and molecules inside the body by coupling the physics of sound waves, nuclear spins and light absorption to unique protein-based materials. These materials, which include air-filled gas vesicles, capsid-like nanocompartments, pigment-producing enzymes and transmembrane transporters, enable new forms of biomolecular and cellular contrast. The ability of these protein-based contrast agents to be genetically encoded and produced by cells creates opportunities for unprecedented in vivo studies of cellular function, while their amenability to genetic engineering enables atomic-level design of their physical, chemical and biological properties.

Fig. 1. Small proteins as genetically encoded contrast agents for non-invasive imaging.

(a) The tetrameric enzyme beta-galactosidase cleaves the galactopyranosyl ring on the synthetic Gd³⁺ chelator EgadMe, leading to increased water binding and T₁-weighted MRI contrast. (b) The heme-binding domain of P450-BM3 was evolved to selectively bind the neurotransmitter dopamine to alter water access to the paramagnetic Fe³⁺, yielding a molecular sensor of dopamine for T₁-weighted MRI. (c) Designed lysine repeat proteins (LRPs) rapidly exchange amide protons with water, thus yielding enhanced contrast in chemical exchange saturation transfer (CEST) MRI. (d) Reporter gene for diffusion-weighted MRI based on increased water diffusion across the cell membrane after overexpression of Aquaporin 1 (AQP1) (e) Hemodynamic contrast mechanism based on local expression and release of vasoactive peptides lead to increased blood flow detectable with fMRI or other imaging techniques sensitive to hemodynamics. (f) Bacterial phytochrome-derived infrared fluorescent proteins (iFPs) can serve as contrast agents for optoacoustic imaging. When absorbing near-infrared laser pulses, the chromophores transform photons into pressure waves detectable with ultrasound. PDB structures 3J7H (β-galactosidase), 4DU2 (BM3h-B7) and 4CQH (iFP 2.0) were visualized using ChimeraX. Adapted from ref., Springer Nature Ltd. (e).

Fig. 2. Proteinaceous nanocompartments as multiscale contrast agents.

Schematic summarizing work on metalloproteins for molecular imaging applications. (a) Genetic constructs for expression of the M. xanthus encapsulin system in mammalian systems consisting of its shell forming monomer MxEncA and a multigene expression cassette for co-expression of its endogenous cargo proteins (MxEncBCD) or engineered cargos such as a soluble bacterial tyrosinase (BmTyr) with a C-terminal encapsulation signal. Cutaway view of the MxEnc nanocompartment (T=3) schematically showing internal cargo proteins either yielding iron oxides for detection in MRI or cryoET or melanin pigments that afford (b) detection by MRI, optoacoustics, and cryo-electron tomography. (c) Genetic constructs for expression of the Q. thermotolerans encapsulin system in mammalian systems consisting of its shell forming monomer QtEnc and its iron-mineralizing cargo protein QtIMEF, or other engineered cargos such as fluorescent proteins. Cutaway view of the larger QtEnc nanocompartment (T=4 icosahedral symmetry) showing a zoom-in onto the pore region at the fivefold symmetry center and docked QtIMEF cargo yielding effective iron biomineralization affording contrast in TEM images of (d) HEK293T cells and T4/5 Drosophila neurons. Structures of BM3h (PDB: 4DU2), ferritin (EMD-2788), Mx Encapsulin (EMD-5917), BmTyr (PDB: 3NM8), Qt Encapsulin (EMD-4879) and QtIMEF (PDB: 6N63) were visualized using ChimeraX. Adapted from Ref., Springer Nature Ltd (b) and Ref., American Chemical Society (d).

Fig. 3. Genetically encodable air-filled protein nanostructures as multimodality contrast agents.

(a) Transmission electron micrograph of a GV, and a diagram of the various material properties used to produce contrast in imaging modalities. Z, acoustic impedance; χ, magnetic susceptibility; n, index of refraction. (b) Engineered bacterial gene cluster, ARG1, comprising genes from A. flos-aquea (orange) and B. megaterium (blue) that encode the heterologous expression of GVs in bacteria. (c) Representative electron micrograph of heterologously expressed GVs in the cytosol of mammalian cells. (d) Synthetic mammalian operon, mARG1, comprising 9 genes originating from B. megaterium that result in GV expression in mammalian cells. (e-g) GVs as genetically encodable contrast agents and reporter genes for in vivo (e) ultrasound imaging, (f) MRI, and (g) OCT. Adapted from ref., AAAS (c,d,e), ref., Springer Nature Ltd. (f).