Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals

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161

Significance Sensory photoreceptors not only enable organisms to derive spatial and temporal cues from incident light but also provide the basis for optogenetics, which denotes the manipulation by light of living systems with supreme spatial and temporal resolution. To expand the scope of optogenetics, we have engineered the light-activated phosphodiesterase LAPD, which degrades the ubiquitous second messengers cAMP and cGMP in a red-light–stimulated manner. Both cAMP and cGMP are key to the regulation of manifold physiological responses, and LAPD now augurs red-light control over these processes. As we demonstrate for two eukaryotic systems, LAPD does not require any additional exogenous factors, and thus permits the light perturbation of living cells in hitherto unrealized ways.

Section: Discussionmentioning

confidence: 99%

Engineering of a red-light–activated human cAMP/cGMP-specific phosphodiesterase

Gasser¹,

Taiber

Yeh

et al. 2014

160

161

“…A significant fraction of NIRW light can pass through several centimeters of human tissues (7-9), which makes NIRW light a promising means of controlling biological processes in animals. Absence of photoreceptors of NIRW light in most animal tissues is an additional advantage that makes NIRW light harmless (10). This is in contrast to blue light, which is absorbed by flavins and porphyrins, and therefore promotes photooxidative damage (11).…”

mentioning

confidence: 99%

“…Further, absorption wavelength maxima of bacteriophytochromes are red shifted compared with the absorption maxima of plant and cyanobacterial phytochromes. This results in a 2-to 10-fold gain in the penetration depth of light through mammalian tissues (7,10). Up to now, bacteriophytochrome engineering for optogenetic applications has lagged behind the engineering of photoreceptors of other types (4,5), including engineering of plant phytochromes (15,16).…”

mentioning

confidence: 99%

Engineering adenylate cyclases regulated by near-infrared window light

Ryu

Kang

Nelson

et al. 2014

108

114

Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochromebased optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.phytochrome | protein engineering | adenylyl cyclase | cAMP | locomotion

“…For fluorescence monitoring in vivo, Verkhusha and coworkers (15) engineered NIR fluorescent proteins from bacterial phytochrome receptors, enabling rapid, inexpensive fluorescence detection and quantification. These NIR proteins were modified in spectra to allow for multicolor imaging and have since proven useful for fluorescence imaging-based tracking of primary and metastatic tumor tissue in vivo, and for flow cytometry-based quantification of fluorescence intensities in tumor cell subpopulations ex vivo (16)(17)(18)(19). Relative to other imaging modalities, such as MRI, PET, and single-photon emission computed tomography this imaging technique is simple and comparatively high-throughput and does not require reporter probes, contrasting agents, or expensive imaging equipment (20).…”

Section: Discussionmentioning

confidence: 99%

Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo

Sita

Kouri

Hurley

et al. 2017

RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAibased nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a nearinfrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O 6 -methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.