Статті в журналах: "Photoactivatable systems"

1

Raymo, Françisco M. "Photoactivatable Fluorophores." ISRN Physical Chemistry 2012 (September 17, 2012): 1–15. http://dx.doi.org/10.5402/2012/619251.

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Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength and then emit after irradiation at an exciting wavelength. The interplay of such activation and excitation events can be exploited to switch fluorescence on in a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence translates into the opportunity to implement imaging and spectroscopic schemes that are not possible with conventional fluorophores. Specifically, photoactivatable fluorophores permit the monitoring of dynamic processes in real time as well as the reconstruction of images with subdiffraction resolution. These promising applications can have a significant impact on the characterization of the structures and functions of biomolecular systems. As a result, strategies to implement mechanisms for fluorescence photoactivation with synthetic fluorophores are particularly valuable. In fact, a number of versatile operating principles have already been identified to activate the fluorescence of numerous members of the main families of synthetic dyes. These methods are based on either the irreversible cleavage of covalent bonds or the reversible opening and closing of rings. This paper overviews the fundamental mechanisms that govern the behavior of these photoresponsive systems, illustrates structural designs for fluorescence photoactivation, and provides representative examples of photoactivatable fluorophores in actions.

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2

Nihongaki, Yuta, Yuichi Furuhata, Takahiro Otabe, Saki Hasegawa, Keitaro Yoshimoto, and Moritoshi Sato. "CRISPR–Cas9-based photoactivatable transcription systems to induce neuronal differentiation." Nature Methods 14, no. 10 (September 11, 2017): 963–66. http://dx.doi.org/10.1038/nmeth.4430.

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3

Smith, Nichola A., and Peter J. Sadler. "Photoactivatable metal complexes: from theory to applications in biotechnology and medicine." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 371, no. 1995 (July 28, 2013): 20120519. http://dx.doi.org/10.1098/rsta.2012.0519.

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This short review highlights some of the exciting new experimental and theoretical developments in the field of photoactivatable metal complexes and their applications in biotechnology and medicine. The examples chosen are based on some of the presentations at the Royal Society Discussion Meeting in June 2012, many of which are featured in more detail in other articles in this issue. This is a young field. Even the photochemistry of well-known systems such as metal–carbonyl complexes is still being elucidated. Striking are the recent developments in theory and computation (e.g. time-dependent density functional theory) and in ultrafast-pulsed radiation techniques which allow photochemical reactions to be followed and their mechanisms to be revealed on picosecond/nanosecond time scales. Not only do some metal complexes (e.g. those of Ru and Ir) possess favourable emission properties which allow functional imaging of cells and tissues (e.g. DNA interactions), but metal complexes can also provide spatially controlled photorelease of bioactive small molecules (e.g. CO and NO)—a novel strategy for site-directed therapy. This extends to cancer therapy, where metal-based precursors offer the prospect of generating excited-state drugs with new mechanisms of action that complement and augment those of current organic photosensitizers.

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4

Snapp, E. L., and P. Lajoie. "Activating Photoactivatable Proteins with Laser Light to Visualize Membrane Systems and Membrane Traffic in Living Cells." Cold Spring Harbor Protocols 2011, no. 11 (November 1, 2011): pdb.prot066571. http://dx.doi.org/10.1101/pdb.prot066571.

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5

Ivanskaya, E. V., M. I. Meschaninova, M. A. Vorobyeva, D. O. Zharkov, and D. S. Novopashina. "The approach to the preparation of cyclic photocleavable RNA for photoactivatable CRISPR/Cas9 System." Биоорганическая химия 50, no. 5 (December 5, 2024): 622–35. https://doi.org/10.31857/s0132342324050051.

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The development of controllable gene editing systems on the base of CRISPR/Cas is an actually problem of modern molecular biology and genetic enginery. Interesting variant of solution of this problem is modification of guide RNA by introduction of photocleavable linkers. We developed the approach to the synthesis of cyclic photocleavable guide crRNA for the CRISPR/Cas9 system with photolinker on the base of 1-(2-nitrophenyl)-1,2-ethanediol (PL). Upon irradiation by UV-light these guide RNA are linearized and CRISPR/Cas9 system is activated. Two chemical methods to the cyclization of RNA were tested: Michael reaction (thiol-maleimide condensation) and Cu-catalyzed azide-alkyne cycloaddition (CuAAC, click-chemistry reaction). For this purpose 5',3'-modified RNA containing reactive groups were prepared. The advantages of CuAAC reaction for cyclic RNA preparation was demonstrated. Effectivity of cyclic RNAs is depends from their secondary structure and ability of reactive groups to draw together. Series of photocleavable and control non-cleavable cyclic crRNA were obtained. It was shown that cyclic crRNAs guide nuclease Cas9 for plasmid cleavage less effective but linearization of photocleavable cyclic crRNA increases extent of plasmid cleavage. Developed approach permits prepare cyclic photocleavable RNA including spatiotemporal activation of guide RNA for gene editing. Photoregulation of gene editing will permit to lower the off-target effects and to carry out the editing more targeting.

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6

Li, Huiying, Qiansen Zhang, Yiran Gu, Yingyin Wu, Yamei Wang, Liren Wang, Shijie Feng, et al. "Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse." Proceedings of the National Academy of Sciences 117, no. 52 (December 14, 2020): 33426–35. http://dx.doi.org/10.1073/pnas.2003991117.

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Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type–specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

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7

Valueva, Anastasia A., Ivan D. Shumov, Anna L. Kaysheva, Irina A. Ivanova, Vadim S. Ziborov, Yuri D. Ivanov, and Tatyana O. Pleshakova. "Covalent Protein Immobilization onto Muscovite Mica Surface with a Photocrosslinker." Minerals 10, no. 5 (May 20, 2020): 464. http://dx.doi.org/10.3390/min10050464.

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Muscovite mica with an amino silane-modified surface is commonly used as a substrate in atomic force microscopy (AFM) studies of biological macromolecules. Herein, the efficiency of two different protein immobilization strategies employing either (N-hydroxysuccinimide ester)-based crosslinker (DSP) or benzophenone-based photoactivatable crosslinker (SuccBB) has been compared using AFM and mass spectrometry analysis. Two proteins with different physicochemical properties—human serum albumin (HSA) and horseradish peroxidase enzyme protein (HRP)—have been used as model objects in the study. In the case of HRP, both crosslinkers exhibited high immobilization efficiency—as opposed to the case with HSA, when sufficient capturing efficiency has only been observed with SuccBB photocrosslinker. The results obtained herein can find their application in commonly employed bioanalytical systems and in the development of novel highly sensitive chip-based diagnostic platforms employing immobilized proteins. The obtained data can also be of interest for other research areas in medicine and biotechnology employing immobilized biomolecules.

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8

Maronde, Erik. "Cyclic Nucleotide (cNMP) Analogues: Past, Present and Future." International Journal of Molecular Sciences 22, no. 23 (November 28, 2021): 12879. http://dx.doi.org/10.3390/ijms222312879.

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Cyclic nucleotides are important second messengers involved in cellular events, and analogues of this type of molecules are promising drug candidates. Some cyclic nucleotide analogues have become standard tools for the investigation of biochemical and physiological signal transduction pathways, such as the Rp-diastereomers of adenosine and guanosine 3′,5′-cyclic monophosphorothioate, which are competitive inhibitors of cAMP- and cGMP-dependent protein kinases. Next generation analogues exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity, or are caged or photoactivatable for fast and/or targeted cellular imaging. Novel specific nucleotide analogues activating or inhibiting cyclic nucleotide-dependent ion channels, EPAC/GEF proteins, and bacterial target molecules have been developed, opening new avenues for basic and applied research. This review provides an overview of the current state of the field, what can be expected in the future and some practical considerations for the use of cyclic nucleotide analogues in biological systems.

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9

Ferenz, Nick P., and Patricia Wadsworth. "Prophase Microtubule Arrays Undergo Flux-like Behavior in Mammalian Cells." Molecular Biology of the Cell 18, no. 10 (October 2007): 3993–4002. http://dx.doi.org/10.1091/mbc.e07-05-0420.

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In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation.

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10

Ben Mihoub, Amina, Ludivine Larue, Albert Moussaron, Zahraa Youssef, Ludovic Colombeau, Francis Baros, Céline Frochot, Régis Vanderesse, and Samir Acherar. "Use of Cyclodextrins in Anticancer Photodynamic Therapy Treatment." Molecules 23, no. 8 (August 2, 2018): 1936. http://dx.doi.org/10.3390/molecules23081936.

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Photodynamic therapy (PDT) is mainly used to destroy cancerous cells; it combines the action of three components: a photoactivatable molecule or photosensitizer (PS), the light of an appropriate wavelength, and naturally occurring molecular oxygen. After light excitation of the PS, the excited PS then reacts with molecular oxygen to produce reactive oxygen species (ROS), leading to cellular damage. One of the drawbacks of PSs is their lack of solubility in water and body tissue fluids, thereby causing low bioavailability, drug-delivery efficiency, therapeutic efficacy, and ROS production. To improve the water-solubility and/or drug delivery of PSs, using cyclodextrins (CDs) is an interesting strategy. This review describes the in vitro or/and in vivo use of natural and derived CDs to improve antitumoral PDT efficiency in aqueous media. To achieve these goals, three types of binding modes of PSs with CDs are developed: non-covalent CD–PS inclusion complexes, covalent CD–PS conjugates, and CD–PS nanoassemblies. This review is divided into three parts: (1) non-covalent CD-PS inclusion complexes, covalent CD–PS conjugates, and CD–PS nanoassemblies, (2) incorporating CD–PS systems into hybrid nanoparticles (NPs) using up-converting or other types of NPs, and (3) CDs with fullerenes as PSs.

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11

White, Paul, Amar Joshi, Patrice Rassam, Nicholas G. Housden, Renata Kaminska, Jonathan D. Goult, Christina Redfield, et al. "Exploitation of an iron transporter for bacterial protein antibiotic import." Proceedings of the National Academy of Sciences 114, no. 45 (October 25, 2017): 12051–56. http://dx.doi.org/10.1073/pnas.1713741114.

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Unlike their descendants, mitochondria and plastids, bacteria do not have dedicated protein import systems. However, paradoxically, import of protein bacteriocins, the mechanisms of which are poorly understood, underpins competition among pathogenic and commensal bacteria alike. Here, using X-ray crystallography, isothermal titration calorimetry, confocal fluorescence microscopy, and in vivo photoactivatable cross-linking of stalled translocation intermediates, we demonstrate how the iron transporter FpvAI in the opportunistic pathogen Pseudomonas aeruginosa is hijacked to translocate the bacteriocin pyocin S2 (pyoS2) across the outer membrane (OM). FpvAI is a TonB-dependent transporter (TBDT) that actively imports the small siderophore ferripyoverdine (Fe-Pvd) by coupling to the proton motive force (PMF) via the inner membrane (IM) protein TonB1. The crystal structure of the N-terminal domain of pyoS2 (pyoS2NTD) bound to FpvAI (Kd = 240 pM) reveals that the pyocin mimics Fe-Pvd, inducing the same conformational changes in the receptor. Mimicry leads to fluorescently labeled pyoS2NTD being imported into FpvAI-expressing P. aeruginosa cells by a process analogous to that used by bona fide TBDT ligands. PyoS2NTD induces unfolding by TonB1 of a force-labile portion of the plug domain that normally occludes the central channel of FpvAI. The pyocin is then dragged through this narrow channel following delivery of its own TonB1-binding epitope to the periplasm. Hence, energized nutrient transporters in bacteria also serve as rudimentary protein import systems, which, in the case of FpvAI, results in a protein antibiotic 60-fold bigger than the transporter’s natural substrate being translocated across the OM.

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12

Jerng, Henry H., Jay M. Patel, Tamor A. Khan, Benjamin R. Arenkiel, and Paul J. Pfaffinger. "Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior." PLOS ONE 16, no. 3 (March 23, 2021): e0248688. http://dx.doi.org/10.1371/journal.pone.0248688.

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Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2–3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.

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13

Vizoso, Miguel, Colin E. J. Pritchard, Lorenzo Bombardelli, Bram van den Broek, Paul Krimpenfort, Roderick L. Beijersbergen, Kees Jalink, and Jacco van Rheenen. "A doxycycline- and light-inducible Cre recombinase mouse model for optogenetic genome editing." Nature Communications 13, no. 1 (October 28, 2022). http://dx.doi.org/10.1038/s41467-022-33863-z.

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AbstractThe experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.

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14

Qiu, Xujun, Eric Pohl, Andre Jung, Qianyu Cai, Haopu Su, Olaf Fuhr, Ute Schepers, and Stefan Bräse. "Modulating the photolysis of aryl azides in supramolecular host to develop photoactivatable fluorophores." Chemical Communications, 2024. http://dx.doi.org/10.1039/d4cc03907f.

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Photolysis of aryl azides is a convenient method to approach more functionalized systems in chemical biology. Here,we present a set of photoactivatable aryl azides that undergo controlled reaction pathways within...

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15

Zhang, Xiao, Yuxin Pan, Shoukai Kang, and Liangcai Gu. "Combinatorial Approaches for Efficient Design of Photoswitchable Protein-Protein Interactions as In Vivo Actuators." Frontiers in Bioengineering and Biotechnology 10 (February 8, 2022). http://dx.doi.org/10.3389/fbioe.2022.844405.

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Light switchable two-component protein dimerization systems offer versatile manipulation and dissection of cellular events in living systems. Over the past 20 years, the field has been driven by the discovery of photoreceptor-based interaction systems, the engineering of light-actuatable binder proteins, and the development of photoactivatable compounds as dimerization inducers. This perspective is to categorize mechanisms and design approaches of these dimerization systems, compare their advantages and limitations, and bridge them to emerging applications. Our goal is to identify new opportunities in combinatorial protein design that can address current engineering challenges and expand in vivo applications.

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16

Song, Kun-Long, Ricardo Meyrelles, Guillaume Pilet, Boris Maryasin, Maurice Médebielle, and Jérémy Merad. "Dication Disulfuranes as Photoactivatable Sources of Radical Organocatalysts." Angewandte Chemie, November 4, 2024. http://dx.doi.org/10.1002/ange.202419751.

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The recent development of photoredox and energy transfer catalysis has led to a significant expansion of visible light‐driven chemical transformations. These methods have demonstrated exceptional efficiency in converting a wide range of substrates into radical intermediates and generating open‐shell catalytic species. However, the simplification of catalytic systems and the direct generation of highly reactive radical organocatalysts through direct visible light irradiation from stable precatalysts remains largely an unrealized goal. This challenge is mainly due to the limited availability of precatalysts that are responsive to visible light. Herein, we introduce a new class of bench‐stable dicationic disulfuranes, which release highly reactive thiyl radicals upon blue light excitation. Spectroscopic and computational studies reveal that this reactivity arises from a combination of structural features and intermolecular interactions. This family of molecules has been employed to catalyze radical cascades previously incompatible with photoredox conditions, enabling the efficient formation of 1,2‐dioxolanes and 1,3‐hydroxyketones in excellent yields and short reaction times.

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17

Song, Kun-Long, Ricardo Meyrelles, Guillaume Pilet, Boris Maryasin, Maurice Médebielle, and Jérémy Merad. "Dication Disulfuranes as Photoactivatable Sources of Radical Organocatalysts." Angewandte Chemie International Edition, November 4, 2024. http://dx.doi.org/10.1002/anie.202419751.

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The recent development of photoredox and energy transfer catalysis has led to a significant expansion of visible light‐driven chemical transformations. These methods have demonstrated exceptional efficiency in converting a wide range of substrates into radical intermediates and generating open‐shell catalytic species. However, the simplification of catalytic systems and the direct generation of highly reactive radical organocatalysts through direct visible light irradiation from stable precatalysts remains largely an unrealized goal. This challenge is mainly due to the limited availability of precatalysts that are responsive to visible light. Herein, we introduce a new class of bench‐stable dicationic disulfuranes, which release highly reactive thiyl radicals upon blue light excitation. Spectroscopic and computational studies reveal that this reactivity arises from a combination of structural features and intermolecular interactions. This family of molecules has been employed to catalyze radical cascades previously incompatible with photoredox conditions, enabling the efficient formation of 1,2‐dioxolanes and 1,3‐hydroxyketones in excellent yields and short reaction times.

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18

Zhang, Lili, Mei Chen, Zhiqiang Wang, Minjuan Zhong, Hong Chen, Ting Li, Linlin Wang, et al. "Spatiotemporal Regulation of Cell Fate in Living Systems Using Photoactivatable Artificial DNA Membraneless Organelles." ACS Central Science, May 21, 2024. http://dx.doi.org/10.1021/acscentsci.4c00380.

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19

Zhao, Hengzhi, Deyu Yi, Lele Li, Yuliang Zhao, and Mengyuan Li. "Modular Weaving DNAzyme in Skeleton of DNA Nanocages for Photoactivatable Catalytic Activity Regulation." Angewandte Chemie International Edition, March 22, 2024. http://dx.doi.org/10.1002/anie.202404064.

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AbstractDNAzymes exhibit tremendous application potentials in the field of biosensing and gene regulation due to its unique catalytic function. However, spatiotemporally controlled regulation of DNAzyme activity remains a daunting challenge, which may cause nonspecific signal leakage or gene silencing of the catalytic systems. Here, we report a photochemical approach via modular weaving active DNAzyme into the skeleton of tetrahedral DNA nanocages (TDN) for light‐triggered on‐demand liberation of DNAzyme and thus conditional control of gene regulation activity. We demonstrate that the direct encoding of DNAzyme in TDN could improve the biostability of DNAzyme and ensure the delivery efficiency, comparing with the conventional surface anchoring strategy. Furthermore, the molecular weaving of the DNA nanostructures allows remote control of DNAzyme‐mediated gene regulation with high spatiotemporal precision of light. In addition, we demonstrate that the approach is applicable for controlled regulation of the gene editing functions of other functional nucleic acids.

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20

Zhao, Hengzhi, Deyu Yi, Lele Li, Yuliang Zhao, and Mengyuan Li. "Modular Weaving DNAzyme in Skeleton of DNA Nanocages for Photoactivatable Catalytic Activity Regulation." Angewandte Chemie, March 22, 2024. http://dx.doi.org/10.1002/ange.202404064.

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AbstractDNAzymes exhibit tremendous application potentials in the field of biosensing and gene regulation due to its unique catalytic function. However, spatiotemporally controlled regulation of DNAzyme activity remains a daunting challenge, which may cause nonspecific signal leakage or gene silencing of the catalytic systems. Here, we report a photochemical approach via modular weaving active DNAzyme into the skeleton of tetrahedral DNA nanocages (TDN) for light‐triggered on‐demand liberation of DNAzyme and thus conditional control of gene regulation activity. We demonstrate that the direct encoding of DNAzyme in TDN could improve the biostability of DNAzyme and ensure the delivery efficiency, comparing with the conventional surface anchoring strategy. Furthermore, the molecular weaving of the DNA nanostructures allows remote control of DNAzyme‐mediated gene regulation with high spatiotemporal precision of light. In addition, we demonstrate that the approach is applicable for controlled regulation of the gene editing functions of other functional nucleic acids.

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21

De Santis, Riccardo, Fred Etoc, Edwin A. Rosado-Olivieri, and Ali H. Brivanlou. "Self-organization of human dorsal-ventral forebrain structures by light induced SHH." Nature Communications 12, no. 1 (November 19, 2021). http://dx.doi.org/10.1038/s41467-021-26881-w.

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AbstractOrganizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body’s axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on human embryonic stem cells (hESCs). We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers.

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22

Fedoryshchak, Roman O., Andrii Gorelik, Mengjie Shen, Maria M. Shchepinova, Inmaculada Perez-Dorado, and Edward William Tate. "Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes." Chemical Science, 2023. http://dx.doi.org/10.1039/d2sc06116c.

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Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in biology. Despite development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches...

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23

Soleimany, Amir, Delaram Kargari Aghmiouni, Masomeh Amirikhah, Mohammad Ali Shokrgozar, Sepideh Khoee, and Bruno Sarmento. "Two‐Photon Mediated Cancer Therapy: A Comprehensive Review on Two‐Photon Photodynamic Therapy and Two‐Photon‐Activated Therapeutic Delivery Systems." Advanced Functional Materials, July 18, 2024. http://dx.doi.org/10.1002/adfm.202408594.

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AbstractTwo‐photon excitation (2PE) represents substantial advantages in biophotonics over traditional one‐photon excitation (1PE), offering enhanced spatial resolution and deeper tissue penetration capabilities. Since its introduction in the 1990s, two‐photon excited microscopy has spurred the rapid emergence of novel 2PE‐based applications, including photodynamic therapy (PDT) and the targeted release of biologically active agents. This review first elucidates the fundamental principles of 2PE before comprehensively examining the evolution of photosensitizers (PSs) for two‐photon PDT (2P‐PDT), covering both nanomaterials and small molecules, categorized based on their respective characteristics. Additionally, it explores advancements in PS development for integrating 2P‐PDT with other therapeutic modalities (e.g., photothermal therapy, chemotherapy, immunotherapy, gene therapy, etc.). Subsequent sections provide an overview of photoactivatable compounds with two‐photon absorption properties for the controlled release of various therapeutic agents (e.g., drugs, genes, and gasotransmitters), emphasizing their potential in anticancer applications. Last, a deep discussion deciphers the prospects and challenges of 2P‐mediated therapy in cancer treatment, particularly concerning their clinical translation.

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24

Bequet‐Ermoy, Etienne, Virginie Silvestre, Stéphane Cuenot, and Eléna Ishow. "Reversible Light‐Triggered Stretching of Small‐Molecule Photochromic Organic Nanoparticles." Small, July 12, 2024. http://dx.doi.org/10.1002/smll.202403912.

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AbstractFunctional organic nanomaterials are nowadays largely spread in the field of nanomedicine. In situ modulation of their morphology is thus expected to considerably impact their interactions with the surroundings. In this context, photoswitchable nanoparticles that are manufactured, amenable to extensive disassembling upon illumination in the visible, and reversible reshaping under UV exposure. Such reversibility turns to be strongly impaired for photochromic nanoparticles in close contact with a substrate. In situ atomic force microscopy investigations at the nanoscale actually reveal progressive disintegration of the organic nanoparticles under successive UV–vis cycles of irradiation, in the absence of intrinsic elastic forces. These results point out the dramatic interactions exerted by surfaces on the cohesion of non‐covalently bonded organic nanoparticles. They invite to harness such systems, often used as biomarkers, to also serve as photoactivatable drug delivery nanocarriers.

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25

Uji, Masanori, Jumpei Kondo, Chikako Hara‐Miyauchi, Saori Akimoto, Rena Haruki, Yoichi Sasaki, Nobuo Kimizuka, Itsuki Ajioka, and Nobuhiro Yanai. "In Vivo Optogenetics Based on Heavy Metal‐Free Photon Upconversion Nanoparticles." Advanced Materials, September 23, 2024. http://dx.doi.org/10.1002/adma.202405509.

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AbstractPhoton upconversion (UC) from red or near‐infrared (NIR) light to blue light is promising for in vivo optogenetics. However, the examples of in vivo optogenetics have been limited to lanthanide inorganic UC nanoparticles, and there have been no examples of optogenetics without using heavy metals. Here the first example of in vivo optogenetics using biocompatible heavy metal‐free TTA‐UC nanoemulsions is shown. A new organic TADF sensitizer, a boron difluoride curcuminoid derivative modified with a bromo group, can promote intersystem crossing to the excited triplet state, significantly improving TTA‐UC efficiency. The TTA‐UC nanoparticles formed from biocompatible surfactants and methyl oleate acquire water dispersibility and remarkable oxygen tolerance. By combining with genome engineering technology using the blue light‐responding photoactivatable Cre‐recombinase (PA‐Cre), TTA‐UC nanoparticles promote Cre‐reporter EGFP expression in neurons in vitro and in vivo. The results open new opportunities toward deep‐tissue control of neural activities based on heavy metal‐free fully organic UC systems.

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Bolonduro, Olurotimi A., Zijing Chen, Corey P. Fucetola, Yan‐Ru Lai, Megan Cote, Rofiat O. Kajola, Akshita A. Rao, Haitao Liu, Emmanuel S. Tzanakakis, and Brian P. Timko. "An Integrated Optogenetic and Bioelectronic Platform for Regulating Cardiomyocyte Function." Advanced Science, July 25, 2024. http://dx.doi.org/10.1002/advs.202402236.

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AbstractBioelectronic medicine is emerging as a powerful approach for restoring lost endogenous functions and addressing life‐altering maladies such as cardiac disorders. Systems that incorporate both modulation of cellular function and recording capabilities can enhance the utility of these approaches and their customization to the needs of each patient. Here is report an integrated optogenetic and bioelectronic platform for stable and long‐term stimulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through the expression of a photoactivatable adenylyl cyclase, that when irradiated with blue light causes a dose‐dependent and time‐limited increase in the secondary messenger cyclic adenosine monophosphate with subsequent rise in autonomous cardiomyocyte beating rate. Bioelectronic readouts are obtained through a multi‐electrode array that measures real‐time electrophysiological responses at 32 spatially‐distinct locations. Irradiation at 27 µW mm−2 results in a 14% elevation of the beating rate within 20–25 min, which remains stable for at least 2 h. The beating rate can be cycled through “on” and “off” light states, and its magnitude is a monotonic function of irradiation intensity. The integrated platform can be extended to stretchable and flexible substrates, and can open new avenues in bioelectronic medicine, including closed‐loop systems for cardiac regulation and intervention, for example, in the context of arrythmias.

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Vogt, Arend, Raik Paulat, Daniel Parthier, Verena Just, Michal Szczepek, Patrick Scheerer, Qianzhao Xu, et al. "Simultaneous spectral illumination of microplates for high-throughput optogenetics and photobiology." Biological Chemistry, September 23, 2024. http://dx.doi.org/10.1515/hsz-2023-0205.

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Abstract The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis of action spectra. Current methods to determine action spectra only allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validated the RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

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Olivi, Lorenzo, Cleo Bagchus, Victor Pool, Ezra Bekkering, Konstantin Speckner, Hidde Offerhaus, Wen Y. Wu, et al. "Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a." Nucleic Acids Research, April 22, 2024. http://dx.doi.org/10.1093/nar/gkae283.

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Abstract CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

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Gabashvili, Anna N., Natalya A. Alexandrushkina, Elizaveta N. Mochalova, Daria V. Goliusova, Ekaterina N. Sapozhnikova, Pavel I. Makarevich, and Petr I. Nikitin. "Internalization of transferrin-tagged Myxococcus xanthus encapsulins into mesenchymal stem cells." Experimental Biology and Medicine 249 (May 7, 2024). http://dx.doi.org/10.3389/ebm.2024.10055.

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Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.

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Cieslik, Patrick A., Simon Klingler, Mirja Nolff, and Jason P. Holland. "Radiolabelled 177Lu‐Bispidine‐Trastuzumab for Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancers." Chemistry – A European Journal, December 8, 2023. http://dx.doi.org/10.1002/chem.202303805.

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Radioimmunotherapy (TRIT) is a promising alternative to conventional treatment options. Here, we present experimental work on the synthesis, radiochemistry, and in vivo performance of a lanthanide‐selective nonadentate bispidine ligand suitable for 177Lu3+ ion complexation. The ligand (bisp,1) was derivatised with a photoactivatable aryl azide (ArN3) group as a bioconjugation handle for light‐induced labelling of proteins. Quantitative radiosynthesis of [177Lu]Lu‐1+ was accomplished in 10 minutes at 40oC. Subsequent incubation [177Lu]Lu‐1+ with trastuzumab, followed by irradiation with light at 365 nm for 15 min, at room temperature and pH8.0–8.3, gave the radiolabelled mAb, [177Lu]Lu‐1‐azepin‐trastuzumab ([177Lu]Lu‐1‐mAb) in a decay‐corrected radiochemical yield of 14%, and radiochemical purity (RCP)>90%. Stability studies and cellular binding assays in vitro using the SK‐OV‐3 human ovarian cancer cells confirmed that [177Lu]Lu‐1‐mAb remained biological active and displayed specific binding to HER2/neu. Experiments in immunocompromised female athymic nude mice bearing subcutaneous xenograft models of SK‐OV‐3 tumours revealed significantly higher tumour uptake in the normal group compared with the control block group (29.8±11.4%ID g‐1 vs. 14.8±6.1%ID g‐1, respectively; P‐value=0.037). The data indicate that bispidine‐based ligand systems are suitable starting points for constructing novel, high‐denticity chelators for specific complexation of larger radiotheranostic metal ion nuclides.

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