Toolkit Items

Focused ultrasound (FUS) is a noninvasive physical delivery and control modality that penetrates deep biological tissues and induces confined mild hyperthermia to activate heat-sensitive genetic modules. In the cited 2023 study, FUS was coupled to heat-sensitive CRISPR, CRISPRa, and CRISPRi systems to enable remote spatiotemporal regulation of genome and epigenome function in live cells and animals.

As a tool component, CIB1 is most directly supported as the cryptochrome-interacting bHLH partner used with CRY2 to create blue-light-controlled protein association systems. In these systems, CIB1 is typically fused to localization, transcriptional, or enzymatic modules so that blue light drives CRY2–CIB1 binding and light withdrawal reverses the interaction.

Subsequently, we delve into cutting-edge applications of nanoparticles to enhance immune protection, including mosaic and cocktail nanoparticle vaccines, surface-modified targeting strategies, and the integration of mRNA technology with virus-like particles (VLPs).

AsLOV2 is the light-oxygen-voltage 2 photosensory domain from Avena sativa phototropin 1 used as a blue-light-responsive actuator in engineered fusion proteins. Blue-light activation drives allosteric conformational extension involving sequential unfolding of the N-terminal A'α helix and the C-terminal Jα helix, enabling conformational uncaging and related optogenetic control.

UNC10245092 is a previously identified linear peptide inhibitor that binds calcium and integrin binding protein 1 (CIB1). It has been used as a reference peptide in structural and in silico studies of CIB1-targeting decoy peptide design.

iLID/SspB is a blue-light-inducible heterodimerization system built from an engineered iLID module and the SspB binding partner. It is used to reversibly recruit proteins in cells for control of localization and signaling, including membrane recruitment, neurotrophin receptor construction, microtubule plus-end targeting, and perturbation of small GTPase pathways.

Prime editing is mentioned in the cited review as part of the broader set of genome-editing approaches considered in bacterial genome engineering. The supplied evidence does not describe its molecular architecture, target scope, or editing outcomes.

Three-dimensional (3D) bioprinting is a rapidly evolving technology that uses complementary biomaterials to emulate native extracellular matrices, enabling the generation of finely patterned, multicellular tissue architectures.

Key ACT modalities include chimeric antigen receptor (CAR) T cells, tumor-infiltrating lymphocytes (TILs), and T cell receptor (TCR)-engineered T cells.

Avena sativa LOV2 domain variants are engineered insertion modules used to build thermosensitive allosteric chimeric proteins. In Escherichia coli, insertion of optimized LOV2 variants into diverse, structurally and functionally unrelated proteins produced potent thermoswitchable variants operating within a narrow 37-41 °C range.

The Light-Oxygen-Voltage (LOV) domain is a small blue-light-sensing protein domain used as an optogenetic input module. It binds flavin nucleotides and undergoes blue-light-induced structural rearrangements that can regulate linked effector domains, including in phototropins where LOV1 and LOV2 are coupled to a C-terminal serine/threonine kinase.

PpSB1-LOV is a bacterial short LOV photosensory domain from Pseudomonas putida KT2440 with a light-induced flavin-cysteinyl photo-adduct and exceptionally slow dark recovery. It has been characterized as a compact LOV building block whose photocycle kinetics can be tuned by conserved hydrophobic-pocket mutation, including the I48T variant that accelerates adduct rupture while remaining structurally and mechanistically benign.

Optical imaging methods covered in this review include... Raman spectroscopy for early-stage cancer detection.

YtvA is a blue-light-sensing LOV-STAS photoreceptor from Bacillus subtilis whose LOV domain has been structurally analyzed for LOV-LOV dimerization and interdomain interactions. Homologous mutations in a conserved LOV hydrophobic pocket alter activation-state kinetics, supporting YtvA as a tunable LOV sensor domain relevant to optogenetic design.

FRASE-bot is an in silico fragment-based hit-finding method for drug discovery against unconventional therapeutic targets. It mines thousands of 3D protein-ligand complex structures to build a fragment-in-structural-environment database, matches target protein environments to that database, and uses machine learning to prioritize seeded fragments as candidate binders.

Lipid-polymer hybrid nanoparticles (LPHNPs) are the next-generation nanocarriers that integrate the mechanical strength and sustained-release capacity of polymeric cores with the biocompatibility and high drug-loading efficiency of lipid shells.

Molecular dynamics simulation is a computational method for modeling atomistic conformational dynamics of proteins and analyzing residue fluctuations and vibrational behavior. In the cited studies, it was used as a noninvasive approach to validate dynamic behavior and to compare PAS-domain dynamics across functional groups.

Nanobody-based CAR-T cells further expand design versatility, offering improved stability, tumor penetration, and reduced immunogenicity compared with single-chain variable fragment constructs.

Chrimson is a red light-activated channelrhodopsin with a reported crystal structure. It provides red-shifted optogenetic excitation and has been used with Chronos to support two-color activation of independent neural populations in mouse brain slice without detectable cross-talk.

Light-induced Fourier transform infrared (FTIR) difference spectroscopy is an assay method for detecting light-triggered structural changes associated with signaling-state formation in photoreceptor proteins. In the cited literature, it was applied to blue-light sensing LOV and BLUF/FAD systems to measure protein- and chromophore-associated spectral changes after illumination.

The Avena sativa LOV2 domain is a blue-light-sensing photosensory domain used as a photoswitchable scaffold for engineered control of protein interactions. In the iLID design, the bacterial SsrA peptide is embedded in the LOV2 C-terminal helix so that blue light triggers helix undocking and enables binding to SspB.

Phototropin is a plant blue-light receptor protein, exemplified by Avena sativa PHOT1/NPH1, that contains two FMN-binding LOV domains and a C-terminal serine/threonine kinase domain. It acts as a light-activated kinase in which LOV2-mediated conformational changes are coupled to kinase activation and signaling.

The Arabidopsis thaliana phototropin 1 LOV2 domain is a blue-light-sensing protein domain from phototropin 1 whose dark-adapted crystal structure has been determined. In this state, the domain is dimeric and contains an N-terminal A'α helix and a C-terminal Jα helix that contribute to coiled-coil-mediated dimerization.

PpSB2-LOV is a compact "short" light, oxygen, voltage (LOV) photosensory protein from Pseudomonas putida KT2440. It forms a light-induced LOV photoadduct and exhibits rapid dark-state thermal recovery, with a reported recovery time of 3.5 min at 20 °C, making it a candidate building block for genetically encoded photoswitches.

Time-resolved serial oscillation crystallography is a synchrotron-based, room-temperature X-ray diffraction method that collects, processes, and merges monochromatic oscillation data from fewer than 100 crystals. It was used to follow light-driven structural changes in a blue-light photoreceptor domain with 63 ms time resolution and to visualize time-dependent rearrangements of both the protein and its chromophore.

Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG) microparticles are injectable yellow-emitting inorganic scintillators used as implanted transducers for X-ray-driven optogenetic control. In the cited study, they converted X-ray irradiation into local light sufficient to activate ChRmine and inhibit via GtACR1, enabling bidirectional modulation of neural activity in mice.

H2B-tKR is a chromatin-targeted phototoxic construct in which histone H2B is fused to tandem KillerRed to control cell division with green light. Upon illumination, it induces nuclear damage-associated mitotic defects, including chromosome nondisjunction during metaphase, and can transiently block proliferation.

their effects on renal functional and structural indices were compared at week 15 with those of diabetic DBA/2J recipients of vehicle alone or of empty vector-transduced DBA/2J ADSCs (EV-ADSCs).

Krokinobacter eikastus rhodopsin 2 (KR2) is the prototypical light-driven sodium pump. Upon illumination, it actively transports small cations across cellular membranes and has been described as a useful optogenetic tool with applications in neuroscience.

CRISPR activation and interference (CRISPRa/CRISPRi) is a CRISPR-based gene regulation method that uses Nab2- or Egr3-targeted single-guide RNAs to modulate transcription. In the cited 2022 study, these tools were used in Neuro2a cells to mimic bidirectional cocaine-associated expression changes in Nab2 and Egr3.

AcrIIC3-LOV2 is an engineered light-switchable anti-CRISPR protein formed by fusing the Neisseria meningitidis Cas9 inhibitor AcrIIC3 to the Avena sativa LOV2 blue-light sensory domain. In mammalian cells, two reported hybrids strongly inhibit NmeCas9 in the dark and allow robust genome editing under blue-light irradiation.

The Aer PAS domain is the FAD-binding sensory domain from the dimeric Escherichia coli aerotaxis receptor Aer. It monitors cellular respiration through a redox-sensitive flavin cofactor and is structurally characterized in the Aer-PAS-GVV variant at 2.4 Å resolution.

The resulting aminated-hyaluronic acid (HAA) scaffolds act as rigid structural backbones in virus-inspired polymer-DNA nanoparticles termed as "Skeletoplexes"

Here, we constructed a hybrid gene cluster of the structural gene cluster from Serratia sp. ATCC 39006 and the accessory gene cluster from Bacillus megaterium in Escherichia coli to synthesize a novel gene-encoded gas vesicle ... termed as ARGS1B.

The review title directly names atomic force microscopy, and the supplied summary states that the review emphasizes AFM modalities for mapping dynamic mechanical properties of biological samples.

We describe the structural features, including solubility, chemical resistance, and modularity that facilitate the advanced development of bispecific, trivalent, and logic-gated CAR forms.

BROAD is a computational protein design method that combines Rosetta-based structure modeling, machine learning, and integer linear programming to improve design search beyond Rosetta sampling alone. It was demonstrated in antibody design to increase the predicted HIV neutralization breadth of VRC23 across a panel of 180 divergent viral strains.

Caspase-2 active-site mutants are engineered variants of human Caspase-2 designed in silico to broaden substrate recognition at the substrate N-terminal amino acid position. In vitro experiments confirmed that two proposed mutants showed enhanced promiscuity, including increased recognition of branched amino acids relative to unmutated Caspase-2.

The supplied review summary explicitly mentions CLARITY for correspondence between CBV-weighted signal spread and microvascular architecture.

the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS)

Here, we introduce an approach for visualizing individual microrobots in real time with color flow mapping ultrasound imaging based on acoustically induced structural oscillations of the microrobot generating a pseudo-Doppler signal.

Here, we report the X-ray crystal structure of plasmid-encoded Csep1P... Transcriptomic analysis revealed that Csep1P induced a chemokine-dominant inflammatory state in macrophages.

Domain insertion permissibility is an experimental engineering paradigm established in the human inward rectifier K+ channel Kir2.1 to identify engineerable allosteric sites. In this framework, sites permissive to insertion of regulatory domains can be converted into functional control points, including light-sensitive regulation when light-switchable domains are inserted.

The Drosophila PERIOD PAS domain fragment is a dPER protein segment comprising the PAS-A and PAS-B domains. Structural and functional analyses indicate that this fragment participates in PAS-mediated protein interactions, with the PAS-B beta-sheet surface mediating heterodimer formation with Drosophila TIMELESS (dTIM).

The EF-III metal-binding site of calcium- and integrin-binding protein 1 (CIB1) is an EF-hand domain characterized as a mixed Mg2+/Ca2+-binding site. Mutational analysis of its metal-coordinating loop indicates that EF-III modulates CIB1 affinity for the integrin alphaIIb domain and tunes metal sensitivity into a range relevant to intracellular Ca2+ sensing.

The elastic network model (ENM) is a computational method for analyzing protein structural dynamics from an elastic network representation. In the cited PAS domain superfamily study, ENM was used to quantify residue fluctuations and vibrational patterns and to relate these dynamic features to sequence conservation, structural features, and biological function.

Through the integration of computational structural biology and enzyme channel engineering, this study successfully elucidated the key intermediates in the stepwise hydroxylation-cleavage catalytic process of Digitalis purpurea-derived DlCYP87A enzyme. Building on this foundation, we implemented structure-guided rational design to precisely engineer the substrate channel and catalytic pocket.

FRASE, also described as FRASE-bot, is a computational fragment-based ligand discovery method that mines 3D ligand–protein complex structures to build a database of fragments in structural environments. It screens this database against a target protein, seeds the target structure with relevant ligand fragments, and uses a neural network to prioritize fragments with the highest likelihood of being native binders.

Free-energy calculations are an in silico prediction method used in the rational design of human Caspase-2 mutants. In the cited study, they were applied alongside sequence and structural comparisons of Caspase-2 and Caspase-3 to predict effects of active-site mutations on substrate recognition and to support engineering of broader amino-acid acceptance.

NoV Virus-like particles (VLPs) composed of the major capsid protein VP1 (~60 kDa) are essential for vaccine development... VP1 from four epidemiologically relevant GI genotypes was expressed using the silkworm-baculovirus system.