Dye Labeling Research Articles

Structural and Functional Plasticity in the Regenerating Olfactory System of the Migratory Locust.

Regeneration after injury is accompanied by transient and lasting changes in the neuroarchitecture of the nervous system and, thus, a form of structural plasticity. In this review, we introduce the olfactory pathway of a particular insect as a convenient model to visualize neural regeneration at an anatomical level and study functional recovery at an electrophysiological level. The olfactory pathway of the locust (Locusta migratoria) is characterized by a multiglomerular innervation of the antennal lobe by olfactory receptor neurons. These olfactory afferents were axotomized by crushing the base of the antenna. The resulting degeneration and regeneration in the antennal lobe could be quantified by size measurements, dye labeling, and immunofluorescence staining of cell surface proteins implicated in axonal guidance during development. Within 3 days post lesion, the antennal lobe volume was reduced by 30% and from then onward regained size back to normal by 2 weeks post injury. The majority of regenerating olfactory receptor axons reinnervated the glomeruli of the antennal lobe. A few regenerating axons project erroneously into the mushroom body on a pathway that is normally chosen by second-order projection neurons. Based on intracellular responses of antennal lobe output neurons to odor stimulation, regenerated fibers establish functional synapses again. Following complete absence after nerve crush, responses to odor stimuli return to control level within 10–14 days. On average, regeneration of afferents, and re-established synaptic connections appear faster in younger fifth instar nymphs than in adults. The initial degeneration of olfactory receptor axons has a trans-synaptic effect on a second order brain center, leading to a transient size reduction of the mushroom body calyx. Odor-evoked oscillating field potentials, absent after nerve crush, were restored in the calyx, indicative of regenerative processes in the network architecture. We conclude that axonal regeneration in the locust olfactory system appears to be possible, precise, and fast, opening an avenue for future mechanistic studies. As a perspective of biomedical importance, the current evidence for nitric oxide/cGMP signaling as positive regulator of axon regeneration in connectives of the ventral nerve cord is considered in light of particular regeneration studies in vertebrate central nervous systems.

Investigation of a Truncated Aptamer for Ofloxacin Detection Using a Rapid FRET-Based Apta-Assay

In this work, we describe the use of a new truncated aptamer for the determination of ofloxacin (OFL), being a principal quinolone commonly used in both human and animal healthcare. Since the affinity of a 72-mer ssDNA sequence has been previously described without further investigations, this paper demonstrates the first computational prediction of the binding motif between this aptamer and OFL through in silico molecular docking studies. Besides, we suggest the application of the characterized recognition mechanism in a simple FRET (Förster Resonance Energy Transfer) pattern for the rapid aptasensing of the quinolone of interest. Accordingly, our approach harnesses the fluorescence quenching of the fluorescein-tagged aptamer (FAM-APT) induced by its partial hybridization to a tetramethyl rhodamine-labelled complementary ssDNA (TAMRA-cDNA). In such a structure, dye labels brought into close proximity act as a FRET pair. Upon ofloxacin addition, an affinity competition occurs to form a more stable FAM-APT/OFL complex, thus unquenching the FAM-APT signal. Interestingly, the recovered fluorescence intensity was found to correlate well with the antibiotic’s concentrations in the range of 0.2–200 μM in HEPES buffer, with a linear response that ranged between 0.2 and 20 μM. The rapid apta-assay achieved limits of detection and quantification of 0.12 and 0.40 μM, respectively. The truncated aptamer has also shown an improved specificity toward OFL than other quinolones, compared to the original full-length aptamer described in previous works. Finally, the practical application of the developed apta-assay was successfully confirmed to detect OFL quinolone in spiked milk samples, with satisfactory recoveries ranging between 97.4% and 111.4%.

Fluorescent Dye Labeling Changes the Biodistribution of Tumor-Targeted Nanoparticles.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution.

Picture perfect: Imaging mitochondrial membrane potential changes in retina slices with minimal stray fluorescence

Mitochondrial membrane potential (Ψm) is a critical parameter that can be used to determine cellular well-being. As it is a direct measure of the cell's ATP generating capability, in recent years, this key component in cell biology has been the subject of thousands of biochemical and biophysical investigations. Membrane-permeant fluorescent dyes, like tetramethylrhodamine ethyl ester (TMRE), have been predominantly employed to monitor ΔΨm in cells. These dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψm. However, the bath loading method practiced for labelling tissue slices with these cationic dyes poses limitations in the form of non-specificity and low signal to noise ratio, which compromises the precision of the results. Therefore, we introduce an alternative way for TMRE loading to image the ΔΨm in tissue slices by utilizing a low resistance glass pipette attached to a pressure injector. This method shows highly precise fluorescent dye labelling of the mitochondria and offers maximum output intensity, in turn enhancing signal to noise ratio.

Mechano-adaptive Responses of Alveolar Bone to Implant Hyper-loading in a pre-clinical in vivo model.

Oral implants transmit biting forces to peri-implant bone. In turn, those forces subject peri-implant bone to mechanical stresses and strains. Here, our objective was to understand how peri-implant bone responded to conditions of normal versus hyper-loading in a mouse model. Sixty-six mice were randomly assigned to 2 groups; both groups underwent bilateral maxillary first molar extraction followed by complete healing. Titanium alloy implants were placed in healed sites and positioned below the occlusal plane. After osseointegration, a composite crown was affixed to the implant so masticatory loading would ensue. In controls, the remaining dentition was left intact but in the hyper-loaded (test) group, the remaining molars were extracted. 3D finite element analysis (FEA) calculated peri-implant strains resulting from normal and hyper-loading. Peri-implant tissues were analyzed at multiple time points using micro-computed tomography (µCT) imaging, histology, enzymatic assays of bone remodeling, and vital dye labeling to evaluate bone accrual. Compared to controls, hyper-loaded implants experienced a 3.6-fold increase in occlusal force, producing higher peri-implant strains. Bone formation and resorption were both significantly elevated around hyper-loaded implants, eventually culminating in a significant increase in peri-implant bone volume/total volume (BV/TV). In our mouse model, masticatory hyper-loading of an osseointegrated implant was associated with increased peri-implant strain, increased peri-implant bone remodeling, and a net gain in bone deposition. Hyper-loading results in bone strain with catabolic and anabolic bone responses, leading to a net gain in bone deposition.

Intravital longitudinal imaging of hepatic lipid droplet accumulation in a murine model for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a rapidly increasing chronic liver disorder worldwide accompanied by hepatic steatosis, inflammation, fibrosis, and severe liver failure. Unfortunately, an effective treatment strategy for NAFLD has not yet been established, which has been hampered by the limited understanding of the pathophysiological drivers for NAFLD. To examine the unknown cellular and molecular mechanisms in the pathogenesis of NAFLD, there is an increasing need for the direct in vivo observation of hepatic microenvironments over extended periods of time. In this work, using a custom-built intravital imaging system and a novel fluorescent lipid droplet labeling dye, Seoul-Fluor 44 (SF44), we established an intravital imaging method to visualize individual lipid droplets and microvasculature simultaneously in the liver of live mice in vivo. In addition, in the nonalcoholic steatosis and steatohepatitis mouse model induced by a methionine and choline-deficient diet, we longitudinally visualized and quantitatively analyzed the development of lipid droplets in hepatocytes and sinusoid at a subcellular resolution during the progression of NAFLD up to 21 days in vivo.

Fluorescence-Labeled Amyloid Beta Monomer: A Molecular Dynamical Study.

The aggregation process of the Amyloidβ (Aβ) peptide is one of the central questions in Alzheimers’s research. Fluorescence-labeled single-molecule detection is a novel technique concerning the early stage investigation of Aβ aggregation, where the labeling dyes are covalently bound to the Aβ monomer. As the influence of the dye on the conformational space of the Aβ monomer can be significant, its effect on the seeding process is an open question. The applied fluorescent molecule continuously switches between an active (ON) and an inactive (OFF) state, where the latter supports an extra rotational restriction at many commercially available dyes. However, only a few theoretical studies simulated the Aβ monomer in the presence of a dye and none of them considered the difference between the ON and the OFF states. Therefore, we examined the impact of a selected fluorescence dye (Alexa 568) on the conformational space of the monomeric Aβ(1–42) peptide in its ON and OFF state by replica exchange molecular dynamic simulations. Investigations on secondary structure elements as well as dye-peptide contact analysis for the monomers are presented. Experimental and theoretical NMR shifts were contrasted to qualify the calculation protocol and theoretical values of the labeled and the non-labeled peptide were also compared. We found that the first five residues have higher helical propensity in the presence of the dye, and electrostatic properties could strongly affect the connection between the dye and the peptide parts.

Comparative cytotoxic effects of methanol, ethanol and DMSO on human cancer cell lines

Introduction: Anti-tumor drug screening is the most popular technique in drug discovery. In this technique, various agents were tested for their cytotoxicity on cell lines, particularly cancer cell lines. Since most of these agents are weakly soluble in water, they can be normally dissolved in lipophilic solvents. To obtain accurate results, the requirement of these solvents are that they be biocompatible and non-toxic to cells. The aim of this study was to investigate the biological effects of the three agents most commonly used as drug vehicles, i.e. dimethyl sulfoxide (DMSO), ethanol and methanol, using cell proliferation measurement techniques.
 Methods: To minimize the errors from the measurement techniques, this study used the xCelligence RTCA system which entails real-time monitoring of cell proliferation without dye labeling. Under the wide range concentration of solvents (from 0% to 100% of solvents by serial dilutions), the cell index (CI) and slope of cell proliferation curves of HepG2, MDA-MB-231, MCF-7 and VNBRCA1 cell lines were analyzed (by the software provided by the xCelligence system).
 Results: The results showed that DMSO had a significant toxicity and inhibition of proliferation on 4 cell lines, at the concentrations of 10%, 5%, 2.5% and 1.25% (p<0.0001). Methanol and ethanol inhibited cellular proliferation at the concentrations of 10% and 5% (p<0.0001). The concentrations of ethanol and methanol ranging from 2.5% to 0.15% concentration were well-tolerated by cells with respect to proliferation. Depending on the extracts or agents, each should be diluted in the suitable vehicle at the proper concentrations.
 Conclusion: Ethanol and methanol are good choices for solvents since they have low toxicity on HepG2, MDA-MB-231, MCF-7 and VNBRCA1 cell lines. However, in the case of agents only dissolvable in DMSO, low concentrations of DMSO from 0.6%-0.015% should be considered.

A Red-Emissive AIE Probe for Targeting Mitochondria in Living Cells

A novel compound has been synthesised and characterised by various spectroscopic techniques. Analysing the crystal structures of the compound shows that multiple molecular interactions exist. The compound exhibits distinct aggregation-induced emission activity in EtOH/H2O accompanying a 2-fold enhancement of fluorescence intensity at ~609nm. In addition, the cytotoxicity assay and confocal microscopy imaging show that the compound is hypotoxic and can be used as a fluorescent labelling dye in the near-infrared region to track mitochondria in living cells.

Controlling Fibronectin Fibrillogenesis Using Visible Light.

We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.

Tuning Cellular Interactions of Carboxylic Acid-Side-Chain-Containing Polyacrylates: The Role of Cyanine Dye Label and Side-Chain Type.

Cellular uptake and intracellular targeting to specific organelles are key events in the cellular processing of nanomaterials. Herein, we perform a detailed structure-property relationship study on carboxylic acid-side-chain-bearing polyacrylates to provide design criteria for the manipulation of their cellular interactions. Redox-initiated reversible addition-fragmentation chain-transfer (RRAFT) polymerization of three tert-butyl-protected N-acylated amino ester-based acrylate monomers of different substitutions and degrees of polymerization (DPs) yielded defined and pH-responsive carboxylic acid-side-chain polymers upon deprotection (N-acetyl, DP 1: P(M1); N-propionyl, DP 1: P(E1), DP 2: P(E2)). Flow cytometry studies revealed time-dependent cell association with P(E2) > P(E1) > P(M1) at any given time point. Importantly, the type of cyanine dye used for labeling was found to significantly influence the cellular processing of the polymers. Changing the dye from Cy5 to its sulfonated version sulfoCy5 resulted in a much lower cellular association. Moreover, Cy5-labeled polymers were targeted to mitochondria, while sulfoCy5 modification caused a significant change in the cellular fate of polymers toward lysosome trafficking. This study highlights the importance of selecting a suitable dye but also demonstrates the possibilities for the rational design of organelle-specific targeting of carboxylated polyacrylates.

Interspecies comparison of alveolar bone biology: Tooth extraction socket healing in mini pigs and mice.

in an effort to identify and validate which animal models are best suited for dental implant research, we used multiscale analyses to examine tooth extraction wound healing in a well-accepted model, the Yucatan mini pig and a more controversial model, the laboratory mouse. first molar extractions were performed in adult, skeletally mature mini pigs and mice. Alveolar bone repair was evaluated at early, intermediate and late timepoints using quantitative micro-computed tomographic (μCT) imaging, histology, molecular, and cellular assays. Vital dye labeling was employed to quantify mineral apposition rates (MAR) in both species. Despite a 3000-fold difference in weight, the relative proportions of the mini pig and murine maxillae and are equivalent. Quantitative μCT demonstrated that within the posterior alveolar bone, the volume of mineralized bone was lower in mini pig than in the mice; during healing, however, the bone volume fraction was equivalent. The histologic appearance of healing sites was also comparable, and alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) staining showed a similar temporal and spatial distribution of bone remodeling. Vital dye labeling indicated equivalent MAR between the species. The absolute duration of the healing period differed: in mice, complete healing was accomplished in ∼21 days. In mini pigs, the same process took four times longer. Extraction socket healing is histologically equivalent between mini pigs and mice, supporting the hypothesis that the underlying mechanisms of alveolar bone healing are conserved among species.

Feasibility of injecting Fluorescent Agent under the Guidance of Electromagnetic Navigation Bronchoscopy in Pulmonary Nodule Resection

背景与目的临床上肺内体积小，位置深且无实性成分的结节在电视辅助胸腔镜手术下切除给外科医生带来很多挑战。本研究的目前在于探讨经磁导航支气管镜（electromagnetic navigation bronchoscope, ENB）引导下肺部结节注入吲哚菁绿（indocyanine green, ICG）定位及在荧光腔镜下肺部小结节切除的可行性。方法2018年12月-2019年8月，我院连续16例共计30个肺周围病变患者接受了荧光胸腔镜下肺部结节切除术。术前均行磁导航引导注入ICG定位。结果所有患者均先行磁导航引导下肺结节定位术，染色完成后立即行手术切除。结节的平均大小为（11.12±3.65）mm。平均导航时间为（12.06±2.74）min，染料标记与肺部手术切除的平均间隔为（25.00±5.29）min。所有病灶均被完全切除，定位成功率100.00%，无出血及其他并发症发生，术后病理结果证实定位的准确性。结论经磁导航引导下注入荧光染剂是一种新颖且有效的方法，可以定位肺部微小不可触及的病灶。这种方法可帮助外科医生更快更方便的识别病灶，实用性强，值得推广。

Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy.

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

Disruption of tmc1/2a/2b Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells.

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENT We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.

Harnessing intrinsic fluorescence for typing of secondary structures of DNA.

High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).

Cultivating DNA Sequencing Technology After the Human Genome Project.

When the Human Genome Project was completed in 2003, automated Sanger DNA sequencing with fluorescent dye labels was the dominant technology. Several nascent alternative methods based on older ideas that had not been fully developed were the focus of technical researchers and companies. Funding agencies recognized the dynamic nature of technology development and that, beyond the Human Genome Project, there were growing opportunities to deploy DNA sequencing in biological research. Consequently, the National Human Genome Research Institute of the National Institutes of Health created a program-widely known as the Advanced Sequencing Technology Program-that stimulated all stages of development of new DNA sequencing methods, from innovation to advanced manufacturing and production testing, with the goal of reducing the cost of sequencing a human genome first to $100,000 and then to $1,000. The events of this period provide a powerful example of how judicious funding of academic and commercial partners can rapidly advance core technology developments that lead to profound advances across the scientific landscape.

Development of a New Nontoxic Thiazole Derivative Dye for Cell Labeling by Using Nanoparticles

Fluorescent labeling of L-929 fibroblast cells was studied by using newly synthesized ligand which was named as 2- N -methylamino-5-(pyrene-1-yl)-thiazole (ABS 40). The maximum fluorescence intensity of ABS 40 was detected at an excitation and emission wavelengths of 350 nm and 400 nm. Fluorescence intensity of the ligand was changed by the addition of DNA from calf thymus (CT-DNA) and human serum albumin (HSA). Both biomolecules and ABS 40 formed complexes that changed the fluorescence intensity of the ligand at the given wavelengths. Cytotoxicity tests were done with XTT analysis. L-929 fibroblast cells were cultured in the presence of ABS 40 to show cell labeling. Results showed that ABS 40 at 318 μM was labeled the cells at an excitation wavelength of 403.7 nm by using confocal microscopy. in vivo cell labeling was also confirmed by using Ct-DNA immobilized iron nanoparticles.

Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates.

The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through trans-cleavage upon activation with a specific target DNA. This shredding activity offered the potential for development of ssDNA-templated probes with fluorescent dye (F) and quencher (Q) labels. However, the formulations of double-stranded DNA (dsDNA)-templated fluorescent probes have not been reported possibly due to unknown (or limited) activity of Cas12a against short dsDNAs. The ssDNA probes have been shown to be powerful for diagnostic applications; however, limiting the probe selections to short ssDNAs could be restrictive from an application and probe diversification standpoint. Here, we report a dsDNA substrate (probe-full) for probing Cas12a trans-cleavage activity upon target detection. A diverse set of Cas12a substrates with alternating dsDNA character were designed and studied using fluorescence spectroscopy. We have observed that probe-full without any nick displayed trans-cleavage performance that was better than that of the form that contains a nick. Different experimental conditions of salt concentration, target concentration, and mismatch tolerance were examined to evaluate the probe performance. The activity of Cas12a was programmed for a dsDNA frame copied from a tobacco curly shoot virus (TCSV) or hepatitis B virus (HepBV) genome by using crRNA against TCSV or HepBV, respectively. While on-target activity offered detection of as little as 10 pM dsDNA target, off-target activity was not observed even at 1 nM control DNAs. This study demonstrates that trans-cleavage of Cas12a is not limited to ssDNA substrates, and Cas12a-based diagnostics can be extended to dsDNA substrates.

In Cell Kinetic FRET Assay to Judge Suitability of Bioorthogonal Dye Labelling Reaction

Genetic code expansion is a versatile tool for labelling of proteins in live cells by incorporating a non-canonical amino acid (ncAA) site-specifically into the protein of interest and subsequently label it with a small, photostable fluorescent dye. The ncAA typically adds only a few atoms to a selected residue side chain in the protein, and is among the smallest possible protein modifications. A wide variety of ncAAs, each with its own strengths and weaknesses, have been presented for this purpose. To identify which strategy and compound choice yields the best labelling efficiency inside mammalian cells has frequently been difficult. To address this complexity, two parameters are decisive, i) how fast is the labelling reaction between the ncAA and the fluorescent dye and ii) the overall yield of the labelled product. We developed a high throughput flow cytometry spectroscopy based FRET assay in live cells, which can assess both of these parameters in a time-resolved manner. We evaluate more than 10 compounds with different click reaction types, with a focus on the strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) between cyclooctynes or a trans-cyclooct-4-ens and tetrazine-dye-conjugate. We revealed clear differences in compound stability and reactivity invivo compared to in vitro studies, leading to different preferences in the choice of the best performing ncAA. Besides presenting severalnew compounds, we identified a so far largely ignored ncAA as the most suitable labelling tool. We present several further improvements to maximize fluorescent labeling efficiency in living cells.