Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery.

Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency. We examined a library of poly(amine-co-ester) (PACE) polymers with different end groups using this probe and observed a strong correlation between endosomal escape and transfection efficiency (R2 = 0.9334). In addition, we found that mRNA encapsulation efficiency and endosomal escape, but not uptake, were determinant factors for transfection efficiency. The polymers with high endosomal escape/transfection efficiency in vitro also showed good transfection efficiency in vivo, and mRNA expression was primarily observed in spleens after intravenous delivery. Together, our study suggests that the luciferase probe can be used as an effective tool to quantitate endosomal escape, which is essential for rational optimization of intracellular drug delivery systems.

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids.

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors.

G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects. Using GPCR conformation-sensitive biosensors, we investigated allosterically induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer. Ligand occupancy of the AT1R induced distinct conformational changes in FP compared with those driven by PGF2α in bioluminescence resonance energy transfer (BRET)-based FP biosensors engineered with Renilla luciferase (RLuc) as an energy donor in the C-tail and fluorescein arsenical hairpin binder (FlAsH)-labeled acceptors at different positions in the intracellular loops. We also found that this allosteric communication is mediated through Gαq and may also involve proximal (phospholipase C) but not distal (protein kinase C) signaling partners. Interestingly, ß-arrestin-biased AT1R agonists could also transmit a Gαq-dependent signal to FP without activation of downstream Gαq signaling. This transmission of information was specific to the AT1R/FP complex, as activation of Gαq by the oxytocin receptor did not recapitulate the same phenomenon. Finally, information flow was asymmetric in the sense that FP activation had negligible effects on AT1R-based conformational biosensors. The identification of partner-induced GPCR conformations may help identify novel allosteric effects when investigating multiprotein receptor signaling complexes.

New insights into the molecular characteristics behind the function of Renilla luciferase.

Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8.

Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems.

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors.

For the highly sensitive near-infrared (NIR) optical detection of epidermal growth factor receptors (EGFRs) expressed on cancer cells, bioluminescence resonance energy transfer (BRET) coupled NIR quantum dots (QDs) are prepared by direct conjugation of his-tagged Renilla luciferase (RLuc) recombinant protein (HisRLuc·GB1) to glutathione-coated CdSeTe/CdS QDs (GSH-QDs). The recombinant protein has two functional groups consisting of a luciferase enzyme and an immunoglobulin binding domain (GB1) of protein G. Recombinant protein (HisRLuc·GB1) conjugated QDs (GB1·RLuc-QDs) show BRET-coupled NIR emission, which results from energy transfer from luciferin to QDs with a high BRET efficiency of ca. 50%. Since the GB1·RLuc-QDs have the GB1 domain at their surface, the QDs have an ability to bind the Fc moiety of immunoglobulin G (IgG). The resulting IgG bound QDs can be used as a molecular imaging probe with NIR fluorescence and BRET-coupled NIR emission. For NIR optical detection of EGFRs on cancer cells, we conjugated anti-EGFR monoclonal antibody to the GB1·RLuc-QDs. Herein, we show that the detection sensitivity of EGFRs by BRET-coupled NIR emission of GB1·RLuc-QDs is at least three times higher than that of the NIR fluorescence of the QDs. The conjugates between anti-EGFR antibody and GB1·RLuc-QDs make it possible to perform BRET-based highly sensitive NIR imaging of EGFRs in living cells.

Expression and characterization of the Renilla luciferase with the cumulative mutation.

Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability. In this study, 5 mutations reported previously [1] and one mutation obtained using site-directed mutagenesis were combined. As a result of this combination, the thermostability effect increased, with the mutant showing approximately 10 °C higher stability. Furthermore, the mutant simultaneously improved a tolerance for protease digestion, e.g. trypsin and proteinase K, and for organic solvent. Residual activity of the mutant after treatment with 10% 2-propanol, 10% DMF and 20% DMSO at 35 °C for 1 h was 29.4, 24.8 and 91.3%, respectively, whereas that of the wild type was 0.4, 0.1 and 24.3%, respectively.

Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure.

Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60°C to 70°C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65°C for 5min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors.

Nanostructured biosensor using bioluminescence quenching technique for glucose detection.

BACKGROUND: Most methods for monitoring glucose level require an external energy source which may limit their application, particularly in vivo test. Bioluminescence technique offers an alternative way to provide emission light without external energy source by using bioluminescent proteins found from firefly or marine vertebrates and invertebrates. For quick and non-invasive detection of glucose, we herein developed a nanostructured biosensor by applying the bioluminescence technique. RESULTS: Luciferase bioluminescence protein (Rluc) is conjugated with ß-cyclodextrin (ß-CD). The bioluminescence intensity of Rluc can be quenched by 8 ± 3 nm gold nanoparticles (Au NPs) when Au NPs covalently bind to ß-CD. In the presence of glucose, Au NPs are replaced and leave far from Rluc through a competitive reaction, which results in the restored bioluminescence intensity of Rluc. A linear relationship is observed between the restored bioluminescence intensity and the logarithmic glucose concentration in the range of 1-100 µM. In addition, the selectivity of this designed sensor has been evaluated. The performance of the senor for determination of the concentration of glucose in the blood of diabetic rats is studied for comparison with that of the concentration of glucose in aqueous. CONCLUSIONS: This study demonstrates the design of a bioluminescence sensor for quickly detecting the concentration of glucose sensitively.

General Bioluminescence Resonance Energy Transfer Homogeneous Immunoassay for Small Molecules Based on Quantum Dots.

Here, we describe a general bioluminescence resonance energy transfer (BRET) homogeneous immunoassay based on quantum dots (QDs) as the acceptor and Renilla luciferase (Rluc) as the donor (QD-BRET) for the determination of small molecules. The ratio of the donor-acceptor that could produce energy transfer varied in the presence of different concentrations of free enrofloxacin (ENR), an important small molecule in food safety. The calculated Förster distance (R0) was 7.86 nm. Under optimized conditions, the half-maximal inhibitory concentration (IC50) for ENR was less than 1 ng/mL and the linear range covered 4 orders of magnitude (0.023 to 25.60 ng/mL). The cross-reactivities (CRs) of seven representative fluoroquinolones (FQs) were similar to the data obtained by an enzyme-linked immunosorbent assay (ELISA). The average intra- and interassay recoveries from spiked milk of were 79.8-118.0%, and the relative standard deviations (RSDs) were less than 10%, meeting the requirement of residue detection, which was a satisfactory result. Furthermore, we compared the influence of different luciferase substrates on the performance of the assay. Considering sensitivity and stability, coelenterazine-h was the most appropriate substrate. The results from this study will enable better-informed decisions on the choice of Rluc substrate for QD-BRET systems. For the future, the QD-BRET immunosensor could easily be extended to other small molecules and thus represents a versatile strategy in food safety, the environment, clinical diagnosis, and other fields.

Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs.

Here we describe the design and construction of an imaging construct with high bioluminescent resonance energy transfer (BRET) efficiency that is composed of multiple quantum dots (QDs; λem = 655 nm) self-assembled onto a bioluminescent protein, Renilla luciferase (Rluc). This is facilitated by the streptavidin-biotin interaction, allowing the facile formation of a hybrid-imaging construct (HIC) comprising up to six QDs (acceptor) grafted onto a light-emitting Rluc (donor) core. The resulting assembly of multiple acceptors surrounding a donor permits this construct to exhibit high resonance energy transfer efficiency (∼64.8%). The HIC was characterized using fluorescence excitation anisotropy measurements and high-resolution transmission electron microscopy. To demonstrate the application of our construct, a generation-5 (G5) polyamidoamine dendrimer (PAMAM) nanocarrier was loaded with our HIC for in vitro and in vivo imaging. We envision that this design of multiple acceptors and bioluminescent donor will lead to the development of new BRET-based systems useful in sensing, imaging, and other bioanalytical applications.

Novel bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution for Renilla luciferase.

Two series of novel coelenterazine analogues (alkynes and triazoles) with imidazopyrazinone C-6 extended substitution have been designed and synthesized successfully for the extension of bioluminescent substrates. After extensive evaluation, some compounds display excellent bioluminescence properties compared with DeepBlueC in cellulo, thus becoming potential molecules for bioluminescence techniques.

The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.

Surface-Tunable Bioluminescence Resonance Energy Transfer via Geometry-Controlled ZnO Nanorod Coordination.

The use of ZnO nanorods (NRs) as an effective coordinator and biosensing platform to create bioluminescence resonance energy transfer (BRET) is reported. Herein, a hydrothermal approach is applied to obtain morphologically controlled ZnO NRs, which are directly bound to luciferase (Luc) and carboxy-modified quantum dot (QD) acting as a donor-acceptor pair for BRET. BRET efficiency varies significantly with the geometry of ZnO NRs, which modulates the coordination between hexahistidine-tagged Luc (Luc-His6 ) and QD, owing to the combined effect of the total surface area consisting of (001) and (100) planes and their surface polarities. Unlike typical QD-BRET reactions with metal ions (e.g., zinc ions), a geometry-controlled ZnO NR platform can facilitate the design of surface-initiated BRET sensors without being supplemented by copious metal ions: the geometry-controlled ZnO NR platform can therefore pave the way for nanostructure-based biosensors with enhanced analytical performance.

In vitro characterization of Fluorescence by Unbound Excitation from Luminescence: broadening the scope of energy transfer.

Energy transfer mechanisms represent the basis for an array of valuable tools to infer interactions in vitro and in vivo, enhance detection or resolve interspecies distances such as with resonance. Based upon our own previously published studies and new results shown here we present a novel framework describing for the first time a model giving a view of the biophysical relationship between Fluorescence by Unbound Excitation from Luminescence (FUEL), a conventional radiative excitation-emission process, and bioluminescence resonance energy transfer. We show here that in homogeneous solutions and in fluorophore-targeted bacteria, FUEL is the dominant mechanism responsible for the production of red-shifted photons. The minor resonance contribution was ascertained by comparing the intensity of the experimental signal to its theoretical resonance counterpart. Distinctive features of the in vitro FUEL signal include a macroscopic depth dependency, a lack of enhancement upon targeting at a constant fluorophore concentration cf and a non-square dependency on cf. Significantly, FUEL is an important, so far overlooked, component of all resonance phenomena which should guide the design of appropriate controls when elucidating interactions. Last, our results highlight the potential for FUEL as a means to enhance in vivo and in vitro detection through complex media while alleviating the need for targeting.

Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV.

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.

BACKGROUND: The fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes. METHODS: Freshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD-) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulus-oocyte complexes (COCs) were matured in the presence of QD- or QD+. Ovarian follicles were microinjected with QD- or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences. RESULTS: All labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD- or QD+. The QD- fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection. CONCLUSION: Findings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.

Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.

We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

Dissipative dynamics of enzymes.

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.