Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes

Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.</p

Design of Bioluminescent Protein-Conjugates with Bioorthogonal Chemistry for Applications in Biomedicine

Bioconjugation is one of the common ways of modifying biomolecules with reporters and other moieties for applications in the development of various sensing systems. However, achieving specific and efficient bioconjugation is challenging. Bioorthogonal chemistry can overcome these challenges as these reactions are designed to work under physiological conditions and target a specific functionality on the target biomolecule. Different functional groups can be introduced genetically or chemically on biomolecules to have a unique reactive group in order to perform bioorthogonal reactions. Herein, work relating to the development of bioluminescent biosensing systems by applying bioorthogonal chemistry between the bioluminescent proteins and other biomolecules is discussed. In particular, antibodies for the development of immunoassays that are labeled bioorthogonally with a bioluminescent protein, Gaussia luciferase were prepared and an immunoassay for the detection of interferon-γ was developed. Also, bioluminescent molecular aptamer beacons utilizing Gaussia luciferase as a reporter is described. In this work, an inhibitor molecule was attached on the beacon to reduce the signal-to-noise ratio and improve the sensitivity. Finally, the prospective research is highlighted as new biosensors can be designed with different bioorthogonal reactions and techniques

Design of Gaussia luciferase-based bioluminescent stem-loop probe for sensitive detection of HIV-1 nucleic acids

Here we describe the design of a bioluminescent stem-loop probe for the sensitive detection of HIV-1 spliced RNA. In this study, we employed Gaussia luciferase (GLuc), a bioluminescent protein that has several advantages over other bioluminescent proteins, including smaller size, higher bioluminescent intensity, and chemical and thermal stability. GLuc was chemically conjugated to the DABCYL-modified stem-loop probe (SLP) and was purified with a 2-step process to remove unconjugated GLuc and SLP. The binding of the target RNA to the loop region of the SLP results in the open conformation separating the stem part of SLP. GLuc conjugated to the stem acts as a reporter that produces light by a chemical reaction upon adding its substrate, coelenterazine in the presence of the target, while DABCYL serves as a quencher of bioluminescence in the closed conformation of SLP in the absence of the target. The optimized GLuc based-SLP assay resulted in a signal-to-background ratio of 47, which is the highest reported with bioluminescent SLPs and is significantly higher compared to traditional fluorescence-based SLPs that yield low signal to background ratio. Moreover, the assay showed an excellent selectivity against a single and double mismatched nucleic acid target, low detection limit, and ability to detect spiked HIV-1 RNA in human serum matrix

Molecular Aptamer Beacons: Molecular Aptamer Beacons and Their Applications in Sensing, Imaging, and Diagnostics (Small 35/2019)

Molecular aptamer beacons represent a new class of antibody‐like recognition elements that have the additional benefit of targetdependent signaling. Besides having high sensitivity and selectivity, aptamer beacons are stable under many conditions that would render protein‐based sensors inert. In article number 1902248, Sapna K. Deo and co‐workers discuss the wide diversity of designs and applications while highlighting the benefits of these unique reporters in assay design

Molecular Aptamer Beacons and Their Applications in Sensing, Imaging, and Diagnostics

The ability to monitor types, concentrations, and activities of different biomolecules is essential to obtain information about the molecular processes within cells. Successful monitoring requires a sensitive and selective tool that can respond to these molecular changes. Molecular aptamer beacon (MAB) is a molecular imaging and detection tool that enables visualization of small or large molecules by combining the selectivity and sensitivity of molecular beacon and aptamer technologies. MAB design leverages structure switching and specific recognition to yield an optical on/off switch in the presence of the target. Various donor–quencher pairs such as fluorescent dyes, quantum dots, carbon‐based materials, and metallic nanoparticles have been employed in the design of MABs. In this work, the diverse biomedical applications of MAB technology are focused on. Different conjugation strategies for the energy donor–acceptor pairs are addressed, and the overall sensitivities of each detection system are discussed. The future potential of this technology in the fields of biomedical research and diagnostics is also highlighted. Molecular aptamer beacons represent a new class of antibody‐like recognition elements that have the additional benefit of target‐dependent signaling. Besides having high sensitivity and selectivity, aptamer beacons are stable under many conditions that would render protein‐based sensors inert. The wide diversity of designs and applications are discussed while highlighting the benefits of these unique reporters in assay design

Expression of a soluble truncated Vargula luciferase in Escherichia coli

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties

Bioluminescent Protein-Inhibitor Pair in the Design of a Molecular Aptamer Beacon Biosensing System

Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ (IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γ and provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems

Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications

Gaussia luciferase (Gluc)-with its many favorable traits such as small size, bright emission, and exceptional stability-has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed

A Bioluminescent Protein-Graphene Oxide Donor-Quencher Pair in DNA Hybridization Assays

Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications - bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we used Gaussia luciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV-1 DNA and IFN-γ. We demonstrated that the incubation of Gluc conjugated HIV-1 and IFN-γ oligonucleotide probes with GO provided for monitoring of probe-target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN-γ and HIV-1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN-γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter

The Effect of NSAID Pretreatment on Aqueous Humor Prostaglandin E2 Concentration in Eyes Undergoing Femtosecond Laser-Assisted Capsulotomy

Purpose. To assess aqueous humor concentration of prostaglandin E2 (PGE2) after capsulotomy creation using a femtosecond laser (FLAC) in patients pretreated with short-term topical ketorolac versus patients without pretreatment. Methods. This prospective study comprised consecutive patients scheduled to undergo cataract surgery using a femtosecond laser platform to perform only capsulotomies. An identical protocol for preoperative mydriasis was used for all the eyes included in the study, while aqueous humor was extracted from the anterior chamber of all patients immediately after the initial side port incision. ELISA was performed to quantify aqueous humor PGE2. The patients were divided into 2 groups; in group 1, the patients received short-term topical ketorolac preoperatively, while the patients in group 2 did not receive NSAID pretreatment. Results. Twenty eyes of 20 patients were included in the study (10 eyes in each group). Mean concentration of aqueous humor PGE2 after FLAC was 392.16 ± 162.00 pg/ml and 622.63 ± 331.84 pg/ml for groups 1 and 2, respectively. A statistically significant difference in aqueous humor PGE2 concentration between the two groups (p<0.05) was demonstrated, with the eyes that received ketorolac pretreatment demonstrating a lower concentration of PGE2. Conclusion. Short-term topical use of ketorolac prior to FLAC seems to prevent excessive release of PGE2 in the anterior chamber of the eyes that received NSAID pretreatment when compared to the eyes that did not receive NSAIDs preoperatively