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.

A paper-based platform for detection of viral RNA.

Viral detection presents a host of challenges for even the most sensitive analytical techniques, and the complexity of common detection platforms typically preclude portability. With these considerations in mind, we designed a paper microzone plate-based virus detection system for the detection of viral genetic material that can be performed with simple instruments. The sensing system can detect viral cDNA reverse-transcribed from total RNA extraction by utilizing a biotinylated capture probe and an Alexa Fluor® 647-labeled reporter probe. The biotinylated capture probe was linked to the paper surface via NeutrAvidin® that was physically adsorbed on the paper. After addition of reverse-transcribed sample and reporter probe in sequence, the reverse-transcribed target captured the reporter probe and tethered it to the capture probe in a bridged format. Fluorescence intensity was imaged using a Western blot imaging system, and higher target concentration was visible by the increased emission intensity from Alexa Fluor® 647. By utilizing paper, this detection setup could also serve as a sample concentration method via evaporation, which could remarkably lower the detection limit if needed. This detection platform used Epstein-Barr virus (EBV) RNA as a proof-of-concept by sensing cDNA resulting from reverse transcription and can be further expanded as a general method for other pathogens. EBV is a well-known human tumor virus, which has also recently been linked to the development of cervical cancer. The assay was accomplished within two hours including the room-temperature RNA extraction and reverse transcription steps. Also, this paper microzone plate-based platform can potentially be applicable for the development of point-of-care (POC) detection kits or devices due to its robust design, convenient interface, and easy portability. The experiment could be stopped after each step, and continued at a later time. The shelf-life of the modified paper plate setup was at least 3 months without a discernible change in signal, and the result from day 1 could be read at 3 months - both of which are important criteria for POC analytical testing tools, especially in resource-poor settings. All of the required assay steps could potentially be performed without any significant equipment using inexpensive paper microzone plates, which will be ideal for further development of POC testing devices. Although, this platform is not at the stage where it can be directly used in a point-of-care setting, it does have fundamental characteristics such as a stable platform, a simple detection method, and relatively common reagents that align closely with a POC system.

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.

Improved visual detection of DNA amplification using pyridylazophenol metal sensing dyes.

Detection of nucleic acid amplification has typically required sophisticated laboratory instrumentation, but as the amplification techniques have moved away from the lab, complementary detection techniques have been implemented to facilitate point-of-care, field, and even at-home applications. Simple visual detection approaches have been widely used for isothermal amplification methods, but have generally displayed weak color changes or been highly sensitive to sample and atmospheric effects. Here we describe the use of pyridylazophenol dyes and binding to manganese ion to produce a strong visible color that changes in response to nucleic acid amplification. This detection approach is easily quantitated with absorbance, rapidly and clearly visible by eye, robust to sample effects, and notably compatible with both isothermal and PCR amplification. Nucleic acid amplification and molecular diagnostic methods are being used in an increasing number of novel applications and settings, and the ability to reliably and sensitively detect them without the need for additional instrumentation will enable even more access to these powerful techniques.

Profiling Thermus thermophilus Argonaute Guide DNA Sequence Preferences by Functional Screening.

Prokaryotic Argonautes (pAgo) are an increasingly well-studied class of guided endonucleases, and the underlying mechanisms by which pAgo generate nucleic acid guides in vivo remains an important topic of investigation. Recent insights into these mechanisms for the Argonaute protein from Thermus thermophilus has drawn attention to global sequence and structural feature preferences involved in oligonucleotide guide selection. In this work, we approach the study of guide sequence preferences in T. thermophilus Argonaute from a functional perspective. Screening a library of 1,968 guides against randomized single- and double-stranded DNA substrates, endonuclease activity associated with each guide was quantified using high-throughput capillary electrophoresis, and localized sequence preferences were identified which can be used to improve guide design for molecular applications. The most notable preferences include: a strong cleavage enhancement from a first position dT independent of target sequence; a significant decrease in activity with dA at position 12; and an impact of GC dinucleotides at positions 10 and 11. While this method has been useful in characterizing unique preferences of T. thermophilus Argonaute and criteria for creating efficient guides, it could be expanded further to rapidly characterize more recent mesophilic variants reported in the literature and drive their utility toward molecular tools in biology and genome editing applications.

Resonance energy transfer methods of RNA detection.

Quantitation of RNA is important in diagnostics, environmental science, and basic biomedical research. RNA is considered a signature for pathogen identification, and its expression profile is linked with disease pathogenesis, allowing for biomarker identification. RNA-based diagnostics is an emerging field of research. This expansion of interest in studying RNA has generated demand for its accurate and sensitive detection. Several methods have therefore been developed to detect RNA. Resonance energy transfer methods of RNA detection are highly promising in terms of simplicity and high sensitivity. In this review, we have focused on the latest developments in resonance energy transfer methods of RNA detection that utilize various probe designs. The probe designs discussed here are molecular beacons, quenched autoligation probes, and linear oligonucleotide probes. Resonance energy transfer methods based on both fluorescence and bioluminescence detection are discussed.

Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro.

Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides, some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however, they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome, specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors, including thermostable single-stranded DNA binding proteins and UvrD-like helicase, in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates, including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.

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.

MicroRNA Detection: Current Technology and Research Strategies.

The relatively new field of microRNA (miR) has experienced rapid growth in methodology associated with its detection and bioanalysis as well as with its role in -omics research, clinical diagnostics, and new therapeutic strategies. The breadth of this area of research and the seemingly exponential increase in number of publications on the subject can present scientists new to the field with a daunting amount of information to evaluate. This review aims to provide a collective overview of miR detection methods by relating conventional, established techniques [such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarray, and Northern blotting (NB)] and relatively recent advancements [such as next-generation sequencing (NGS), highly sensitive biosensors, and computational prediction of microRNA/targets] to common miR research strategies. This should guide interested readers toward a more focused study of miR research and the surrounding technology.

Bioluminescent stem-loop probes for highly sensitive nucleic acid detection.

Here, we report the first bioluminescent stem-loop probe, which is 50 times more sensitive and able to achieve a LOD 25 times lower than fluorescent stem-loop probes. Chemical generation of a signal from Renilla luciferase reduces background noise for improved quantitative utility in nucleic acid biomarker detection.

Bioluminescence-based detection of microRNA, miR21 in breast cancer cells.

A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death. miRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the determination of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell extracts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early molecular markers and their detection methods in cancer diagnosis is tremendous. The characteristics of the assay developed in this work show its suitability for early cancer diagnosis based on miRNA as a biomarker.