Internal Quencher Research Articles

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detectionmethod, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the5' endof the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10min faster than several otherprobe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

The Internal Structure of Lanthanide-Doped Nanoparticles and the Effect of High-Temperature Annealing on Their Luminescent Properties

Up-converting nanoparticles are widely studied for a wide range of applications based on their unique optical properties, with NaYF4 nanoparticles doped with Yb3+ and Er3+ receiving particular attention. While developing this material in nanoparticle form extends their potential applications, the resulting nanoparticles have proven less efficient up-converters than their bulk counterpart. Reported up-conversion quantum yields are significantly lower, even when very thick shells were grown to eliminate quenching by surface defects and surface-bound molecules. This raises the question whether the internal structure of these particles contributes to the lower quantum yield. In our work, we investigated the internal structure of NaYF4:Yb3+ and Er3+ NPs using high-resolution scanning energy dispersive X-ray spectroscopy, generating two-dimensional elemental maps. We deduced that the ions are not distributed homogeneously in the nanoparticles as made via a colloidal synthesis route. Heating the nanoparticles to temperatures used to anneal bulk crystals resulted in a homogeneous distribution, but an increase in emission intensity under similar measurement conditions was not observed. Vibrational spectroscopy showed the presence of OH– in dried nanoparticles, which might act as an internal quencher.

Evaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection.

Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II.

The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.

Designing a Thermostable Switch-Based Biosensor

There is a critical need for developing new diagnostic chemical reporter tools that are stable, cost effective, and capable of sensitive detection. Fluorescent protein biosensors relay changes in fluorescence based on the physical interaction between a protein and the ligand. The thermostable arginine binding protein (ArgBP) from the Gram-negative, hyperthermophilic bacterium, Thermotoga maritima, has been modified to create a reagentless fluorescent biosensor. Typically, single Cys mutants are constructed and covalently modified with environmentally-sensitive fluorescent probes. However, no theoretical framework exists to accurately model placement of the probe to the protein sensor. In this work, site-directed mutations were engineered whereby a significant fluorescence quenching (>80%) is observed based on a photo-induced electron transfer (PET) mechanism, which occurs after TmArgBP binds with increasing concentrations of arginine. This method gave significantly better fluorescence response to arginine and responded in a predictable distance-dependent manner when compared with construction of single Cys mutants of TmArgBP. Using the PET method, a dissociation constant of 0.86 microMolar was determined to be consistent with previously published data for this protein. Time-resolved fluorescence lifetime measurements indicated that fluorescence deactivation of the TmArgBP biosensor is a result of static quenching, which is consistent with the formation of ground state complexes between the fluorescent probe and an internal quencher. In addition to the development of a thermostable fluorescent protein sensor, this investigation provides a generalizable spectroscopic tool for the study of conformational movements within a single polypeptide chain.

Quencher-free Linear Beacon Systems with Dual Fluorene-labeled Deoxyuridines

The identification of natural genetic variation in human populations is arguably one of the most important spin-offs of the effort toward the completion of the Human Genome Project. Single nucleotide polymorphisms (SNPs) are the most abundant natural genetic variations in the human genome. To satisfy the demand for molecular diagnostic assays for identifying SNPs, Tyagi and Kramer developed fluorogenic hairpin-shaped DNA probes, called “molecular beacons in 1996. Conventional molecular beacons are single-stranded oligodeoxynucleotides (ODNs) probes that exist in solution as stable stem-and-loop structures in which a fluorophore is covalently linked to one end and a quencher is covalently linked to the other end. The stem keeps the fluorophore and quencher of the molecular beacon in extremely close proximity to each other, when they are not bound to their targets; this causes the fluorescence of the fluorophore to be quenched via the fluorescence resonance energy transfer. By contrast, the conformational change induced by binding to a target nucleic acid causes the fluorophore and the quencher to move away from each other, thereby restoring the fluorescence. Therefore, the presence of the target nucleic acid is characterized by a sharp increase in fluorescence intensity. Recently, we reported that fluorescent hairpin oligonucleotides can function as molecular beacons even without the attachment of additional quencher moieties. Such probes can be called “quencher-free molecular beacons. A quencher-free molecular beacon consists of only a single fluorophore (2-ethynylfluorene-labeled deoxyuridine, U) positioned in the hairpin loop. When this probe undergoes DNA hybridization, it can discriminate between fully matched and single-base-mismatched sequences by exhibiting a strong dA-selective fluorescence. When the fluorene unit is intercalated within a DNA duplex as a result of mismatched base pairing, the fluorescence of the U unit is quenched as a result of a photoinduced charge transfer originating from interactions with neighboring nucleobases. In particular, the quencher-free molecular beacon probe having C-flanking bases (C-FBs) shows more efficient fluorescence ON/OFF behavior than do any other combinations of FBs. The only drawback of this quencher-free molecular beacon system is its poor detection sensitivity due to its relatively high intrinsic emission intensity. To solve this problem, we recently positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite the U unit to reduce the intrinsic fluorescence on hybridization with a probe. A short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective in reducing the intrinsic fluorescence of a linear beacon. In another effort to eliminate this drawback, we employed an ODN containing dual U units at specific distances (Figure 1). We observed that using this dual-labeled linear beacon system resulted in a clear improvement in the detection sensitivity for the perfectly matched target. The modified nucleotide U was synthesized through Sonogashira coupling of a 2-ethynylfluorene unit at the 5position of a 2-deoxyuridine base. Incorporations of the fluorene-labeled deoxyuridine U into the central position of the ODN 1 and of the two U units into the positions with one deoxycytosine spacer unit were effected using standard protocols of automated DNA synthesis (Figure 2). The fluorescence properties of fluorophore-containing DNA are strongly dependent on the electron injection and transfer properties of the FBs. In particular, fluorescent ODNs having C-FBs produce efficient fluorescence ON/OFF systems and have higher sensitivity as probes than do any other combinations of FBs. Therefore, we positioned two deoxy-

A Linear Beacon System Featuring an Internal Deoxyguanine Quencher Allows Highly Selective Detection of Single Base Mismatches

The fluorescence intensity of a single-stranded oligonucleotide containing a fluorene-labeled deoxyuridine (U FI ) unit increases by only 1.5-fold upon formation of its perfectly matched duplex. To increase the fluorescence signal during hybridization, we positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite to the U FI unit to reduce the intrinsic fluorescence upon hybridization with a probe. From an investigation of the optimal length of the quencher strand and the effect of the neighboring base sequence, we found that a short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective at reducing the intrinsic fluorescence of a linear beacon; it also exhibited high total discrimination factors for the formation of perfectly matched and single base-mismatched duplexes. Such assays that function based on clear changes in fluorescence in response to single-base nucleotide mutations would be useful tools for accelerating diagnoses related to various diseases.

3-Nitro-Tyrosine as an Internal Quencher of Autofluorescence Enhances the Compatibility of Fluorescence Based Screening of OBOC Combinatorial Libraries

In the one-bead-one-compound (OBOC) combinatorial method, compounds are constructed on bead resin via split-mix library synthesis such that multiple copies of the same compound are displayed on each bead. These libraries are rapidly screened with enzyme-linked colorimetric, fluorescent, radiometric, or whole-cell binding assays. While fluorescence-based probes are powerful tools in OBOC screening, their utility is greatly limited by the intrinsic fluorescence of many commonly used solid supports, (e.g. TentaGel), residual coupling reagents, and library compounds. To overcome this problem, we topologically partitioned TentaGel resin with a thin Fmoc-protected outer layer and an unprotected inner core. The inner core was derivatized with 3-nitro-tyrosine, followed by random peptide library construction. Spectral scans from a confocal microscope showed a dramatic decrease in the autofluorescence of blank beads and OBOC peptide libraries across a broad range of the optical spectrum. The quenching capacity of 3-nitro-tyrosine was also visualized in fluorescent micrographs. Using biotin/streptavidin as a model ligand/receptor system, we demonstrated a marked increase in visibility of three commercially available fluorescent probes binding to quenched beads, and increased feasibility of using a robust and efficient fluorescence-based, bead sorting platform known as COPAS. These data show that using 3-nitro-tyrosine as an internal quencher greatly enhances the compatibility of fluorescence-based applications and OBOC combinatorial screening.

Turn-On Fluorescence Detection of Cyanide in Water: Activation of Latent Fluorophores through Remote Hydrogen Bonds That Mimic Peptide β-Turn Motif

A molecular probe was prepared that selectively responds to cyanide in aqueous solutions by fluorescence enhancement. Using the peptide beta-turn as a structural template, we designed a series of diphenylacetylene derivatives in which the pi-conjugated backbone was functionalized with an aldehyde group to render the molecule nonfluorescent. The N-H...O hydrogen bond across the 2,2'-functionalized diphenylacetylene turn motif activates the carbonyl group toward nucleophilic attack, and chemical transformation of this internal quencher site by reaction with CN(-) elicits a rapid (k = 72 M(-1) s(-1)) enhancement in the emission at lambda(max) = 375 nm. Tethering of an ammonium group to the hydrogen bond donor fragment significantly increased both the response kinetics and the intensity of the fluorescence signal. In addition to providing electrostatic attraction toward the CN(-) ion, this positively charged R-NH(3)(+) fragment can engage in a secondary hydrogen bond to facilitate the formation of the cyanohydrin adduct responsible for the signaling event. The structurally optimized molecular probe 3 responds exclusively to microM-level cyanide in neutral aqueous solutions, with no interference from other common anions including F(-) and AcO(-).

Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

A continuous fluorimetric assay for aminopeptidase P detailed analysis of product inhibition

Aminopeptidase P (APP; EC 3.4.11.9) is a proline-specific peptidase hydrolyzing N-terminal Xaa-Pro peptide bonds. On the basis of its unique substrate specificity it is difficult to determine enzyme activity and to estimate potential enzyme inhibitors. In this report, we describe the synthesis of a new fluorogenic substrate to assay APP. The substrate was characterized using Escherichia coli APP and rat intestine APP. The compound contains the fluorogenic 2-aminobenzoyl residue and 4-nitroanilide as internal quencher. Both enzymes hydrolyze the substrate Lys( N ε -2-aminobenzoyl)-Pro-Pro-4-nitroanilide at the Lys-Pro peptide bond with K m values in the micromolar range. Lys( N ε -2-aminobenzoyl)-Pro-Pro-4-nitroanilide is the best substrate of APP from rat intestine that is known with a K m value of 3.54 μM and a second-order rate constant of 1,142,000 M −1 s −1. Unfortunately, product inhibition occurs. Inhibition studies using the hydrolysis product Pro-Pro-4-nitroanilide demonstrate a linear mixed-type mechanism with a K i value of 20.8 μM and an α value of 5.1 for rat APP and a K i value of 76.1 μM and an α value of 0.4 for E. coli APP, respectively. Furthermore, the hydrolysis of the fluorogenic APP substrate catalyzed by prolyl oligopeptidase was investigated.

Intraprotein electron transfer in a ruthenium-modified Tyr83-His plastocyanin mutant: evidence for strong electronic coupling

A site-directed mutant of spinach plastocyanin, Pc(Tyr83-His), has been modified by covalent attachment of a photoactive [Ru(bpy)2(im)]2+ complex to the His83 residue. The residue is surface exposed and located about 10–12 A from the copper ion at the entrance of a proposed natural electron transfer pathway from cytochrome f. Electron transfer within the Ru-Pc complex has been studied with time-resolved optical spectroscopy using two different approaches. In the first, the fully reduced [Cu(I), Ru(II)] protein was photoexcited and subsequently oxidized by an external quencher, forming the [Cu(I), Ru(III)] protein. This was followed by an electron transfer from reduced Cu(I) to Ru(III). In the second method, the initially oxidized Cu(II) ion acted as an internal quencher for excited Ru(II) and the photoinduced reduction of the Cu(II) ion was followed by a thermal recombination with the Ru(III) ion. The reoxidation of the Cu ion, which has an estimated driving force of 0.56 eV, occured with a rate constant k et = (9.5±1.0)×106 s–1, observed with both methods. The results suggest a strong electronic coupling (H DA>0.3 cm–1) along the Ru-His(83)-Cys(84)-Cu pathway.

Studies of Two-Dimensional Morphology at Surface-Functionalized Polyethylene Films

Polyethylene simultaneously functionalized with both pyrene labels and amine quencher groups has been studied to determine the extent to which self-organization and segregation of functional groups occurred during formation of a surface-bifunctionalized polymer. By measuring pyrene fluorescence in the presence of surface-bound amine quencher, pyrene fluorescence after the surface bound quenchers had been protonated, and the total amount of polymer-bound pyrene based on the amount of quenching seen with a large excess of an external quencher, we have been able to show that the mixture of functional groups at the surface is not highly segregated. The suggested integrated morphology was further supported by studies where the ratio of pyrene to internal quencher was varied over approximately 2 orders of magnitude. These studies of a bifunctional surface-modified polymer also showed that the percentage of bound pyrene that was internally quenched varied in a roughly linear way with the molar ratio of pyrene to internal quencher and that mild annealing did not change the observed two-dimensional morphology.

Protein fluorescence quenching by small molecules: protein penetration versus solvent exposure.

Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pattern of fluorescence quenching exhibited by quenching molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves protein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quenching, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For several proteins having tryptophans known to be buried, no quenching occurs. These results are not consistent with the view that the kinds of small molecules studied can quite generally penetrate into and diffuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion.