Capture, detection and purification of dsDNA amplicons using a DNA binding protein on magnetic beads.

Magnetic separation has been widely exploited for capture and detection of nucleic acids, including amplicons. Streptavidin-magnetic beads (SA-MB) are typically employed for this purpose, as well as in biosensing applications. However, remaining biotinylated primer in the amplification reaction can compete with labeled amplicon for binding to the beads. Also, the harsh conditions needed for elution of bound amplicons restrict their use for purification purposes. Herein we show that a sequence-specific DNA binding protein immobilized on magnetic beads can serve as an alternative to SA-MB for these applications. This is enabled by the high binding affinity of scCro DNA binding protein for its specific sequence and its ability to bind dsDNA but not ssDNA. This specific sequence is easily incorporated in the amplicon during amplification with an extended primer. The scCro-MB exhibited higher amplicon binding capacity and detection sensitivity compared to SA-MB when both synthetic and genomic DNA were used as templates for PCR. This resulted not only from increased protein load on the beads but also from minimized interference of excess labeled primer remaining in the unpurified amplification reactions. Finally, a proof-of-concept was provided for the use of the scCro-MB for PCR amplicon purification under mild elution conditions using salt.

Nucleic acid lateral flow dipstick assay for the duplex detection of Gambierdiscus australes and Gambierdiscus excentricus.

The proliferation of harmful microalgae endangers aquatic ecosystems and can have serious economic implications on a global level. Harmful microalgae and their associated toxins also pose a threat to human health since they can cause seafood-borne diseases such as ciguatera. Implementation of DNA-based molecular methods together with appropriate detection strategies in monitoring programs can support the efforts for effective prevention of potential outbreaks. A PCR-lateral flow assay (PCR-LFA) in dipstick format was developed in this work for the detection of two Gambierdiscus species, G. australes and G. excentricus, which are known to produce highly potent neurotoxins known as ciguatoxins and have been associated with ciguatera outbreaks. Duplex PCR amplification of genomic DNA from strains of these species utilizing species-specific ssDNA tailed primers and a common primer containing the binding sequence of scCro DNA binding protein resulted in the generation of hybrid ssDNA-dsDNA amplicons. These were captured on the dipsticks via hybridization with complementary probes and detected with a scCro/carbon nanoparticle (scCro/CNPs) conjugate. The two different test zones on the dipsticks allowed the discrimination of the two species and the assay exhibited high sensitivity, 6.3 pg/µL of genomic DNA from both G. australes and G. excentricus. The specificity of the approach was also demonstrated using genomic DNA from non-target Gambierdiscus species and other microalgae genera which did not produce any signals. The possibility to use cells directly for amplification instead of purified genomic DNA suggested the compatibility of the approach with field sample testing. Future work is required to further explore the potential use of the strategy for on-site analysis and its applicability to other toxic species.

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

Nucleic acid lateral flow assays using a conjugate of a DNA binding protein and carbon nanoparticles.

Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 103 CFU·mL-1. The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence. Graphical abstract Schematic presentation of the lateral flow-based fluorometric detection of DNA amplicons using conjugates of scCro DNA binding protein with (A) carbon nanoparticles, (B) HRP and (C) HRP and carbon nanoparticles.

Sandwich-type aptasensor employing modified aptamers and enzyme-DNA binding protein conjugates.

The use of aptamers in various analytical applications as molecular recognition elements and alternative to antibodies has led to the development of various platforms that facilitate the sensitive and specific detection of targets ranging from small molecules and proteins to whole cells. The goal of this work was to design a universal and adaptable sandwich-type aptasensor exploiting the unique properties of DNA binding proteins. Specifically, two different enzyme-DNA binding protein conjugates, GOx-dHP and HRP-scCro, were used for the direct detection of a protein using two aptamers for target capture and detection. The specific dsDNA binding sequence for each DNA binding protein tag was incorporated in the form of a hairpin at one end of each aptamer sequence during the synthesis step. Detection was accomplished by an enzymatic (GOx/HRP) cascade reaction after the binding of each enzyme conjugate to its corresponding binding sequence on each aptamer. The proposed sandwich-type aptasensor was validated for the detection of thrombin, which is one of the most commonly used model targets with known dual aptamers. The limit of detection accomplished was 0.92 nM which is comparable with other colorimetric platforms reported in the literature. The sensitivity of the aptasensor was easily modulated by changing the number of dsDNA binding sites incorporated in the aptamer sequences, thus controlling the enzyme stoichiometry. Finally, the potential use of the proposed sensing approach for real sample testing was demonstrated using spiked human plasma and no significant matrix effects were observed when up to 2% plasma was used.

Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the conjugates. Graphical abstract DNA sensing platform based on an immobilized ssDNA capture probe and a hybrid ssDNA-dsDNA detection probe that specifically hybridize with the ssDNA target. The hybrid ssDNA-dsDNA detection probe also provides the binding sites for the enzyme-DNA binding protein conjugates (HRP-scCro and GOx-dHP) that generate the colorimetric signal.

Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags.

The need for sensitive detection of DNA is growing as more specific DNA sequences are being correlated to gene markers for disease diagnosis, food safety and other security related applications. Detection in hybridization-based assays is usually achieved with target-specific ssDNA probes conjugated directly to enzyme labels like HRP that provide signal amplification or with nanoparticles functionalized with DNA and multiple HRP molecules. In order to overcome some of the drawbacks presented by these approaches, we developed a unique DNA sensing platform based on an HRP-DNA binding protein tag conjugate and a hybrid ssDNA-dsDNA detection probe. Specifically, in this work we describe the preparation and characterization of an HRP conjugate with scCro DNA binding protein tag and its application for the detection of a model ssDNA target sequence. By using the HRP-scCro conjugate together with a hybrid detection probe containing three scCro-specific dsDNA binding sites, we demonstrate an improvement by over 3-fold in both sensitivity and limit of detection of high-risk human papillomavirus (HPV16), compared to the standard ssDNA-HRP conjugate. These results show that the HRP-DNA binding protein tag conjugate can be used as an alternative and universal tool for signal enhancement in enzyme-linked assays suitable for integration in point-of-care systems.

Disulfide bond formation by exported glutaredoxin indicates glutathione's presence in the E. coli periplasm.

Organisms have evolved elaborate systems that ensure the homeostasis of the thiol redox environment in their intracellular compartments. In Escherichia coli, the cytoplasm is kept under reducing conditions by the thioredoxins with the help of thioredoxin reductase and the glutaredoxins with the small molecule glutathione and glutathione reductase. As a result, disulfide bonds are constantly resolved in this compartment. In contrast to the cytoplasm, the periplasm of E. coli is maintained in an oxidized state by DsbA, which is recycled by DsbB. Thioredoxin 1, when exported to the periplasm turns from a disulfide bond reductase to an oxidase that, like DsbA, is dependent on DsbB. In this study we set out to investigate whether a subclass of the thioredoxin superfamily, the glutaredoxins, can become disulfide bond-formation catalysts when they are exported to the periplasm. We find that glutaredoxins can promote disulfide bond formation in the periplasm. However, contrary to the behavior of thioredoxin 1 in this environment, the glutaredoxins do so independently of DsbB. Furthermore, we show that glutaredoxin 3 requires the glutathione biosynthesis pathway for its function and can oxidize substrates with only a single active-site cysteine. Our data provides in vivo evidence suggesting that oxidized glutathione is present in the E. coli periplasm in biologically significant concentrations.

Laboratory evolution of Escherichia coli thioredoxin for enhanced catalysis of protein oxidation in the periplasm reveals a phylogenetically conserved substrate specificity determinant.

Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA(-) cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA(-) cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.

Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1.

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron-sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron-sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron-sulfur biosynthesis is discussed with respect to their intracellular localization.

The many faces of glutathione in bacteria.

Glutathione is one of the most abundant thiols present in cyanobacteria and proteobacteria, and in all mitochondria or chloroplast-bearing eukaryotes. In bacteria, in addition to its key role in maintaining the proper oxidation state of protein thiols, glutathione also serves a key function in protecting the cell from the action of low pH, chlorine compounds, and oxidative and osmotic stresses. Moreover, glutathione has emerged as a posttranslational regulator of protein function under conditions of oxidative stress, by the direct modification of proteins via glutathionylation. This review summarizes the biosynthesis and function of glutathione in bacteria from physiological and biotechnological standpoints.

An engineered pathway for the formation of protein disulfide bonds.

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.