Ligands with polyfluorophenyl moieties promote a local structural rearrangement in the Spinach2 and Broccoli aptamers that increases ligand affinities.

The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand how nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI [(Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one] that reach out from the Spinach2 ligand binding pocket. Studies of the interaction of these compounds with the aptamers revealed that polyfluorophenyl-modified ligands initiate a slow change in aptamer affinity that takes an extended time (half-life of ∼40 min) to achieve. The change in affinity appears to involve an initial disruption of the entrance to the ligand binding pocket followed by a gradual transition to a more defined structure for which the most likely driving force is an interaction of the gateway adenine with a nearby 2'OH group. These results suggest that polyfluorophenyl modifications might increase the ability of small molecule drugs to disrupt local structure and promote RNA remodeling.

Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1,2 and Their Application in Electrochemical Sensing.

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2'FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.

The Magnetosome Protein, Mms6 from Magnetospirillum magneticum Strain AMB-1, Is a Lipid-Activated Ferric Reductase.

Magnetosomes of magnetotactic bacteria consist of magnetic nanocrystals with defined morphologies enclosed in vesicles originated from cytoplasmic membrane invaginations. Although many proteins are involved in creating magnetosomes, a single magnetosome protein, Mms6 from Magnetospirillum magneticum strain AMB-1, can direct the crystallization of magnetite nanoparticles in vitro. The in vivo role of Mms6 in magnetosome formation is debated, and the observation that Mms6 binds Fe3+ more tightly than Fe2+ raises the question of how, in a magnetosome environment dominated by Fe3+, Mms6 promotes the crystallization of magnetite, which contains both Fe3+ and Fe2+. Here we show that Mms6 is a ferric reductase that reduces Fe3+ to Fe2+ using NADH and FAD as electron donor and cofactor, respectively. Reductase activity is elevated when Mms6 is integrated into either liposomes or bicelles. Analysis of Mms6 mutants suggests that the C-terminal domain binds iron and the N-terminal domain contains the catalytic site. Although Mms6 forms multimers that involve C-terminal and N-terminal domain interactions, a fusion protein with ubiquitin remains a monomer and displays reductase activity, which suggests that the catalytic site is fully in the monomer. However, the quaternary structure of Mms6 appears to alter the iron binding characteristics of the C-terminal domain. These results are consistent with a hypothesis that Mms6, a membrane protein, promotes the formation of magnetite in vivo by a mechanism that involves reducing iron.

Common Secondary and Tertiary Structural Features of Aptamer-Ligand Interaction Shared by RNA Aptamers with Different Primary Sequences.

Aptamer selection can yield many oligonucleotides with different sequences and affinities for the target molecule. Here, we have combined computational and experimental approaches to understand if aptamers with different sequences but the same molecular target share structural and dynamical features. NEO1A, with a known NMR-solved structure, displays a flexible loop that interacts differently with individual aminoglycosides, its ligand affinities and specificities are responsive to ionic strength, and it possesses an adenosine in the loop that is critical for high-affinity ligand binding. NEO2A was obtained from the same selection and, although they are only 43% identical in overall sequence, NEO1A and NEO2A share similar loop sequences. Experimental analysis by 1D NMR and 2-aminopurine reporters combined with molecular dynamics modeling revealed similar structural and dynamical characteristics in both aptamers. These results are consistent with the hypothesis that the target ligand drives aptamer structure and also selects relevant dynamical characteristics for high-affinity aptamer-ligand interaction. Furthermore, they suggest that it might be possible to "migrate" structural and dynamical features between aptamer group members with different primary sequences but with the same target ligand.

Creating metamaterial building blocks with directed photochemical metallization of silver onto DNA origami templates.

DNA origami can be used to create a variety of complex and geometrically unique nanostructures that can be further modified to produce building blocks for applications such as in optical metamaterials. We describe a method for creating metal-coated nanostructures using DNA origami templates and a photochemical metallization technique. Triangular DNA origami forms were fabricated and coated with a thin metal layer by photochemical silver reduction while in solution or supported on a surface. The DNA origami template serves as a localized photosensitizer to facilitate reduction of silver ions directly from solution onto the DNA surface. The metallizing process is shown to result in a conformal metal coating, which grows in height to a self-limiting value with increasing photoreduction steps. Although this coating process results in a slight decrease in the triangle dimensions, the overall template shape is retained. Notably, this coating method exhibits characteristics of self-limiting and defect-filling growth, which results in a metal nanostructure that maps the shape of the original DNA template with a continuous and uniform metal layer and stops growing once all available DNA sites are exhausted.

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells.

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of "light-up" aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl.

The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand.

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures.

Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a well-structured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment.

Aptamers in analytics.

Nucleic acid aptamers are promising alternatives to antibodies in analytics. They are generally obtained through an iterative SELEX protocol that enriches a population of synthetic oligonucleotides to a subset that can recognize the chosen target molecule specifically and avidly. A wide range of targets is recognized by aptamers. Once identified and optimized for performance, aptamers can be reproducibly synthesized and offer other key features, like small size, low cost, sensitivity, specificity, rapid response, stability, and reusability. This makes them excellent options for sensory units in a variety of analytical platforms including those with electrochemical, optical, and mass sensitive transduction detection. Many novel sensing strategies have been developed by rational design to take advantage of the tendency of aptamers to undergo conformational changes upon target/analyte binding and employing the principles of base complementarity that can drive the nucleic acid structure. Despite their many advantages over antibodies, surprisingly few aptamers have yet been integrated into commercially available analytical devices. In this review, we discuss how to select and engineer aptamers for their identified application(s), some of the challenges faced in developing aptamers for analytics and many examples of their reported successful performance as sensors in a variety of analytical platforms.

Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

Morphological transformations in the magnetite biomineralizing protein Mms6 in iron solutions: a small-angle X-ray scattering study.

Magnetotactic bacteria that produce magnetic nanocrystals of uniform size and well-defined morphologies have inspired the use of biomineralization protein Mms6 to promote formation of uniform magnetic nanocrystals in vitro. Small angle X-ray scattering (SAXS) studies in physiological solutions reveal that Mms6 forms compact globular three-dimensional (3D) micelles (approximately 10 nm in diameter) that are, to a large extent, independent of concentration. In the presence of iron ions in the solutions, the general micellar morphology is preserved, however, with associations among micelles that are induced by iron ions. Compared with Mms6, the m2Mms6 mutant (with the sequence of hydroxyl/carboxyl containing residues in the C-terminal domain shuffled) exhibits subtle morphological changes in the presence of iron ions in solutions. The analysis of the SAXS data is consistent with a hierarchical core-corona micellar structure similar to that found in amphiphilic polymers. The addition of ferric and ferrous iron ions to the protein solution induces morphological changes in the micellar structure by transforming the 3D micelles into objects of reduced dimensionality of 2, with fractal-like characteristics (including Gaussian-chain-like) or, alternatively, platelet-like structures.

Investigating the malleability of RNA aptamers.

Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics. Here we discuss the integration of biochemical analysis with NMR spectroscopy and computational modeling to explore the relation between ligand binding and structural malleability of some well-studied aptamers. Several methods for determining aptamer binding affinity and specificity are discussed, including isothermal titration calorimetry, steady state fluorescence of 2-aminopurine substituted aptamers, and dye displacement assays. Also considered are aspects of molecular dynamics simulations specific to aptamers including adding ions and simulating aptamer structure in the absence of ligand when NMR spectroscopy or X-ray crystallography structures of the unoccupied aptamer are not available. We focus specifically on RNA aptamers that bind small molecule ligands as would be applied in sensors or integrated into riboswitches such as to measure the products of metabolic activity.

A computational study of alternate SELEX.

Systematic evolution of ligands by exponential enrichment (SELEX) is a procedure for identifying nucleic acid (NA) molecules with affinities for specific target species, such as proteins, peptides, or small organic molecules. Here, we extend the work in Seo et al. (Bull Math Biol 72:1623-1665, 2010) (multiple-target SELEX or positive SELEX) and examine an alternate SELEX process with multiple targets by incorporating negative selection into a positive SELEX protocol. The alternate SELEX process is done iteratively by alternating several positive selection rounds with several negative selection rounds. At the end of each positive selection round, NAs are eluted from the bound product and amplified by polymerase chain reaction (PCR) to increase the size of the pool of NA species that bind preferentially to the given positive target vector. The enriched population of NAs is then exposed to the negative targets (undesired targets). The free NA species (instead of the bound NA species being eluted) are retained and amplified by PCR (negative selection). The goal is to minimize an enrichment of nonspecifically binding NAs against multiple targets. While positive selection alone results in a pool of NAs that bind tightly to a given target vector, negative selection results in the subset of the NAs that bind best to the nontarget vectors that are also present. By alternating the two processes, we eventually obtain a refined population of nucleic acids that bind to the desired target(s) with high "selectivity" and "specificity." In the present paper, we give formulations of the negative and alternate selection processes and define their efficiencies in a meaningful way. We study the asymptotic behavior of alternate SELEX system as a discrete-time dynamical system. To do this, we use the chemical potential to examine how alternate SELEX leads to the selection of NAs with more specific interactions when the ratio of the number of positive selection rounds to the number of negative selection rounds is fixed. Alternate SELEX is said to be globally asymptotically stable if, given the initial target vector and a fixed ratio, the distribution of the limiting NA fractions does not depend on the relative concentrations of the NAs in the initial pool (provided that all of the NA species are initially present in the initial pool). We state conditions on the matrix of NA-target affinities that determine when the alternate SELEX process is globally asymptotically stable in this sense and illustrate these results computationally.

Plant root associated chitinases: structures and functions.

Chitinases degrade chitin, a linear homopolymer of ß-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type ß and epidermal growth factor.

The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type ß (TGFß) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFß regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFß also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFß pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFß are able to promote large and rapid changes in PAI-1 expression.

Integrated self-assembly of the Mms6 magnetosome protein to form an iron-responsive structure.

A common feature of biomineralization proteins is their self-assembly to produce a surface consistent in size with the inorganic crystals that they produce. Mms6, a small protein of 60 amino acids from Magnetospirillum magneticum strain AMB-1 that promotes the in vitro growth of superparamagnetic magnetite nanocrystals, assembles in aqueous solution to form spherical micelles that could be visualized by TEM and AFM. The results reported here are consistent with the view that the N and C-terminal domains interact with each other within one polypeptide chain and across protein units in the assembly. From studies to determine the amino acid residues important for self-assembly, we identified the unique GL repeat in the N-terminal domain with additional contributions from amino acids in other positions, throughout the molecule. Analysis by CD spectroscopy identified a structural change in the iron-binding C-terminal domain in the presence of Fe3+. A change in the intrinsic fluorescence of tryptophan in the N-terminal domain showed that this structural change is transmitted through the protein. Thus, self-assembly of Mms6 involves an interlaced structure of intra- and inter-molecular interactions that results in a coordinated structural change in the protein assembly with iron binding.

An RNA aptamer-based microcantilever sensor to detect the inflammatory marker, mouse lipocalin-2.

Lipocalin-2 (Lcn2) is a biomarker for many inflammatory-based diseases, including acute kidney injury, cardiovascular stress, diabetes, and various cancers. Inflammatory transitions occur rapidly in kidney and cardiovascular disease, for which an in-line monitor could be beneficial. Microcantilever devices with aptamers as recognition elements can be effective and rapidly responsive sensors. Here, we have selected and characterized an RNA aptamer that specifically binds mouse Lcn2 (mLcn2) with a dissociation constant of 340 ± 70 nM in solution and 38 ± 22 nM when immobilized on a surface. The higher apparent affinity of the immobilized aptamer may result from its effective multivalency that decreases the off-rate. The aptamer competes with a catechol iron-siderophore, the natural ligand of mLcn2. This and the results of studies with mLcn2 mutants demonstrate that the aptamer binds to the siderophore binding pocket of the protein. A differential interferometer-based microcantilever sensor was developed with the aptamer as the recognition element in which the differential response between two adjacent cantilevers (a sensing/reference pair) is utilized to detect the binding between mLcn2 and the aptamer, ensuring that sensor response is independent of environmental influences, distance between sensing surface and detector and nonspecific binding. The system showed a detection limit of 4 nM. This novel microcantilever aptasensor has potential for development as an in-line monitoring system for mLcn2 in studies of animal models of acute diseases such as kidney and cardiac failure.

Interfacial properties and iron binding to bacterial proteins that promote the growth of magnetite nanocrystals: X-ray reflectivity and surface spectroscopy studies.

Surface sensitive X-ray scattering and spectroscopic studies have been conducted to determine structural properties of Mms6, the protein in Magnetospirillum magneticum AMB-1 that is implicated as promoter of magnetite nanocrystals growth. Surface pressure versus molecular area isotherms indicate Mms6 forms stable monolayers at the aqueous/vapor interface that are strongly affected by ionic conditions of the subphase. Analysis of X-ray reflectivity from the monolayers shows that the protein conformation at the interface depends on surface pressure and on the presence of ions in the solutions, in particular of iron ions and its complexes. X-ray fluorescence at grazing angles of incidence from the same monolayers allows quantitative determination of surface bound ions to the protein showing that ferric iron binds to Mms6 at higher densities compared to other ions such as Fe(2+) or La(3+) under similar buffer conditions.

Self-assembly and biphasic iron-binding characteristics of Mms6, a bacterial protein that promotes the formation of superparamagnetic magnetite nanoparticles of uniform size and shape.

Highly ordered mineralized structures created by living organisms are often hierarchical in structure with fundamental structural elements at nanometer scales. Proteins have been found responsible for forming many of these structures, but the mechanisms by which these biomineralization proteins function are generally poorly understood. To better understand its role in biomineralization, the magnetotactic bacterial protein, Mms6, which promotes the formation in vitro of superparamagnetic magnetite nanoparticles of uniform size and shape, was studied for its structure and function. Mms6 is shown to have two phases of iron binding: one high affinity and stoichiometric and the other low affinity, high capacity, and cooperative with respect to iron. The protein is amphipathic with a hydrophobic N-terminal domain and hydrophilic C-terminal domain. It self-assembles to form a micelle, with most particles consisting of 20-40 monomers, with the hydrophilic C-termini exposed on the outside. Studies of proteins with mutated C-terminal domains show that the C-terminal domain contributes to the stability of this multisubunit particle and binds iron by a mechanism that is sensitive to the arrangement of carboxyl/hydroxyl groups in this domain.