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.

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.

Computational Modeling of RNA Aptamers: Structure Prediction of the Apo State.

RNA aptamers are single-stranded oligonucleotides that bind to specific molecular targets with high affinity and specificity. To design aptamers for new applications, it is critical to understand the ligand binding mechanism in terms of the structure and dynamics of the ligand-bound and apo states. The problem is that most of the NMR or X-ray crystal structures available for RNA aptamers are for ligand-bound states. Available apo state structures, mostly characterized by crystallization under nonphysiological conditions or probed by low resolution techniques, might fail to represent the diverse structural variations of the apo state in solution. Here, we develop an approach to obtain a representative ensemble of apo structures that are based on in silico RNA 3D structure prediction and in vitro experiments that characterize base stacking. Using the neomycin-B aptamer as a case study, an ensemble of structures for the aptamer in the apo (unbound) state are validated and then used to investigate the ligand-binding mechanism for the aptamer in complex with neomycin-B.

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.

Aptamer Applications in Neuroscience.

Being the predominant cause of disability, neurological diseases have received much attention from the global health community. Over a billion people suffer from one of the following neurological disorders: dementia, epilepsy, stroke, migraine, meningitis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, prion disease, or brain tumors. The diagnosis and treatment options are limited for many of these diseases. Aptamers, being small and non-immunogenic nucleic acid molecules that are easy to chemically modify, offer potential diagnostic and theragnostic applications to meet these needs. This review covers pioneering studies in applying aptamers, which shows promise for future diagnostics and treatments of neurological disorders that pose increasingly dire worldwide health challenges.

Sampling Performance of Multiple Independent Molecular Dynamics Simulations of an RNA Aptamer.

Using multiple independent simulations instead of one long simulation has been shown to improve the sampling performance attained with the molecular dynamics (MD) simulation method. However, it is generally not known how long each independent simulation should be, how many independent simulations should be used, or to what extent either of these factors affects the overall sampling performance achieved for a given system. The goal of the present study was to assess the sampling performance of multiple independent MD simulations, where each independent simulation begins from a different initial molecular conformation. For this purpose, we used an RNA aptamer that is 25 nucleotides long as a case study. The initial conformations of the aptamer are derived from six de novo predicted 3D structures. Each of the six de novo predicted structures is energy minimized in solution and equilibrated with MD simulations at high temperature. Ten conformations from these six high-temperature equilibration runs are selected as initial conformations for further simulations at ambient temperature. In total, we conducted 60 independent MD simulations, each with a duration of 100 ns, to study the conformation and dynamics of the aptamer. For each group of 10 independent simulations that originated from a particular de novo predicted structure, we evaluated the potential energy distribution of the RNA and used recurrence quantification analysis to examine the sampling of RNA conformational transitions. To assess the impact of starting from different de novo predicted structures, we computed the density of structure projection on principal components to compare the regions sampled by the different groups of ten independent simulations. The recurrence rate and dependence of initial conformation among the groups were also compared. We stress the necessity of using different initial configurations as simulation starting points by showing long simulations from different initial structures suffer from being trapped in different states. Finally, we summarized the sampling efficiency for the complete set of 60 independent simulations and determined regions of under-sampling on the potential energy landscape. The results suggest that conducting multiple independent simulations using a diverse set of de novo predicted structures is a promising approach to achieve sufficient sampling. This approach avoids undesirable outcomes, such as the problem of the RNA aptamer being trapped in a local minimum. For others wishing to conduct multiple independent simulations, the analysis protocol presented in this study is a guide for examining overall sampling and determining if more simulations are necessary for sufficient sampling.

The Lipocalin2 Gene is Regulated in Mammary Epithelial Cells by NFκB and C/EBP In Response to Mycoplasma.

Lcn2 gene expression increases in response to cell stress signals, particularly in cells involved in the innate immune response. Human Lcn2 (NGAL) is increased in the blood and tissues in response to many stressors including microbial infection and in response to LPS in myeloid and epithelial cells. Here we extend the microbial activators of Lcn2 to mycoplasma and describe studies in which the mechanism of Lcn2 gene regulation by MALP-2 and mycoplasma infection was investigated in mouse mammary epithelial cells. As for the LPS response of myeloid cells, Lcn2 expression in epithelial cells is preceded by increased TNFα, IL-6 and IκBζ expression and selective reduction of IκBζ reduces Lcn2 promoter activity. Lcn2 promoter activation remains elevated well beyond the period of exposure to MALP-2 and is persistently elevated in mycoplasma infected cells. Activation of either the human or the mouse Lcn2 promoter requires both NFκB and C/EBP for activation. Thus, Lcn2 is strongly and enduringly activated by mycoplasma components that stimulate the innate immune response with the same basic regulatory mechanism for the human and mouse genes.

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.

Characterization of the Photophysical Behavior of DFHBI Derivatives: Fluorogenic Molecules that Illuminate the Spinach RNA Aptamer.

( Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one (DFHBI) and its analogues are fluorogenic molecules that bind the Spinach aptamer (a small RNA molecule), which was selected for imaging RNA. They are extremely weakly fluorescent in liquid solvents. It had been hypothesized that photoisomerization is a very efficient nonradiative process of deactivation. We show, consistent with the results of other studies, that if the isomerization is impeded, the fluorescence signal is enhanced significantly. In addition, we provide a thorough characterization of the photophysical behavior of DFHBI and its derivatives, notably that of ( Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-3-((perfluorophenyl)methyl)-3,5-dihydro-4 H-imidazol-4-one (PFP-DFHBI) in various solvent environments. Solvent-dependent studies were performed with various mixtures of solvents. The results suggest that hydrogen bonding or strong interactions of the solvents with the phenolic-OH group change the absorption band near 420-460 nm and the nature of emission near 430 and 500 nm through various degrees of stabilization and the transformation between the neutral and the anionic species at both ground and excited states. These observations are confirmed by using a methoxy-substituted molecule (( Z)-5-(4-methoxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one), where the 420-460 nm band is absent in the presence of methanol and the spectra are similar to those of PFP-DFHBI in noninteracting solvents, such as acetonitrile and dichloromethane. Thus, in addition to the major role of photoisomerization as a nonradiative process of deactivation of the excited state, the fluorescence of DFHBI-type molecules is very sensitively dependent upon the pH of the medium as well as upon solvent-specific interactions, such as hydrogen-bonding ability and polarity.

Correction: Protein patterns template arrays of magnetic nanoparticles.

[This corrects the article DOI: 10.1039/C6RA07662A.].

Label free thrombin detection in presence of high concentration of albumin using an aptamer-functionalized nanoporous membrane.

Nanoporous alumina membranes have become a ubiquitous biosensing platform for a variety of applications and aptamers are being increasingly utilized as recognition elements in protein sensing devices. Combining the advantages of the two, we report label-free sensitive detection of human α-thrombin by an aptamer-functionalized nanoporous alumina membrane using a four-electrode electrochemical cell. The sensor response to α-thrombin was determined in the presence of a high concentration (500 µM) of human serum albumin (HSA) as an interfering protein in the background. The sensor sensitivity was also characterized against γ-thrombin, which is a modified α-thrombin lacking the aptamer binding epitope. The detection limit, within an appreciable signal/noise ratio, was 10 pM of α-thrombin in presence of 500 µM HSA. The proposed scheme involves the use of minimum reagents/sample preparation steps, has appreciable response in presence of high concentrations of interfering molecules and is readily amenable to miniaturization by association with existing-chip based electrical systems for application in point-of-care diagnostic devices.

Aptamer-enabled uptake of small molecule ligands.

The relative ease of isolating aptamers with high specificity for target molecules suggests that molecular recognition may be common in the folds of natural RNAs. We show here that, when expressed in cells, aptamers can increase the intracellular concentrations of their small molecule ligands. We have named these aptamers as DRAGINs (Drug Binding Aptamers for Growing Intracellular Numbers). The DRAGIN property, assessed here by the ability to enhance the toxicity of their ligands, was found for some, but not all, aminoglycoside aptamers. One aptamer protected cells against killing by its ligand. Another aptamer promoted killing as a singlemer and protected against killing as a tandemer. Based on a mathematical model, cell protection vs. killing is proposed as governed by aptamer affinity and access to the inner surface of the cell membrane, with the latter being a critical determinant. With RNA molecules proposed as the earliest functional polymers to drive the evolution of life, we suggest that RNA aptamer-like structures present in primitive cells might have selectively concentrated precursors for polymer synthesis. Riboswitches may be the evolved forms of these ancient aptamer-like "nutrient procurers". Aptamers with DRAGIN capability in the modern world could be applied for imaging cells, in synthetic cell constructs, or to draw drugs into cells to make "undruggable" targets accessible to small molecule inhibitors.

A 2'FY-RNA Motif Defines an Aptamer for Ebolavirus Secreted Protein.

With properties such as stability to long-term storage and amenability to repetitive use, nucleic acid aptamers are compatible with many sensing/transducing platforms intended for use in remote locations. Sensors with these properties are important for quickly identifying ebolavirus outbreaks, which frequently start in locations that lack sophisticated equipment. Soluble glycoprotein (sGP), an excellent biomarker for ebolaviruses, is produced from the same gene as the ebolavirus glycoprotein GP1,2 that decorates the surface of the viral particle and is secreted in abundance into the blood stream even during the early stages of infection. Here, we report the selection and properties of a 2'fluoro pyrimidine (2'FY)-modified RNA aptamer, 39SGP1A, that specifically binds sGP. We demonstrate by computational and biochemical analysis that the recognition motif of 39SGP1A is a novel polypyrimidine-rich sequence. Replacement of -F by -OH in the 2' position of the ribose resulted in complete loss of affinity for sGP. The protein motif to which the aptamer binds requires an intact sGP dimer and binds to an epitope conserved between Ebola virus (EBOV) and Sudan virus (SUDV) sGP, the most divergent Ebolavirus species. This identifies 39SGP1A as an excellent option for integration on a sensor platform to detect ebolavirus infections.

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.

Data on IL-10R neutralization-induced chronic colitis in Lipocalin 2 deficient mice on BALB/c background.

The data herein is related to the research article entitled "Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis" (Singh et al., 2016) [1] where we have demonstrated that C57BL/6 Lipocalin 2 deficient mice (Lcn2KO) developed chronic colitis upon anti-interleukin-10 receptor (αIL-10R) monoclonal antibody administration. In the present article, we evaluated the susceptibility of BALB/c Lcn2KO mice and their WT littermates to the αIL-10R neutralization-induced chronic colitis. Our data showed that αIL-10R mAb-treated BALB/c Lcn2KO mice exhibited severe chronic colitis (i.e., splenomegaly, colomegaly, colonic pathology, and incidence of rectal prolapse) when compared to WT mice.

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.

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.

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.