Structural analyses of an RNA stability element interacting with poly(A).

Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (∼24 kDa and ∼33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENE-poly(A) interaction involves a motif that protects the 3'-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi et al., Science 371, eabe6523 (2021)]. Identification of additional modes of poly(A)-RNA interaction opens new venues for better understanding of poly(A) tail biology.

tRNA-like leader-trailer interaction promotes 3'-end maturation of MALAT1.

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a nuclear long noncoding RNA (lncRNA) that is highly overexpressed in many cancer tissues and plays important roles in tumor progression and metastasis. The MALAT1 primary transcript contains evolutionarily conserved structural elements in its 3'-terminal region: a triple helix forming element called element for nuclear expression (ENE) and a downstream tRNA-like structure called mascRNA. Instead of being polyadenylated, mature MALAT1 is generated by recognition and processing of the mascRNA by RNase P. A genomically encoded A-rich tract at the new 3' end of MALAT1, which is generated upon RNase P cleavage, forms a triple helical structure with the upstream ENE. Triplex formation is vital for stabilization of the mature transcript and for subsequent accumulation and oncogenic activity of MALAT1. Here, we demonstrate that efficient 3'-end maturation of MALAT1 is dependent on an interaction between the A-rich tract and the mascRNA 3' trailer. Using mutational analyses of cell-based reporter accumulation, we show that an extended mascRNA acceptor stem and formation of a single bulged A 5' to the RNase P cleavage site are required for efficient maturation of the nascent MALAT1 3' end. Our results should benefit the development of therapeutic approaches to cancer through targeting MALAT1.

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing.

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na(+)), has remained underdeveloped, even though Na(+) is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na(+)-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (ko(bs)) ∼ 0.1 min(-1)], and the transformation of this DNAzyme into a fluorescent sensor for Na(+) by labeling the enzyme strand with a quencher at the 3' end, and the DNA substrate strand with a fluorophore and a quencher at the 5' and 3' ends, respectively. The presence of Na(+) catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na(+) over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na(+)-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na(+) in living cells.

Identification of the Same Na(+)-Specific DNAzyme Motif from Two In Vitro Selections Under Different Conditions.

We report an investigation of the functional relationship between two independently selected RNA-cleaving DNAzymes, NaA43, and Ce13, through in vitro selection. The NaA43 DNAzyme was obtained through a combination of gel-based and column-based in vitro selection in the presence of Na(+) and reported to be highly selective for Na(+) over other metal ions. The Ce13 DNAzyme was isolated via a gel-based method in the presence of Ce(4+) and found to be active with trivalent lanthanides, Y(3+) and Pb(2+). Despite completely different activities reported for the two DNAzymes, they share a high level of sequence similarity (~60% sequence identity). In this work, we systematically analyzed the activity of both DNAzymes to elucidate their potential functional relationship. We found that Na(+) is an obligate cofactor of the Ce13 DNAzyme and lanthanides cannot initiate the cleavage reaction in the absence of Na(+). Hence, we conclude that the Ce13 DNAzyme is a variant of the NaA43 DNAzyme that catalyzes reaction in the presence Na(+) and also utilizes lanthanides in a potentially allosteric manner. These results have identified a new DNAzyme motif that is not only remarkably Na(+)-specific, but also allows for design of novel allosteric DNAzymes for different biotechnological applications.

Simultaneous Fe2+/Fe3+ imaging shows Fe3+ over Fe2+ enrichment in Alzheimer's disease mouse brain.

Visualizing redox-active metal ions, such as Fe2+ and Fe3+ ions, are essential for understanding their roles in biological processes and human diseases. Despite the development of imaging probes and techniques, imaging both Fe2+ and Fe3+ simultaneously in living cells with high selectivity and sensitivity has not been reported. Here, we selected and developed DNAzyme-based fluorescent turn-on sensors that are selective for either Fe2+ or Fe3+, revealing a decreased Fe3+/Fe2+ ratio during ferroptosis and an increased Fe3+/Fe2+ ratio in Alzheimer's disease mouse brain. The elevated Fe3+/Fe2+ ratio was mainly observed in amyloid plaque regions, suggesting a correlation between amyloid plaques and the accumulation of Fe3+ and/or conversion of Fe2+ to Fe3+. Our sensors can provide deep insights into the biological roles of labile iron redox cycling.

RNA stabilization by a poly(A) tail 3'-end binding pocket and other modes of poly(A)-RNA interaction.

Polyadenylate [poly(A)] tail addition to the 3' end of a wide range of RNAs is a highly conserved modification that plays a central role in cellular RNA function. Elements for nuclear expression (ENEs) are cis-acting RNA elements that stabilize poly(A) tails by sequestering them in RNA triplex structures. A crystal structure of a double ENE from a rice hAT transposon messenger RNA complexed with poly(A)28 at a resolution of 2.89 angstroms reveals multiple modes of interaction with poly(A), including major-groove triple helices, extended minor-groove interactions with RNA double helices, a quintuple-base motif that transitions poly(A) from minor-groove associations to major-groove triple helices, and a poly(A) 3'-end binding pocket. Our findings both expand the repertoire of motifs involved in long-range RNA interactions and provide insights into how polyadenylation can protect an RNA's extreme 3' end.

Covalent immobilization of Drosophila acetylcholinesterase for biosensor applications.

The synthesized cDNA coding for AChE (acetylcholinesterase) was subcloned in pENTR/D-TOPO plasmid and expressed using baculovirus expression vector and Sf9 insect cells as host. Purified enzyme (specific activity 36374 micromol x min(-1) x mg(-1)) was immobilized on pre-activated perlite (a porous silica matrix) by silanization and glutaraldehyde treatment. The total enzyme immobilized was then measured, and total and specific activity of immobilized AChE was compared with that of soluble enzyme. Using this perlite support not only resulted in a great amount of maintained immobilized enzyme activity (more than 70%, specific activity 26238 micromolx min(-1) x mg(-1)), but also significantly improved stability against temperature (8.7- and 17.7-fold at 50 and 60 degrees C respectively), urea (2.7-fold) and acetonitrile (1.7-fold). Kinetic studies showed that the K(m) value for immobilized enzyme is very similar to the soluble one (0.088 and 0.081 mM respectively). In addition, immobilized enzymes retained 80% of their initial activity after 16 consecutive reactor batch cycles. A comparison of the inhibitory effect of paraoxon on soluble and immobilized AChE showed that immobilization increased the linearity of the inhibition plot particularly in the range 0.1 nM-0.1 microM.

Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization.

In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization.

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained∼50% of the free enzyme activity at 6.4mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.

Functional DNA nanomaterials for sensing and imaging in living cells.

Recent developments in integrating high selectivity of functional DNA, such as DNAzymes and aptamers, with efficient DNA delivery into cells by gold nanoparticles or superior near-infrared optical properties of upconversion nanoparticles are reviewed. Their applications in sensing and imaging small organic metabolites, toxins, metal ions, pH, DNA, RNA, proteins, and pathogens are summarized. The advantages and future directions of these functional DNA materials are discussed.

DNA Aptamer Technology for Personalized Medicine.

This review highlights recent progress in developing DNA aptamers for personalized medicine, with more focus on in vivo studies for potential clinical applications. Examples include design of aptamers in combination with DNA nanostructures, nanomaterials, or microfluidic devices as diagnostic probes or therapeutic agents for cancers and other diseases. The use of aptamers as targeting agents in drug delivery is also covered. The advantages and future directions of such DNA aptamer-based technology for the continued development of personalized medicine are discussed.

Small-molecule diagnostics based on functional DNA nanotechnology: a dipstick test for mercury.

Detecting small molecular targets such as metal ions is just as important as detecting large molecules such as DNA, RNA and proteins, but the field of metal ion sensors has not yet been well developed. A good example of a metal ion target is mercury, which is highly toxic, widely distributed in the environment and affects human health. To develop a diagnostic platform for metal ions, we demonstrate that functional DNA-linked gold nanoparticles (AuNPs) can quickly and simply detect and quantify Hg(2+) ions in aqueous solution, with high sensitivity and selectivity over competing metal ions. A linker DNA molecule containing thymine residues and sequences complementary to the DNA on the AuNPs was designed to aggregate DNA-functionalized AuNPs. When Hg(2+) ions were introduced into this system, they induced the linker DNA to fold by forming thymine-Hg(2+)-thymine bonds. The linker DNA's folding caused the AuNPs to rapidly disassemble, which caused a discernable color change in the solution from purple to red. The limit of detection for Hg(2+) in the present method is 5.4 nM, which is below the 10 nM maximum contaminant level defined by the US Environmental Protection Agency (EPA) for drinking water. Our results show that this Hg20 detection method has excellent selectivity over other divalent metal ions (e.g. Pb(2+), Cu(2+), Mn(2+), Co(2+), Zn(2+), Cd(2+), Mg(2+), Ca(2+), and Ba(2+)). This system has been converted into a dipstick test using lateral-flow devices, making it even more practical for point-of-care diagnostics.

Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase.

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.

Optimization of peroxidase-catalyzed oxidative coupling process for phenol removal from wastewater using response surface methodology.

Hydroxylated aromatic compounds (HACs) are considered to be primary pollutants in a wide variety of industrial wastewaters. Horseradish peroxidase (HRP) is suitable for the removal of these toxic substances. However, development of a mathematical model and optimization of the HRP-based treatment considering the economical issues by novel methods is a necessity. In the present study, optimization of phenol removal from wastewater by horseradish peroxidase (HRP) was carried out using response surface methodology (RSM) and central composite design (CCD). As the initial experimental design, 2(4-1) half-fraction factorial design (H-FFD) is accomplished in triplicate at two levels to select the most significant factors and interactions in the phenol removal procedure. Temperature (degrees C), pH, concentration of enzyme (unit mL(-1)), and H202 (mM) were determined as the most effective independent variables. Finally, a fourfactor-five coded level CCD, 30 runs, was performed in order to fit a second-order polynomial function to the results and calculate the economically optimum conditions of the reaction. The goodness of the model was checked by different criteria including the coefficient of determination (R2 = 0.93), the corresponding analysis of variance ((Pmodel > F) < 0.0001) and parity plot (r = 0.96). These analyses indicated that the fitted model is appropriate for this enzymatic system. With the assumption that the minimum enzyme concentration was 0.26 unit mL(-1), the analysis of the response surface contour and surface plots defined the optimum conditions as follows: pH = 7.12, hydrogen peroxide concentration 1.72 mM, and 10 degrees C. This work improves phenol removal operation economically by applying minimum enzyme concentration and highest removal in comparison with previous studies.