Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers

Xeno nucleic acids (XNAs) are analogues of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addition, XNA oligomers can be used as biological tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a negatively charged backbone is preferable to provide sufficient water solubility; however, acyclic XNAs containing polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.     

Orthogonal Genetic Systems

Xenobiology is an emerging area of synthetic biology that aims to safeguard genetically engineered cells by storing synthetic biology information in xeno-nucleic acid polymers (XNAs). Critical to the success of this effort is the need to establish cellular systems that can maintain an XNA chromosome in actively dividing cells. This viewpoint discusses the structural parameters of the nucleic acid backbone that should be considered when designing an orthogonal genetic system that can replicate without interference from the endogenous genome. In addition to practical value, these studies have the potential to provide new fundamental insight into the structure and function properties of unnatural nucleic acid polymers.     

Design of biomimetic catalysts by molecular imprinting in synthetic polymers: the role of transition state stabilization

The impressive efficiency and selectivity of biological catalysts has engendered a long-standing effort to understand the details of enzyme action. It is widely accepted that enzymes accelerate reactions through their steric and electronic complementarity to the reactants in the rate-determining transition states. Thus, tight binding to the transition state of a reactant (rather than to the corresponding substrate) lowers the activation energy of the reaction, providing strong catalytic activity. Debates concerning the fundamentals of enzyme catalysis continue, however, and non-natural enzyme mimics offer important additional insight in this area. Molecular structures that mimic enzymes through the design of a predetermined binding site that stabilizes the transition state of a desired reaction are invaluable in this regard. Catalytic antibodies, which can be quite active when raised against stable transition state analogues of the corresponding reaction, represent particularly successful examples. Recently, synthetic chemistry has begun to match nature's ability to produce antibody-like binding sites with high affinities for the transition state. Thus, synthetic, molecularly imprinted polymers have been engineered to provide enzyme-like specificity and activity, and they now represent a powerful tool for creating highly efficient catalysts. In this Account, we review recent efforts to develop enzyme models through the concept of transition state stabilization. In particular, models for carboxypeptidase A were prepared through the molecular imprinting of synthetic polymers. On the basis of successful experiments with phosphonic esters as templates to arrange amidinium groups in the active site, the method was further improved by combining the concept of transition state stabilization with the introduction of special catalytic moieties, such as metal ions in a defined orientation in the active site. In this way, the imprinted polymers were able to provide both an electrostatic stabilization for the transition state through the amidinium group as well as a synergism of transition state recognition and metal ion catalysis. The result was an excellent catalyst for carbonate hydrolysis. These enzyme mimics represent the most active catalysts ever prepared through the molecular imprinting strategy. Their catalytic activity, catalytic efficiency, and catalytic proficiency clearly surpass those of the corresponding catalytic antibodies. The active structures in natural enzymes evolve within soluble proteins, typically by the refining of the folding of one polypeptide chain. To incorporate these characteristics into synthetic polymers, we used the concept of transition state stabilization to develop soluble, nanosized carboxypeptidase A models using a new polymerization method we term the "post-dilution polymerization method". With this methodology, we were able to prepare soluble, highly cross-linked, single-molecule nanoparticles. These particles have controlled molecular weights (39 kDa, for example) and, on average, one catalytically active site per particle. Our strategies have made it possible to obtain efficient new enzyme models and further advance the structural and functional analogy with natural enzymes. Moreover, this bioinspired design based on molecular imprinting in synthetic polymers offers further support for the concept of transition state stabilization in catalysis.     

Artificial enzyme with magnetic properties and peroxidase activity on indoleamine metabolite tumor marker

Stable and easily-handled synthetic materials mimicking natural enzymes activity would find important biotechnological applications. This article describes the synthesis and characterization of magnetic molecularly imprinted catalytic polymers that exhibit peroxidase-like activity towards 5-hydroxyindole-3-acetic acid (5-HIAA) oxidation. This multifunctional material is obtained from highly crystalline magnetite nuclei coated with a silica layer to protect the iron nucleus from oxidation and to provide anchoring for hydroxyl surface groups. After acrylic functionalization via sol–gel process, a molecularly imprinted polymer with hemin as catalytic center and 5-HIAA as template has been successfully attached to the structure. The resulting hybrid composite is magnetically separable and possesses excellent catalytic ability for the selective oxidation of the indoleamine metabolite tumor marker, showing Michaelis–Menten kinetics with this molecule but not towards other structural analogs. Therefore, it can be considered an artificial peroxidase enzyme.     

Aminoacyl-tRNA Synthetases as Valuable Targets for Antimicrobial Drug Discovery

Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of tRNA with a cognate amino acid and are essential enzymes in all three kingdoms of life. Due to their important role in the translation of the genetic code, aaRSs have been recognized as suitable targets for the development of small molecule anti-infectives. In this review, following a concise discussion of aaRS catalytic and proof-reading activities, the various inhibitory mechanisms of reported natural and synthetic aaRS inhibitors are discussed. Using the expanding repository of ligand-bound X-ray crystal structures, we classified these compounds based on their binding sites, focusing on their ability to compete with the association of one, or more of the canonical aaRS substrates. In parallel, we examined the determinants of species-selectivity and discuss potential resistance mechanisms of some of the inhibitor classes. Combined, this structural perspective highlights the opportunities for further exploration of the aaRS enzyme family as antimicrobial targets.     

Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.     

Expanding the catalytic repertoire of DNAzymes by modified nucleosides

Modified nucleoside triphosphates (dNTPs) are a versatile platform for the generation of high-density functionalized nucleic acids. The enzymatic polymerization of dNTPs allows the introduction of sensible functionalities that might not be compatible with the standard automated synthesis of oligonucleotides. Their application to in vitro selections, an elegant chemical approach to Darwinian evolution, delivers modified aptamers and catalytic nucleic acids with potentially enhanced properties. This review article highlights some recent synthetic examples of dNTPs bearing functionalities that are either found in the active site of protein enzymes or have been employed in organocatalysis and further underscores their usefulness in the development of some modified catalytic nucleic acids with special emphasis on M(2+)-independent RNA-cleaving DNAzymes.     

Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H

A series of DNA-linked RNases H, in which the 15-mer DNA iscross-linked to the Thermus thermophilus RNase HI (TRNH) variantsat positions 135, 136, 137 and 138, were constructed and analyzedfor their abilities to cleave the complementary 15-mer RNA.Of these, that with the DNA adduct at position 135 most efficientlycleaved the RNA substrate, indicating that position 135 is themost appropriate cross-linking site among those examined. Toexamine whether DNA-linked RNase H also site-specifically cleavesa highly structured natural RNA, DNA-linked TRNHs with a seriesof DNA adducts varying in size at position 135 were constructedand analyzed for their abilities to cleave MS2 RNA. These DNAadducts were designed such that DNA-linked enzymes cleave MS2RNA at a loop around residue 2790. Of the four DNA-linked TRNHswith the 8-, 12-, 16- and 20-mer DNA adducts, only that withthe 16-mer DNA adduct efficiently and site-specifically cleavedMS2 RNA. Primer extension revealed that this DNA-linked TRNHcleaved MS2 RNA within the target sequence.     

Replacing Mg2+ by Fe2+ for RNA-Cleaving DNAzymes

It has been proposed that Mg²⁺ and Fe²⁺ are very similar in interacting with ribozymes and some protein-based enzymes, but their activities with DNAzymes have yet to be studied. Here, the activity of Fe²⁺ as cofactor for a few RNA-cleaving DNAzymes is investigated. 17E is a well-studied DNAzyme that is active in the presence of many different divalent metal ions; it is highly active with Fe²⁺ with an apparent K_d of 29.7±2.3 μm and a k_obs of 1.12±0.11 min⁻¹ in the presence of 1 mm Fe²⁺ at pH 7.5. Fe²⁺ has 21-fold higher activity than Mg²⁺. Six different DNAzymes are then tested, and only the DNAzymes active with Mg²⁺ (17E, 8–17, and E5) are active with Fe²⁺. Fe²⁺ has 25 and one- to twofold higher activity than Mg²⁺ for the 8–17 and E5 DNAzymes, respectively. In pH>7 buffer and in presence of air, 1 mm Fe²⁺ results in a nonspecific degradation of the DNA strand due to reactive oxygen species (ROS). Cleavage reactions in anoxic environment and antioxidant ascorbate can be used to overcome the effect of oxidation. The findings provide insights for potential DNAzyme catalysis in the early Earth, and they further support the similarity between Mg²⁺ and Fe²⁺ in enzyme catalysis.     

Kemp Elimination Catalyzed by Naturally Occurring Aldoxime Dehydratases

Recently, the Kemp elimination reaction has been extensively studied in computational enzyme design of new catalysts, as no natural enzyme has evolved to catalyze this reaction. In contrast to in silico enzyme design, we were interested in searching for Kemp eliminase activity in natural enzymes with catalytic promiscuity. Based on similarities of substrate structures and reaction mechanisms, we assumed that the active sites of naturally abundant aldoxime dehydratases have the potential to catalyze the non‐natural Kemp elimination reaction. We found several aldoxime dehydratases that are efficient catalysts of this reaction. Although a few natural enzymes have been identified with promiscuous Kemp eliminase activity, to the best of our knowledge, this is a rare example of Kemp elimination catalyzed by naturally occurring enzymes with high catalytic efficiency.     

Optimization of electrochemical and peroxide-driven oxidation of styrene with ultrathin polyion films containing cytochrome P450cam and myoglobin

The catalytic and electrochemical properties of myoglobin and cytochrome P450(cam) in films constructed with alternate polyion layers were optimized with respect to film thickness, polyion type, and pH. Electrochemical and hydrogen peroxide driven epoxidation of styrene catalyzed by the proteins was used as the test reaction. Ionic synthetic organic polymers such as poly(styrene sulfonate), as opposed to SiO(2) nanoparticles or DNA, supported the best catalytic and electrochemical performance. Charge transport involving the iron heme proteins was achieved over 40-320 nm depending on the polyion material and is likely to involve electron hopping facilitated by extensive interlayer mixing. However, very thin films (ca. 12-25 nm) gave the largest turnover rates for the catalytic epoxidation of styrene, and thicker films were subject to reactant transport limitations. Classical bell-shaped activity/pH profiles and turnover rates similar to those obtained in solution suggest that films grown layer-by-layer are applicable to turnover rate studies of enzymes for organic oxidations. Major advantages include enhanced enzyme stability and the tiny amount of protein required.     

Zn2+-Dependent DNAzymes: From Solution Chemistry to Analytical,Materials and Therapeutic Applications

Since 1994, deoxyribozymes or DNAzymes have been in vitro selected to catalyze various types of reactions. Metal ions play a critical role in DNAzyme catalysis, and Zn²⁺ is a very important one among them. Zn²⁺ has good biocompatibility and can be used for intracellular applications. Chemically, Zn²⁺ is a Lewis acid and it can bind to both the phosphate backbone and the nucleobases of DNA. Zn²⁺ undergoes hydrolysis even at neutral pH, and the partially hydrolyzed polynuclear complexes can affect the interactions with DNA. These features have made Zn²⁺ a unique cofactor for DNAzyme reactions. This review summarizes Zn²⁺-dependent DNAzymes with an emphasis on RNA-/DNA-cleaving reactions. A key feature is the sharp Zn²⁺ concentration and pH-dependent activity for many of the DNAzymes. The applications of these DNAzymes as biosensors for Zn²⁺, as therapeutic agents to cleave intracellular RNA, and as chemical biology tools to manipulate DNA are discussed. Future studies can focus on the selection of new DNAzymes with improved performance and detailed biochemical characterizations to understand the role of Zn²⁺, which can facilitate practical applications of Zn²⁺-dependent DNAzymes.     

Cleavage of phosphodiesters and of DNA by a bis(guanidinium)naphthol acting as a metal-free anion receptor

Phosphoric acid diesters form anions at neutral pH. As a result of charge repulsion they are notoriously resistant to hydrolysis. Nucleophilic attack, however, can be promoted by different types of electrophilic catalysts that bind to the anions and reduce their negative charge density. Although in most cases phosphodiester-cleaving enzymes and synthetic catalysts rely on Lewis acidic metal ions, some exploit the guanidinium residues of arginine as metal-free electrophiles. Here we report that a combination of two guanidines and a hydroxy group yields highly reactive receptor molecules that can attack a broad range of phosphodiester substrates by nucleophilic displacement at phosphorus in a single-turnover mode. Some stable O-phosphates were isolated and characterized further by NMR spectroscopy. The bis(guanidinium)naphthols also cleave plasmid DNA, presumably by a transphosphorylation mechanism.     

A Versatile Endoribonuclease Mimic Made of DNA: Characteristics and Applications of the 8–17 RNA‐Cleaving DNAzyme

Enzymes play a crucial role in all living organisms by accelerating the rates of a myriad of biochemical reactions that are necessary to sustain life. Although the vast majority of known enzymes are made of protein, in recent years it has become increasingly apparent that other molecular formats, like nucleic acids, can also serve in this capacity. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid. These DNA‐based enzymes offer the prospect of significant commercial utility, because they are exceptionally stable and can be produced very easily and inexpensively. The study of one particular DNAzyme, known as “8–17”, has enhanced our understanding of DNAzyme‐mediated catalysis. Moreover, the function of 8–17 has been regarded with special importance because it can catalyze sequence‐specific cleavage of RNA, a reaction that has broad implications in biotechnology and biomedical fields. In this review, we explore the creation, characterization, and application of the 8–17 RNA‐cleaving DNAzyme.     

Review Oligosaccharide Synthesis Using Glycosidases*

A wide range of strategies may be considered for the synthesis of oligosaccharides in vitro using enzymes, all of which present significant challenges to the enzyme technologist. Many simple oligosaccharides may be produced by the hydrolysis of readily-available polysaccharides using specific enzymes. However, to produce the complex branched hetero-oligosaccharides of the types which occur N-linked to glycoproteins is more taxing. Materials of this type may be synthesised using the natural synthetic enzymes which employ sugar nucleotides as substrates. These enzymes are highly specific but they are costly to use due to their instability and to the cost of their substrates. It has been demonstrated that glycosidases are capable of synthesising hetero-oligosaccharides when provided with underivatised sugars in conditions of low water activity but that the specificity of synthetic reactions is apparently not high and that yields of material are low. Approaches to these problems are discussed, including the use of immobilised enzymes in packed-bed reactors to allow the ‘ping’ stage of the synthetic reaction to be separated in time from the ‘pong’ stage, and the application of aqueous two-phase systems which may be ‘tailored’ to separate the enzyme and the substrates from the final product. The ability to synthesise a range of oligosaccharides is dependent on the availability of appropriate glycosidases with differing specificities. There is a clear importance of ‘biodiversity’ in providing knowledge of sources of these.     

Proteolytic Antibodies

Catalytic activities are known to arise naturally in antibodies. Several naturally-occurring peptides, synthetic protease substrates, DNA, and esters are known to be cleaved by antibodies. There is increased production of antigen-specific catalytic antibodies in autoimmune disease. Polyreactive catalytic antibodies are present in unimmunized donors. Antibody light chains isolated from multiple myeloma patients frequently express proteolytic activity. Immunization protocols using as immunogens the ground state of a naturally-occurring polypeptide, anti-enzyme antibodies, or the enzyme itself are known to provoke catalytic antibody synthesis. Active site residues in the light chain subunit serve as the catalytic residues in a model antibody with peptide bond cleaving activity. A split-site model in which distinct amino acids serve as the essential catalytic residues and substrate ground-state recognition residues has been developed from mutagenesis studies. Engineering of the available antibodies could potentially generate improved catalysts. The possible mechanisms underlying proteolysis by natural antibodies and evolution of the catalytic activity are reviewed.     

The Two Classic Pb2+-Selective DNAzymes Are Related: Rational Evolution for Understanding Metal Selectivity

In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb²⁺ for its RNA cleavage activity. Three years later, the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb²⁺, although other divalent metals can work as well. GR5 and 17E have the same substrate sequence, and their catalytic loops in the enzyme strands also have a few similar and conserved nucleotides. Considering these, we hypothesized that 17E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17E. By using the activity ratio in the presence of Pb²⁺ and Mg²⁺ for defining these two DNAzymes, the critical nucleotide was identified to be T₁₂ in 17E for metal specificity. In addition, G₉ in GR5 is a position not found in most 17E or 8-17 DNAzymes, and G₉ needs to be added to rescue GR5 activity if T₁₂ becomes a cytosine. This study highlights the links between these two classic and widely used DNAzymes, and offers new insight into the sequence–activity relationship related to metal selectivity.     

Investigation of synthetic lipase and its use in transesterification reactions

A novel synthetic polymer selective for p-nitrophenylpalmitate was synthesized by molecular imprinting technique. We have combined the principle of molecular imprinting with the ability of histidine, glutamic acid and serine to form a catalytic cavity that can promote the catalytic degradation of p-nitrophenyl palmitate.For the creation of such catalytic sites we first synthesized appropriate monomers and used p-nitrophenyl palmitate as a template to synthesize the imprinted polymers and the binding characteristics of the polymers were evaluated. The optimum pH was determined by evaluating different pH values and the hydrolytic activity of synthetic lipase was evaluated in the framework of Micheaelis–Menten kinetics. In addition, the values of maximal rate: Vm (0.68 mM/min) and Michaelis–Menten constant, Km (1.4 × 10^?2 mM) were obtained from Lineweaver–Burk plots for the imprinted polymeric catalyst.