Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization

Engineering the specificity of DNA-modifying enzymes has proven extremely challenging, as sequence recognition by these enzymes is poorly understood. Here we used directed evolution to generate a variant of HaeIII methyltransferase that efficiently methylates a novel target site. M.HaeIII methylates the internal cytosine of the canonical sequence GGCC, but there is promiscuous methylation of a variety of non-canonical sites, notably AGCC, at a reduced rate. Using in vitro compartmentalization (IVC), libraries of M.HaeIII genes were selected for the ability to efficiently methylate AGCC. A two-step mutagenesis strategy, involving initial randomization of DNA-contacting residues followed by randomization of the loop that lies behind these residues, yielded a mutant with a 670-fold improvement in catalytic efficiency (k(cat)/K(m)(DNA)) using AGCC and a preference for AGCC over GGCC. The mutant methylates three sites efficiently (AGCC, CGCC and GGCC). Indeed, it methylates CGCC slightly more efficiently than AGCC. However, the mutant discriminates against other non-canonical sites, including TGCC, as effectively as the wild-type enzyme. This study provides a rare example of a laboratory-evolved enzyme whose catalytic efficiency surpasses that of the wild-type enzyme with the principal substrate.     

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.     

Two-Step Validation Approach for Tools To Study the DNA Repair Enzyme SNM1A

We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide–alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.     

Nucleoside-Driven Specificity of DNA Methyltransferase**

We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l -methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l -methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.     

2D Enzyme Cascade Network with Efficient Substrate Channeling by Swinging Arms

In living cells, compartmentalized or membrane‐associated enzymes are often assembled into large networks to cooperatively catalyze cascade reaction pathways essential for cellular metabolism. Here, we report the assembly of an artificial 2D enzyme network of two cascade enzymes—glucose‐6‐phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH)—on a wireframe DNA origami template. Swinging arms were used to facilitate the transport of the redox intermediate of NAD⁺/NADH between enzyme pairs on the array. The assemblies of 2D enzyme networks were characterized by gel electrophoresis and visualized by atomic force microscopy (AFM). The spatial arrangements of multiple enzyme pairs were optimized to facilitate efficient substrate channeling by exploiting the programmability of DNA origami to manipulate the key parameters of swinging arm length and stoichiometry. Compared with a single enzyme pair, the 2D organized enzyme systems exhibited higher reaction efficiency due to the promoted transfer of intermediates within the network.     

Molecular drivers of base flipping during sequence-specific DNA methylation

One step at a time: Substrates containing nucleotide analogues lacking sequence-specific contacts to the C5 methyltransferase M.HhaI were used to probe the role of individual interactions in effecting conformational transitions during base flipping. A segregation of duties, that is, specific recognition and chemomechanical force for base flipping and active site assembly, within the enzyme is confirmed.     

Giant vesicles as microreactors for enzymatic mRNA synthesis

Giant vesicles have attracted much attention as possible microreactors for the conduction of enzymatic reactions in an artificial, cell-sized compartment. In this context, we demonstrated in the first part of the present work that giant vesicles formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in an alternating electric field can be made more permeable to Ca(2+) ions or nucleotide triphosphates by addition of ethanol. This methodology is then applied in a second step whereby these giant vesicles are used as microreactors in which mRNA synthesis can occur. The macromolecules (the DNA template and the enzyme T7 RNA polymerase) are microinjected into a selected giant vesicle, while the substrate molecules (nucleotide triphosphates) are added from the external medium. The fact that mRNA synthesis can be detected is a further step towards our aim: the design of a microreactor that can be seen as a model for a protocell.     

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA‐triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid‐programmed ligation reactions. A DNA‐triggered ligation–cyclization sequence (“cycligation”) of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA‐thioesters and isocysteinyl‐PNA‐cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product‐template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur‐halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2–3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25–0.01 equiv). We believe that cyclization of products from DNA‐templated reactions could ultimately afford systems that completely overcome product inhibition.     

酶活性部位柔性的分形分析

引言蛋白质三维结构的研究一直是生物学上一个重要而复杂的问题。通过X晶体衍射和核磁共振技术,人们已经积累了大量的蛋白质结构数据,但进一步解析蛋白质结构与功能间的关系,尚存在一定难度。作为非线性科学分支的分形理论(fractaltheory),以其独特的研究方法揭示了自然界中非线性过程内在的随机性及其所具有的特殊规律性。虽然分形理论在20世纪70年代才首次提出[1],但     

DNA‐Templated Synthesis of Perylenediimide Stacks Utilizing Abasic Sites as Binding Pockets and Reactive Sites

DNA is considered to be a promising biomolecule as a template and scaffold for arranging and organizing functional molecules on the nanoscale. The construction and evaluation of DNAs containing multiple functional molecules that are useful for optoelectronic devices and sensors has been studied. In this paper we report the efficient incorporation of perylenediimide (PDI) units into DNA by using abasic sites both as binding sites and as reactive sites and the construction of PDI stacks within the DNA structure, accomplished through the preorganization of the PDI units in the hydrophobic pocket within the DNA. Our approach could become a valuable method for construction of DNA/chromophore hybrid structures potentially useful for the design of DNA‐based devices and biosensors.     

Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology

The use of polymerase enzymes in biotechnology has allowed us to gain unprecedented control over the manipulation of DNA, opening up new and exciting applications in areas such as biosensing, polynucleotide synthesis, and DNA storage, aptamer development and DNA-nanotechnology. One of the most intriguing enzymes which has gained prominence in the last decade is terminal deoxynucleotidyl transferase (TdT), which is one of the only polymerase enzymes capable of catalysing the template independent stepwise addition of nucleotides onto an oligonucleotide chain. This unique enzyme has seen a significant increase in a variety of different applications. In this review, we give a comprehensive discussion of the unique properties and applications of TdT as a biotechnology tool, and the application in the enzymatic synthesis of poly/oligonucleotides. Finally, we look at the increasing role of TdT enzyme in biosensing, DNA storage, synthesis of DNA nanostructures and aptamer development, and give a future outlook for this technology.     

Flexible Assembly of an Enzyme Cascade on a DNA Triangle Prism Nanostructure for the Controlled Biomimetic Generation of Nitric Oxide

Spatial organization of multiple enzymes at specific positions for a controlled reaction cascade has attracted wide attention in recent years. Here, we report the construction of a biomimetic enzyme cascade organized on DNA triangle prism (TP) nanostructures to enable the efficient catalytic production of nitric oxide (NO) on a single microbead. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), were assembled at adjacent locations on a DNA TP nanostructure by using DNA‐binding protein adaptors with small interenzyme distances. In the cascade, the first enzyme, GOx, converts glucose into gluconic acid in the presence of oxygen. The produced H₂O₂ intermediate is rapidly transported to the second enzyme, HRP, which oxides hydroxyurea into NO and other nitroxyl species. The pH near the surface of the negatively charged DNA nanostructures is believed to be lower than that in the bulk solution; this creates an optimal pH environment for the anchored enzymes, which results in higher yields of the NO product. Furthermore, the multienzyme system was immobilized on a microbead mediated by a DNA adaptor, and this enabled the efficient catalytic generation of gas molecules in the microreactor. Therefore, this work provides an alternative route for the biomimetic generation of NO through enzyme cascades. In particular, the dynamic binding capability of the DNA sequence enabled the positions of the protein enzyme and the DNA nanostructure to be reversed, which allowed the cascade catalysis to be modulated.     

Why enzymes are proficient catalysts: beyond the Pauling paradigm

Pauling proposed that "enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyze, ..., [rather than] entering into reactions". This paradigm has dominated thinking in the field. While complementarity of the type proposed by Pauling can account for acceleration up to 11 orders of magnitude, most enzymes exceed that proficiency. Enzymes with proficiencies ((k(cat)/K(M))/k(uncat)) > 10(11) M(-1) achieve over 15 kcal/mol of "transition state binding" not merely by a concatenation of noncovalent effects but by covalent bond formation between enzyme or cofactor and transition state, involving a change in mechanism from that in aqueous solution. Enzymes enter into reactions with substrates and do not merely complement the transition states of the uncatalyzed reactions.     

Magnetic and conductive magnetite nanowires by DNA-templating

The synthesis of nanowires made of magnetite (Fe(3)O(4)) phase iron oxide was achieved using DNA as a template to direct formation of the metal oxide and confine its growth in two dimensions. This simple solution-based approach involves initial association of Fe(2+) and Fe(3+) to the DNA "template" molecules, and subsequent co-precipitation of the Fe(3)O(4) material, upon increasing the solution pH, to give the final metal oxide nanowires. Analysis of the DNA-templated material, using a combination of FTIR, XRD, XPS, and Raman spectroscopy, confirmed the iron oxide formed to be the Fe(3)O(4) crystal phase. Investigation of the structural character of the nanowires, carried out by AFM, revealed the metal oxide to form regular coatings of nanometre-scale thickness around the DNA templates. Statistical analysis showed the size distribution of the nanowires to follow a trimodal model, with the modal diameter values identified as 5-6 nm, 14-15 nm, and 23-24 nm. Additional scanning probe microscopy techniques (SCM, MFM) were also used to verify that the nanowire structures are electrically conducting and exhibit magnetic behaviour. Such properties, coupled with the small dimensions of these materials, make them potentially good candidates for application in a host of future nanoscale device technologies.     

Mechanisms of Enhanced Catalysis in Enzyme–DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme–DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9‐ and 2.4‐fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4‐aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems.     

Real-time detection of nucleotide incorporation during complementary DNA strand synthesis

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.     

Intensification of the bio‐processing of cotton textiles by combined enzyme/ultrasound treatment

Utilization of highly specific enzymes for various textile‐processing applications is becoming increasingly popular because of their ability to replace harsh organic/inorganic chemicals currently used by the textile industry. Thus, a significant decrease in the amount and toxicity of textile wastewater effluents is achievable. It was established that ultrasound does not inactivate the complex structures of enzyme molecules and as a consequence there was significant improvement in the performance of both cellulase and pectinase enzymes. The experimental data indicate that the maximum benefit provided by sonication occurs at relatively low enzyme concentrations. Ultrasonic energy significantly intensified enzymatic activity on various types of cotton fabrics, but it did not contribute to a decrease in tensile strength. The combined enzyme/sonication treatment of cellulosic textiles offers significant advantages such as less consumption of expensive enzymes, shorter processing time, less fiber damage and better uniformity of treatment. Published in 2002 for SCI by John Wiley & Sons, Ltd     

Nanopore detection of DNA molecules in magnesium chloride solutions

High translocation speed of a DNA strand through a nanopore is a major bottleneck for nanopore detection of DNA molecules. Here, we choose MgCl₂ electrolyte as salt solution to control DNA mobility. Experimental results demonstrate that the duration time for straight state translocation events in 1 M MgCl₂ solution is about 1.3 ms which is about three times longer than that for the same DNA in 1 M KCl solution. This is because Mg²⁺ ions can effectively reduce the surface charge density of the negative DNA strands and then lead to the decrease of the DNA electrophoretic speed. It is also found that the Mg²⁺ ions can induce the DNA molecules binding together and reduce the probability of straight DNA translocation events. The nanopore with small diameter can break off the bound DNA strands and increase the occurrence probability of straight DNA translocation events.     

Improvement of chymotrypsin enzyme stability as single enzyme nanoparticles

Individual enzyme molecules were coated with a nanometer-scale polymer network in order to increase their stability, providing longer lifetime of enzymes for biochemical processes. This polymer nanolayer is thin and porous enough [Kim, J., Grate, J.W., 2003. Single-enzyme nanoparticles armored by a nanometer-scale organic/inorganic network. Nano Letters 3, 1219-1222] to allow practically unhindered diffusion of the substrate from the solution to the active site of the enzyme, and unhindered transfer of the product from the active site to the solution. During the three-step preparation of the individual enzyme nanoparticles, the chymotrypsin enzyme investigated can lose 30-50% of its original activity. The main cause for a decrease in the enzyme's activity is the UV-light irradiation used during the polymerization step of the pretreatment. The UV-light can destroy the tertiary structure of the enzyme, and this can lead to reduced activity. The enzyme modification and solubilization steps of the treatment methodology do not essentially lower enzyme activity. The morphology and size of enzyme nanoparticles prepared was examined by transmission electron microscopy and demonstrated in this paper. The activity's change of the free and the covered enzyme was investigated at different temperatures, shaking frequencies and pH values. It has been proved that the preparation of enzyme nanoparticles can essentially stabilize the enzyme. Its activity changes much slower than that of native enzyme. The pretreated enzyme can have relatively high residual activity under extreme pH values and temperature, as well. The essence of the results presented is that stability of the enzyme can be increased significantly by covering individual enzymes with a thin, porous polymer layer.     

Lipolysis and heterogeneous catalysis. A new concept for expressing the substrate concentration

A new concept is proposed for quantifying the substrate concentration during heterogeneous catalysis of the kind which occurs during lipolysis. The number of molecules of protein (enzyme) adsorbable to the lipid substrate interface per unit of volume was evaluated and defined as a volumetric concentration of protein (enzyme) binding site (PEBS). Using porcine pancreatic lipase (EC 3.1.1.3) as a model enzyme, the maximal PEBS concentration was measured under various assay conditions by determining the saturation of the lipid substrate with the enzyme. Abacuses correlating the lipid substrate concentration (M) with the PEBS concentration (M) under each experimental conditions were used to express the kinetic data in terms of a volumetric concentration of PEBS. Comparisons could thus be made between data obtained with various enzymes and lipid interfaces because they were expressed with the same unit. In the case of pancreatic lipase, using triolein and tributyrylglycerol as substrates,K _m values of 2.7 and 7.5 nM PEBS were obtained, respectively, andK _D values ranging around 9 nM PEBS were also obtained from Scatchard plots. In addition, the average superficial density of PEBS was found to be 10×10¹¹ molecules·cm⁻², which is a value commonly obtained with structural proteins and enzymes adsorbed to an acylglyceride-water interface, this finding supports the idea that the PEBS concept represents the room in which the protein molecule adsorbs at the lipidic interface.