Ligating DNA with DNA

Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes. We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1). Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated.     

DNA polymerase-catalyzed DNA network growth

The distinct base pairing property of DNA is an advantageous phenomenon that has been exploited in the usage of DNA as scaffold for directed self-organization to form nanometer-sized objects in a desirable fashion. Herein we report the construction of three-dimensional DNA-based networks that can be generated and amplified by the DNA polymerase chain reaction (PCR). The approach is flexible allowing tuning of the meshes of the network by variation of the size of the template. Additionally, further diversification can be introduced by employment of chemically modified nucleotides in PCR allowing the introduction of functionalities and reporter moieties.     

DNA origami: the art of folding DNA

The advent of DNA origami technology greatly simplified the design and construction of nanometer-sized DNA objects. The self-assembly of a DNA-origami structure is a straightforward process in which a long single-stranded scaffold (often from the phage M13mp18) is folded into basically any desired shape with the help of a multitude of short helper strands. This approach enables the ready generation of objects with an addressable surface area of a few thousand nm(2) and with a single "pixel" resolution of about 6 nm. The process is rapid, puts low demands on experimental conditions, and delivers target products in high yields. These features make DNA origami the method of choice in structural DNA nanotechnology when two- and three-dimensional objects are desired. This Minireview summarizes recent advances in the design of DNA origami nanostructures, which open the door to numerous exciting applications.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

DNA loops and semicatenated DNA junctions

Background  

Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized.     

Electrontransfer through DNA and metal-containing DNA

DNA is currently explored as a new material for functional, molecular nano-architectures. In this respect, one major question is to transform DNA into a conducting material which has the potential for self-assembly into electronically active networks. The article covers recent insight into how DNA transports positive (holes) and negative (excess electrons) charges. It was found that holes move through DNA over significant distances using a G- and to a lesser extent also A-based hopping mechanism. EPR studies and recent investigations with model systems show that excess electrons can also hop through the duplex. The second part of the article describes how DNA is currently modified, particularly coated with metals, in order to increase the conductivity.     

Voltammetric determination of DNA based on regulation of DNA strand displacement using an allosteric DNA toehold

An electrochemical biosensor for determination of DNA is described that is based on the reaction of regulated DNA (reg-DNA) first with substrated DNA (subs-DNA) to form a reaction intermediate. The intermediate binds target DNA (T) by hybridization and initiates a branch migration leading to the production of complex of substrated DNA and target DNA (TC). Once TC is produced, it reacts with assisted DNA (ass-DNA) through a toehold exchange mechanism, yielding the product complex of substrated DNA and assisted DNA (CS). The target is then released back into the solution and and catalyzes the next cycle of toehold-exchange with the reaction intermediate of substrated DNA and regulated DNA (CPR). Unlike in a conventional DNA toehold that is hardwired with the branch migration domain, the allosteric DNA toehold is designed into a reg-DNA which is independent of the branch migration domain. Under the optimal experimental conditions and at a working potential as low as 0.18 V, response to DNA is linear in the 1 fM to 1000 pM concentration range, and the detection limit is 0.83 fM. The assay is highly specific and can discriminate target DNA even from a single-base mismatch. It was applied to the analysis of DNA spiked plasma samples.

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Graphical abstract Schematic illustration of the electrochemical strategy for target DNA detection based on regulation of DNA strand displacement using an allosteric DNA toehold strategy. It can be used to analyze DNA-spiked plasma samples and has a low detection limit of 0.83 fM.

     

DNA分析仪测定聚合酶活性——介绍一个化学生物学实验

把科研成果转化为实验教学内容,不仅会丰富课堂内容,同时也拓宽并延展了学生的知识面。该实验项目最初源于作者的科研课题,综合蛋白的结构、荧光标记DNA与DNA分析仪在酶动力学方面的应用,比较不同DNA聚合酶的结构差异对其复制功能的影响。本实验属于综合化学类实验,可以将科研成果与实验教学有机地结合,提高学生的综合实践能力以及动手能力。     

Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.     

Electron-transfer oxidation properties of DNA bases and DNA oligomers

Kinetics for the thermal and photoinduced electron-transfer oxidation of a series of DNA bases with various oxidants having the known one-electron reduction potentials (E(red)) in an aqueous solution at 298 K were examined, and the resulting electron-transfer rate constants (k(et)) were evaluated in light of the free energy relationship of electron transfer to determine the one-electron oxidation potentials (E(ox)) of DNA bases and the intrinsic barrier of the electron transfer. Although the E(ox) value of GMP at pH 7 is the lowest (1.07 V vs SCE) among the four DNA bases, the highest E(ox) value (CMP) is only 0.19 V higher than that of GMP. The selective oxidation of GMP in the thermal electron-transfer oxidation of GMP results from a significant decrease in the pH dependent oxidation potential due to the deprotonation of GMP*+. The one-electron reduced species of the photosensitizer produced by photoinduced electron transfer are observed as the transient absorption spectra when the free energy change of electron transfer is negative. The rate constants of electron-transfer oxidation of the guanine moieties in DNA oligomers with Fe(bpy)3(3+) and Ru(bpy)3(3+) were also determined using DNA oligomers containing different guanine (G) sequences from 1 to 10 G. The rate constants of electron-transfer oxidation of the guanine moieties in single- and double-stranded DNA oligomers with Fe(bpy)3(2+) and Ru(bpy)3(3+) are dependent on the number of sequential guanine molecules as well as on pH.     

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.     

Manipulating DNA writhe through varying DNA sequences

It is demonstrated that the shapes and magnitudes of DNA writhe can be precisely manipulated solely through maneuvering the nucleotide sequence of DNA and without the assistance of topoisomerases.     

New DNA Technology and the DNA Biosensor

Abstract

Recently there has been an increasing demand to produce systems for the detection of specific DNA sequences that are amenable to non-specialised laboratories. This demand has led to many innovative and novel approaches to DNA analysis which may collectively be termed ′new DNA technology′. Here, we review these advances in relation to their applicability for the production of a DNA biosensor. The present state of the art is described and future possibilities are considered.     

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage,DNA Cleavage Inhibition,and Formation of Ligand–DNA Adducts

Ligands interacting with abasic (AP) sites in DNA may generate roadblocks in base-excision DNA repair (BER) due to indirect inhibition of DNA repair enzymes (e.g., APE1) and/or formation of toxic byproducts, resulting from ligand-induced strand cleavage or covalent cross-links. Herein, a series of 12 putative AP-site ligands, sharing the common naphthalenophane scaffold, but endowed with a variety of substituents, have been prepared and systematically studied. The results demonstrate that most naphthalenophanes bind to AP sites in DNA and inhibit the APE1-induced hydrolysis of the latter in vitro. Remarkably, their APE1 inhibitory activity, as characterized by IC₅₀ and K_I values, can be directly related to their affinity and selectivity to AP sites, as assessed by means of fluorescence melting experiments. On the other hand, the molecular design of naphthalenophanes has a crucial influence on their intrinsic AP-site cleavage activity (i.e., ligand-catalyzed β- and β,δ-elimination reactions at the AP site), as illustrated by the compounds either having an exceptionally high AP-site cleavage activity (e.g., 2,7-BisNP-S , 125-fold more efficacious than spermine) or being totally devoid of this activity (four compounds). Finally, the unprecedented formation of a stable covalent DNA adduct upon reaction of one ligand ( 2,7-BisNP-NH ) with its own product of the AP-site cleavage is revealed.