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Nanoscale Sep 2019DNA origami structures have developed into versatile tools in molecular sciences and nanotechnology. Currently, however, many potential applications are hindered by...
DNA origami structures have developed into versatile tools in molecular sciences and nanotechnology. Currently, however, many potential applications are hindered by their poor stability, especially under denaturing conditions. Here we present and evaluate two simple approaches to enhance DNA origami stability. In the first approach, we elevated the melting temperature of nine critical staple strands by merging the oligonucleotides with adjacent sequences. In the second approach, we increased the global stability by enzymatically ligating all accessible staple strand ends directly. By monitoring the gradual urea-induced denaturation of a prototype triangular DNA origami modified by these approaches using atomic force microscopy, we show that rational redesign of a few, critical staple strands leads to a considerable increase in overall stability at high denaturant concentration and elevated temperatures. In addition, enzymatic ligation yields DNA nanostructures with superior stability at up to 37 °C and in the presence of 6 M urea without impairing their shape. This bio-orthogonal approach is readily adaptable to other DNA origami structures without the need for synthetic nucleotide modifications when structural integrity under harsh conditions is required.
Topics: DNA; Nanostructures; Nucleic Acid Conformation; Oligonucleotides; Urea
PubMed: 31455950
DOI: 10.1039/c9nr04460d -
Analytical Biochemistry Jul 2018The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are...
The typical products of enzymatic circularization of DNA, using DNA ligase or recombinase, are covalently closed and mostly relaxed DNA circles. Because they are difficult to analyze on conventional gels, they are often converted to nicked circles prior to electrophoresis. Herein, we present a sensitive and quantitative procedure for directly analyzing ligated closed circle DNA on agarose gels without additional treatments. Specifically, inclusion of GelStar dye in the gel allowed detection of ligated closed circle DNAs, which were likely super-twisted by being intercalated by GelStar, as discrete bands with good separation from linear DNA of the same sizes.
Topics: DNA Ligases; DNA, Circular; Electrophoresis, Agar Gel; Fluorescence; Nucleic Acid Conformation
PubMed: 29856979
DOI: 10.1016/j.ab.2018.05.026 -
Nature Jan 2016Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic...
Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.
Topics: Base Sequence; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; DNA, Catalytic; Deoxyribose; Kinetics; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Nucleotides; Polynucleotide Ligases; RNA; RNA Folding; Substrate Specificity
PubMed: 26735012
DOI: 10.1038/nature16471 -
Organic & Biomolecular Chemistry Dec 2017DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial...
DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.
Topics: Combinatorial Chemistry Techniques; Crystallography, X-Ray; DNA; Humans; Ligands; Models, Molecular; Molecular Structure; Small Molecule Libraries; Thrombin
PubMed: 29215120
DOI: 10.1039/c7ob02119d -
New Biotechnology Sep 2015We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without...
We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.
Topics: DNA; Ligation; Limit of Detection; Reproducibility of Results
PubMed: 25681158
DOI: 10.1016/j.nbt.2015.01.011 -
Organic Letters Feb 2003[reaction: see text] An efficient synthesis of a medium-sized DNA lariat through the chemical ligation of a Y-shaped dumbbell precursor is described. The methodology...
[reaction: see text] An efficient synthesis of a medium-sized DNA lariat through the chemical ligation of a Y-shaped dumbbell precursor is described. The methodology requires only commercially available phosphoramidites and reagents and affords regioisomerically pure lariat molecules. Characterization of the lariat by T(m) analysis reveals that the molecule displays markedly enhanced thermal stability and unimolecular association-dissociation kinetics, consistent with DNA dumbbell behavior.
Topics: Base Sequence; DNA; Nucleic Acid Conformation
PubMed: 12556170
DOI: 10.1021/ol027227e -
Analytical Biochemistry Mar 2021The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile...
The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4'-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5'-DMT-protected ONs could allow producing both symmetric and asymmetric 5'-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5'-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5'-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5'-DMT modification of dsDNA does not affect activity of T5 5',3'-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5'-DMT-modification or search of ways to separate long 5'-DMT-ssDNA and 5'-OH-ssDNA could allow finding application of 5'-DMT-modified oligo- and polynucleotides.
Topics: DNA Ligases; DNA, Single-Stranded; Exodeoxyribonucleases
PubMed: 33508272
DOI: 10.1016/j.ab.2021.114115 -
Current Issues in Molecular Biology 1999Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction...
Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.
Topics: Cloning, Molecular; DNA, Recombinant; Deoxyribonucleases, Type II Site-Specific; Genetic Engineering; Humans; Polymerase Chain Reaction
PubMed: 11475698
DOI: No ID Found -
Journal of Biotechnology Aug 2002The most widely used technique for preventing self-ligation (self-circularization and concatenation) of DNA is dephosphorylation of the 5'-end, which stops DNA ligase...
The most widely used technique for preventing self-ligation (self-circularization and concatenation) of DNA is dephosphorylation of the 5'-end, which stops DNA ligase from catalyzing the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate residues at the DNA ends. The 5'-dephosphorylation technique cannot be applied to both DNA species to be ligated and thus, the untreated DNA species remains capable of self-ligation. To prevent this self-ligation, we replaced the 2'-deoxyribose at the 3'-end of the untreated DNA species with a 2',3'-dideoxyribose. Self-ligation was prevented at the replaced 3'-end, while the 5'-phosphate remaining at the 5'-end permitted ligation with the 3'-hydroxyl end of the 5'-dephosphorylated DNA strand. We successfully applied this 3'-replacement technique to gene cloning, adapter-mediated polymerase chain reaction and messenger RNA fingerprinting. The 3'-replacement technique is simple and not restricted by sequence or conformation of the DNA termini and is thus applicable to a wide variety of methods involving ligation.
Topics: Cloning, Molecular; DNA; DNA Damage; DNA Fingerprinting; DNA Ligases; Gene Expression Regulation; Models, Chemical; Models, Genetic; Polymerase Chain Reaction; RNA, Messenger
PubMed: 12084479
DOI: 10.1016/s0168-1656(02)00107-4 -
PloS One 2014DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional...
DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method's utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.
Topics: Cloning, Molecular; DNA; Genetic Engineering; Oligonucleotides; Plasmids
PubMed: 25226397
DOI: 10.1371/journal.pone.0107329