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Nano Letters Jul 2006DNA nanotubes are crystalline self-assemblies of DNA tiles approximately 10 nm in diameter that readily grow tens of micrometers in length. Easy assembly,...
DNA nanotubes are crystalline self-assemblies of DNA tiles approximately 10 nm in diameter that readily grow tens of micrometers in length. Easy assembly, programmability, and stiffness make them interesting for many applications, but DNA nanotubes begin to melt at temperatures below 40 degrees C, break open when deposited on mica or scanned by AFM, and disintegrate in deionized water. These weaknesses can be traced to the presence of discontinuities in the phosphate backbone, called nicks. The nanotubes studied here have five nicks, one in the core of a tile and one at each corner. We report the successful ligation of all four corner nicks by T4 DNA ligase. Although ligation does not change the nanotubes' stiffness, ligated nanotubes withstand temperatures over 70 degrees C, resist breaking during AFM, and are stable in pure water for over a month. Ligated DNA nanotubes are thus physically and chemically sturdy enough to withstand the manipulations necessary for many technological applications.
Topics: Aluminum Silicates; Base Sequence; DNA; DNA Ligases; Microscopy, Atomic Force; Molecular Sequence Data; Nanotubes; Substrate Specificity; Surface Properties
PubMed: 16834415
DOI: 10.1021/nl0603505 -
Nucleic Acids Research Nov 1988Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for four types of reactions: (a) a homogeneous population of singly...
Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for four types of reactions: (a) a homogeneous population of singly restricted DNA fragments, (b) insertion of singly restricted insert into vector, (c) forced directional insertion of doubly restricted insert into vector, and (d) insertion of singly restricted insert into phosphatased vector. The effects of varying vector and insert sizes, starting concentrations, and phosphatase treatment on the yield of circular 1:1 recombinants were analyzed. Selected theoretical predictions were experimentally tested and verified. Our suggestions on optimizing ligation reactions in several cases are at variance with common practice. For example, optimum conditions in case (b) and (d) ligations are best specified as individual insert and vector concentrations rather than as insert/vector molar ratios, except in case (d) ligations involving very small insert size. In case (c) ligations, highest efficiencies are obtained when both vector and insert are at relatively low concentration.
Topics: Cloning, Molecular; Computer Simulation; DNA; DNA Transposable Elements; Genetic Vectors; Kinetics; Mathematics; Models, Theoretical
PubMed: 2848221
DOI: 10.1093/nar/16.21.10301 -
Journal of the American Chemical Society Jun 2012A generic fluorescence sensing platform for analyzing DNA by the Zn(2+)-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the...
A generic fluorescence sensing platform for analyzing DNA by the Zn(2+)-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a "helper" nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components ("helper" sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn(2+)-dependent ligation DNAzyme-driven activation of the Mg(2+)-dependent DNAzyme. According to this method, the Mg(2+)-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay-Sachs genetic disorder mutant.
Topics: Chemistry Techniques, Analytical; DNA; DNA, Catalytic; Fluorescence; Limit of Detection; Models, Biological; Zinc
PubMed: 22612395
DOI: 10.1021/ja3037838 -
Nucleic Acids Research Jun 1992Nonenzymatic ligation of double-stranded DNA has been performed using an alternate-strand binding oligodeoxyribonucleotide template to juxtapose the duplex termini in a...
Nonenzymatic ligation of double-stranded DNA has been performed using an alternate-strand binding oligodeoxyribonucleotide template to juxtapose the duplex termini in a triple helical complex. The template associates with the duplex termini by Hoogsteen hydrogen bonding to alternate strands on opposite sides of the ligation site. Intermolecular and intramolecular ligation of linearized plasmid DNA are observed in the reaction, which depends on the template oligodeoxyribonucleotide and a condensing agent, N-cyanoimidazole. Intramolecular ligation products include those in which both strands are covalently closed in a circle. Ligation of the two strands is sequential and occurs at comparable rates for the first and second strands ligating. The covalent linkages formed in the reaction can be cleaved by the restriction endonuclease Stu I, supporting their identification as phosphodiesters.
Topics: Base Sequence; DNA; Deoxyribonucleases, Type II Site-Specific; Hydrogen Bonding; Imidazoles; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Plasmids
PubMed: 1620596
DOI: 10.1093/nar/20.12.3005 -
Molecular and Cellular Biology Feb 1984An uninterrupted avian sarcoma viral genome terminated by viral long terminal repeat sequences was cloned into a pBR322 plasmid. After introduction into a cultured avian...
An uninterrupted avian sarcoma viral genome terminated by viral long terminal repeat sequences was cloned into a pBR322 plasmid. After introduction into a cultured avian cell, transcription of either the circular plasmid molecule or one linearized within the pBR322 sequences could initiate and terminate at long terminal repeat sequences, yielding full-sized viral RNA. A plasmid DNA molecule linearized by cleavage within the viral pol gene, on the other hand, would have to undergo ligation to yield full-sized viral RNA. Microinjection of each of these three types of DNA into the nuclei of quail cells promoted the release of similar virus titers, indicating that the plasmid DNA cleaved within the viral pol gene had been efficiently and accurately ligated. When plasmid DNA was transfected into quail cells, circular and pBR322-cleaved molecules directed the synthesis of similar virus titers, indicating that they were similarly taken up and utilized by the cells. Compared with these results, plasmid DNA cleaved within the pol gene was reduced in activity over 95% after transfection. This reduction did not result from inefficient ligation but from the generation of mutations (of limited size) during ligation of the transfected molecules. Mutations were not observed after microinjection even into the cytoplasm. Consistent with these findings, transfected DNA termini were found to be joined regardless of their structure, whereas ligation after microinjection required that single-stranded protruding DNA termini be complementary.
Topics: Animals; Avian Sarcoma Viruses; Cell Line; Cell Nucleus; Chick Embryo; Cytoplasm; DNA; DNA Ligases; DNA, Viral; Microinjections; Quail; Transfection
PubMed: 6321956
DOI: 10.1128/mcb.4.2.240-246.1984 -
Chembiochem : a European Journal of... Oct 2018The copper(I)-mediated azide-alkyne cycloaddition (CuAAC) of 3'-propargyl ether and 5'-azide oligonucleotides is a particularly promising ligation system because it...
The copper(I)-mediated azide-alkyne cycloaddition (CuAAC) of 3'-propargyl ether and 5'-azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate-sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA-templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k value of 1.1 min , comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3'-propargyl ether and 5'-azide-terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.
Topics: Alkynes; Azides; Click Chemistry; Copper; Cycloaddition Reaction; DNA; DNA Ligases; DNA Replication; Ethers; Kinetics; Oligonucleotides; Substrate Specificity
PubMed: 30059599
DOI: 10.1002/cbic.201800305 -
Bioorganic & Medicinal Chemistry Jan 2008DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive...
DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.
Topics: Base Sequence; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Molecular Probe Techniques; Peptide Nucleic Acids
PubMed: 17499998
DOI: 10.1016/j.bmc.2007.04.059 -
Nucleic Acids Research 2007The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities....
The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Ligase ATP; DNA Ligases; DNA Repair Enzymes; DNA, Single-Stranded; DNA-Binding Proteins; Humans; Magnesium
PubMed: 17717001
DOI: 10.1093/nar/gkm579 -
Plant Molecular Biology Jun 1990The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could...
The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.
Topics: Chloramphenicol O-Acetyltransferase; DNA; DNA, Circular; Escherichia coli; Gene Expression; Nucleic Acid Conformation; Plants; Plasmids; Protoplasts; Time Factors; Transfection
PubMed: 2102875
DOI: 10.1007/BF00019388 -
Methods in Molecular Biology (Clifton,... 2022Chemical ligation can be used to install encoding tags during the synthesis of DNA-encoded chemical libraries and can present a number of advantages. Here we describe...
Chemical ligation can be used to install encoding tags during the synthesis of DNA-encoded chemical libraries and can present a number of advantages. Here we describe methods to generate polymerase-readable oligonucleotide junctions and for the polymerase-mediated amplification of oligonucleotides ligated with these chemistries, including triazole junctions generated from 2'-ribo-3'-propargyl and 5'-azido oligonucleotides and from 2'-deoxy-3'-propargyl and 5'-azido oligonucleotides. We also present methods for the synthesis of phosphorothioate junctions from 3'-thiophospho and 5'-iodo oligonucleotides and for the synthesis of phosphodiester junctions from both 3'-hydroxy and 5'-phospho- and 3'-phospho and 5'-hydroxy oligonucleotides using 1-cyanoimidazole.
Topics: DNA; Gene Library; Oligonucleotides; Small Molecule Libraries
PubMed: 36083539
DOI: 10.1007/978-1-0716-2545-3_4