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Analytical Biochemistry Feb 2006Ligation of an adapter oligonucleotide to a single-stranded cDNA is central to many molecular biology techniques. Current single-stranded ligation approaches suffer from...
Ligation of an adapter oligonucleotide to a single-stranded cDNA is central to many molecular biology techniques. Current single-stranded ligation approaches suffer from low efficiencies and are strongly inhibited by preexisting DNA secondary structure. We develop an approach for ligating low concentrations of single-stranded DNAs to a DNA adapter with near-quantitative efficiency, unaffected by secondary structure in the target DNA. This efficient DNA ligation reaction will facilitate development of robust procedures for quantifying small amounts of highly structured cDNAs and their RNA templates.
Topics: Base Sequence; DNA Primers; DNA, Single-Stranded; Nucleic Acid Conformation
PubMed: 16325753
DOI: 10.1016/j.ab.2005.11.002 -
Methods in Molecular Biology (Clifton,... 2022Libraries of DNA-encoded compounds (DELs) are a validated screening technology for drug discovery. Here we describe a library synthesis strategy that starts with a solid...
Libraries of DNA-encoded compounds (DELs) are a validated screening technology for drug discovery. Here we describe a library synthesis strategy that starts with a solid phase-bound, chemically very stable hexathymidine DNA sequence "hexT." Different heterocycle conjugates of the hexT oligonucleotide were synthesized from simple starting materials using metal or acid catalysts. The hexT conjugates were isolated, characterized, and ligated to coding DNA sequences.
Topics: Combinatorial Chemistry Techniques; DNA; Drug Discovery; Gene Library; Oligonucleotides; Small Molecule Libraries
PubMed: 36083549
DOI: 10.1007/978-1-0716-2545-3_14 -
Nature Protocols 2007This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique....
This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique. DNA fragments in ligation reactions are capable of combining to produce numerous products. The ELAN method uses judicious choice of restriction enzyme sites coupled with simultaneous digestion and ligation reactions to create just one product, by converting off-pathway products back into substrate. The experimental parameters critical for a successful ELAN reaction are discussed, and the ordered, one-tube assembly of four DNA fragments in the presence of eight restriction enzymes is demonstrated. This technique will be useful to those performing gene construction, DNA computing, biophysics and even standard molecular cloning. Starting with reactant fragments, the protocol takes 4-16 h to produce nanogram to microgram yields, depending on the complexity of the reaction.
Topics: DNA; DNA Ligases; DNA Restriction Enzymes; Genetic Techniques
PubMed: 17853876
DOI: 10.1038/nprot.2007.325 -
Nature Communications Feb 2024Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that...
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.
Topics: DNA Ligase ATP; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA Ligases; DNA
PubMed: 38341432
DOI: 10.1038/s41467-024-45553-z -
Bioconjugate Chemistry Oct 2012We report the preparation and use of an N-methyl picolinium carbamate protecting group for applications in a phototriggered nonenzymatic DNA phosphoramidate ligation...
We report the preparation and use of an N-methyl picolinium carbamate protecting group for applications in a phototriggered nonenzymatic DNA phosphoramidate ligation reaction. Selective 5'-amino protection of a modified 13-mer oligonucleotide is achieved in aqueous solution by reaction with an N-methyl-4-picolinium carbonyl imidazole triflate protecting group precursor. Deprotection is carried out by photoinduced electron transfer from Ru(bpy)(3)(2+) using visible light photolysis and ascorbic acid as a sacrificial electron donor. Phototriggered 5'- amino oligonucleotide deprotection is used to initiate a nonenzymatic ligation of the 13-mer to an imidazole activated 3'-phospho-hairpin template to generate a ligated product with a phosphoramidate linkage. We demonstrate that this methodology offers a simple way to exert control over reaction initiation and rates in nonenzymatic DNA ligation for potential applications in the study of model protocellular systems and prebiotic nucleic acid synthesis.
Topics: Amides; Amines; Base Sequence; DNA; Imidazoles; Oligodeoxyribonucleotides; Phosphates; Phosphoric Acids; Photochemical Processes
PubMed: 22985338
DOI: 10.1021/bc300093y -
Current Biology : CB Jun 2021Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of...
Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a 'hybrid nick' with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA Ligases; DNA Replication; DNA-Directed DNA Polymerase; Escherichia coli; Genome; Multienzyme Complexes; Replication Origin; Yeasts
PubMed: 34102115
DOI: 10.1016/j.cub.2021.03.043 -
Chembiochem : a European Journal of... Dec 2021Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical...
Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.
Topics: DNA; Gene Silencing; HeLa Cells; Humans
PubMed: 34519401
DOI: 10.1002/cbic.202100312 -
Analytical Chemistry Apr 2018A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation...
A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3'-OH termini exposed outside. Then terminal deoxynucleotidyl transferase (TdT), a sequence-independent and template-free polymerase, would specifically catalyze the DNA polymerization along these 3'-OH termini on the MBs, forming poly(T) tails up to thousands of nucleotides long. Each poly(T) tail allows specific binding of numerous 6-carboxyfluorescein (FAM)-labeled poly(A)25 oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. By integrating the advantages of CCNAL-TEP for highly efficient signal amplification and robust MBs signal readout with powerful flow cytometer, high sensitivity is achieved and the detection limit of plant miRNA has been pushed down to a low level of 5 fM with high specificity to well discriminate even single-base difference between miRNA targets.
Topics: Arabidopsis; Click Chemistry; DNA; DNA Nucleotidylexotransferase; Flow Cytometry; Methylation; MicroRNAs; Polymerization
PubMed: 29600844
DOI: 10.1021/acs.analchem.8b00589 -
Reducing product inhibition in nucleic acid-templated ligation reactions: DNA-templated cycligation.Chembiochem : a European Journal of... Nov 2013Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups....
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.
Topics: Cyclization; DNA; Peptide Nucleic Acids; Templates, Genetic
PubMed: 24243697
DOI: 10.1002/cbic.201300516 -
The Journal of Biological Chemistry Jan 1994The capacity of eukaryotic topoisomerase I to catalyze intra- and intermolecular DNA strand transfer via a two-step cleavage/ligation reaction was investigated by use of...
The capacity of eukaryotic topoisomerase I to catalyze intra- and intermolecular DNA strand transfer via a two-step cleavage/ligation reaction was investigated by use of purified enzyme and defined DNA substrates. Topoisomerase I-mediated cleavage requires separate interaction with a duplex region encompassing the cleavage site (region A) and a duplex region located on the side holding the 5'-OH end generated by cleavage (region B). Cleaved topoisomerase I-DNA complexes containing enzyme covalently attached at internal and terminal positions were employed to characterize the intra- and intermolecular ligation reactions. Enzyme attached covalently at an internal position of a partially single-stranded DNA molecule is able to catalyze ligation of a complementary dinucleotide within region A in the absence of interaction with region B. Moreover, the dinucleotide confines the minimal DNA acceptor for intramolecular ligation. Topoisomerase I attached covalently to DNA at a terminal position can ligate the cleaved strand to heterologous duplex DNA regardless of sequence, whereas ligation does not proceed with single-stranded DNA. When these features are considered together with the observation that intermolecular ligation is inhibited by 1 M NaCl, it suggests that the reaction requires bipartite DNA interaction. A model is proposed that relates the bipartite DNA binding of eukaryotic topoisomerase I to the catalytic functions.
Topics: Base Sequence; Binding Sites; Catalysis; DNA; DNA Topoisomerases, Type I; Humans; Molecular Sequence Data; Nucleic Acid Conformation
PubMed: 8276874
DOI: No ID Found