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Biotechnology Journal Mar 2024DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on...
DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.
Topics: DNA Ligases; Ligases; DNA; Base Sequence; MicroRNAs
PubMed: 38528369
DOI: 10.1002/biot.202300711 -
ACS Nano May 2019DNA-mediated self-assembly of nanoparticles has been of great interest because it enables access to nanoparticle superstructures that cannot be synthesized otherwise....
DNA-mediated self-assembly of nanoparticles has been of great interest because it enables access to nanoparticle superstructures that cannot be synthesized otherwise. However, the programmability of higher order nanoparticle structures can be easily lost under DNA denaturing conditions. Here, we demonstrate that light can be employed as an external stimulus to master the stability of nanoparticle superlattices (SLs) via the promotion of a reversible photoligation of DNA in SLs. The oligonucleotides attached to the nanoparticles are encoded to ligate using 365 nm light, effectively locking the SLs and rendering them stable under DNA denaturing conditions. The reversible process of unlocking these structures is possible by irradiation with light at 315 nm, recovering the structures to their natural state. Our work inspires an alternative research direction toward postassembly manipulation of nanoparticle superstructures using external stimuli as a tool to enrich the library of additional material forms and their application in different media and environments.
Topics: DNA; Gold; Metal Nanoparticles; Microscopy, Electron, Transmission; Nanotechnology; Oligonucleotides
PubMed: 30958671
DOI: 10.1021/acsnano.9b01294 -
Bio Systems Oct 1999DNA computing is a novel method of computing proposed by Adleman (1994), in which the data is encoded in the sequences of oligonucleotides. Massively parallel reactions...
DNA computing is a novel method of computing proposed by Adleman (1994), in which the data is encoded in the sequences of oligonucleotides. Massively parallel reactions between oligonucleotides are expected to make it possible to solve huge problems. In this study, reliability of the ligation process employed in the DNA computing is tested by estimating the error rate at which wrong oligonucleotides are ligated. Ligation of wrong oligonucleotides would result in a wrong answer in the DNA computing. The dependence of the error rate on the number of mismatches between oligonucleotides and on the combination of bases is investigated.
Topics: Animals; Computational Biology; Computer Simulation; DNA; Humans; Oligonucleotides
PubMed: 10636043
DOI: 10.1016/s0303-2647(99)00045-3 -
Bioconjugate Chemistry Jan 2008Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA...
Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.
Topics: Base Pair Mismatch; Base Sequence; DNA; Hot Temperature; Iodoacetates; Nucleic Acid Amplification Techniques; Nucleic Acid Denaturation; Organothiophosphorus Compounds; RNA
PubMed: 17990846
DOI: 10.1021/bc700244s -
Chembiochem : a European Journal of... May 2023DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects...
Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation.
DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent "RPA-Spy-UNG2" complex could identify and excise uracil bases in duplex areas next to ssDNA-dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA-Spy-UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections. Conversely, the enzymes preferred uracil sites in ssDNA where RPA strongly enhanced uracil excision by UNG2 regardless of ssDNA length. Finally, RPA was found to promote UNG2 excision of two uracil sites positioned across a ssDNA-dsDNA junction, and dissociation of UNG2 from RPA enhanced this process. Our approach of ligating together RPA and UNG2 to reveal how complex formation affects enzyme function could be applied to examine other assemblies of DNA repair proteins.
Topics: Humans; DNA; DNA Repair; DNA Replication; DNA, Single-Stranded; Kinetics; Replication Protein A; Uracil; Uracil-DNA Glycosidase
PubMed: 36883884
DOI: 10.1002/cbic.202200765 -
Mikrochimica Acta Nov 2023Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive...
Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 μU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.
Topics: Humans; CRISPR-Cas Systems; DNA Glycosylases; DNA; Guanine
PubMed: 37968435
DOI: 10.1007/s00604-023-06050-0 -
PloS One 2013Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of...
Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.
Topics: Artifacts; DNA Damage; DNA, Bacterial; Gene Library; Genomics; High-Throughput Nucleotide Sequencing
PubMed: 24205269
DOI: 10.1371/journal.pone.0078575 -
DNA Repair Oct 2023DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the... (Review)
Review
DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.
Topics: Animals; DNA End-Joining Repair; DNA; DNA-Binding Proteins; DNA Breaks, Double-Stranded; Chromosome Pairing; DNA Repair
PubMed: 37572577
DOI: 10.1016/j.dnarep.2023.103553 -
Accounts of Chemical Research Apr 2017Bioresponsive hydrogels can respond to various biological stimuli by a macroscopic change of physical state or by converting biochemical inputs into biological or... (Review)
Review
Bioresponsive hydrogels can respond to various biological stimuli by a macroscopic change of physical state or by converting biochemical inputs into biological or mechanical outputs. These materials are playing an increasingly important role in a wide variety of applications, especially in the biological and biomedical fields. However, the design and engineering of intriguing bioresponsive materials with adequate biocompatibility and biodegradability have proven to be a great challenge. DNA, on the other hand, possesses many unique and fascinating properties, including its indispensable genetic function, broad biocompatibility, precise molecular recognition capability, tunable multifunctionality, and convenient programmability. Therefore, DNA has provided crucial prerequisites for the exploration of novel bioresponsive hydrogels and has since become an ideal building block for the construction of novel materials. In this Account, we describe our efforts over more than a decade to develop DNA-based materials including bioresponsive hydrogels. These DNA hydrogels were created through either chemical cross-linking or physical entanglement among DNA chains. We further divided them into two categories: pure DNA-based and hybrid DNA-based hydrogels. For the pure DNA-based hydrogels, we developed the first bulk DNA hydrogel entirely from branched DNA by using enzymatic ligation. Certain drugs were encapsulated in such hydrogels in situ and released in a controllable manner under the stimulation of environmental factors such as nucleases and/or changes in ionic strength. Furthermore, we prepared a protein-producing hydrogel (termed a "P-gel") by ligating X-shaped DNA (X-DNA) and linear plasmids. Following the central dogma of molecular biology, this hydrogel responded to enzymes and substrate and converted them into proteins. This was the first example showing that a hydrogel could be employed to produce proteins without the involvement of live cells. This might also be the first attempt to create cell-like hydrogels that will be ultimately bioresponsive. In addition, we also constructed a DNA physical hydrogel via entanglement of DNA chains elongated by a special polymerase: Phi29. This hydrogel (termed a "meta-hydrogel") exhibited a "meta" property: freely reversible change between liquidlike and solidlike states through stimulation by water molecules. Besides these pure DNA-based hydrogels, we also created a hybrid DNA-based hydrogel: a DNA-clay hybrid hydrogel utilizing electrostatic interactions between DNA and clay nanocrystals. We discovered a synergistic responsiveness in biochemical reaction in this hydrogel, suggesting that a DNA-clay hydrogel might be the environment for the origination of life and that DNA and clay might have been coevolving during early evolution. In summary, DNA links the nonbiological world with biological processes by virtue of its bioresponsiveness. We envision that bioresponsive DNA hydrogels will play an irreplaceable part in the development of future evolvable materials such as soft robots and artificial cells.
Topics: Biocompatible Materials; DNA; Hydrogels
PubMed: 28186723
DOI: 10.1021/acs.accounts.6b00581 -
DNA Research : An International Journal... Feb 2019Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various genomic tools have been developed using the...
Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various genomic tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to insert 5' phosphorylated blunt-end double-stranded DNA to DNA breaks at 3'-recessive ends, gaps, or nicks to form a Y-shaped 3'-branch structure. Therefore, this base pairing-independent ligation is termed 3'-branch ligation (3'BL). In an extensive study of optimal ligation conditions, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency to more than 80%. Ligation efficiency was slightly varied between different donor and acceptor sequences. More interestingly, we discovered that T4 DNA ligase efficiently ligated DNA to the 3'-recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, suggesting a ternary complex formation preference of T4 DNA ligase. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important genomic applications, such as 3'-end labelling by adding a universal sequence; directional tagmentation for NGS library construction that achieve theoretical 100% template usage; and targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.
Topics: DNA; DNA Ligases; Genetic Engineering; High-Throughput Nucleotide Sequencing; Humans; RNA
PubMed: 30428014
DOI: 10.1093/dnares/dsy037