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Journal of the American Chemical Society Oct 2019Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has...
Engineered 3D DNA crystals are promising scaffolds for bottom-up construction of three-dimensional, macroscopic devices from the molecular level. Nevertheless, this has been hindered by the highly constrained conditions for DNA crystals to be stable. Here we report a method to prepare robust 3D DNA crystals by postassembly ligation to remove this constraint. Specifically, sticky ends at crystal contacts were enzymatically ligated, and the covalent bonds significantly enhanced crystal stability, e.g., being stable at 65 °C. This method also enabled the fabrication of DNA crystals with complex architectures including crystal shell, core-shell, and matryoshka dolls. Furthermore, we have demonstrated the applications of the robust DNA crystals in biocatalysis and protein entrapment. Our study removes one key obstacle for the applications of DNA crystals and offers many new opportunities in DNA nanotechnology.
Topics: Crystallization; DNA; DNA Ligases; Microscopy, Electron, Transmission; Nanotechnology; Nucleic Acid Conformation; Stress, Mechanical; X-Ray Diffraction
PubMed: 31553173
DOI: 10.1021/jacs.9b06613 -
Small Methods Jan 2024There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical...
There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical and biological environments. Here, two near quantitative ligation methods are demonstrated for the native backbone linkage at the nicks in origami: i) a cosolvent dimethyl sulfoxide (DMSO)-assisted enzymatic ligation and ii) enzyme-free chemical ligation by CNBr. Both methods achieved over 90% ligation in 2D origami, only CNBr-method resulted in ≈80% ligation in 3D origami, while the enzyme-alone yielded 31-55% (2D) or 22-36% (3D) ligation. Only CNBr-method worked efficiently for 3D origami. The CNBr-mediated reaction is completed within 5 min, while DMSO-method took overnight. Ligation by these methods improved the structural stability up to 30 °C, stability during the electrophoresis and subsequent extraction, and against nuclease and cell lysate. These methods are straightforward, non-tedious, and superior in terms of cost, reaction time, and efficiency.
Topics: Nanostructures; Dimethyl Sulfoxide; Nucleic Acid Conformation; DNA; Endonucleases
PubMed: 37736703
DOI: 10.1002/smtd.202300999 -
BMC Genomics Dec 2019Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression....
BACKGROUND
Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming.
RESULTS
Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding.
CONCLUSIONS
SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.
Topics: Cell-Free Nucleic Acids; DNA, Single-Stranded; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Oligonucleotides; Sequence Analysis, DNA
PubMed: 31881841
DOI: 10.1186/s12864-019-6355-0 -
Analytical and Bioanalytical Chemistry Jul 2014DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for...
DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.
Topics: DNA; DNA Ligases; Electrochemical Techniques; Enzyme Assays; Enzymes, Immobilized; Escherichia coli; Escherichia coli Proteins; Magnetics
PubMed: 24820061
DOI: 10.1007/s00216-014-7811-y -
Accounts of Chemical Research Aug 2012Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1... (Review)
Review
Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have potential therapeutic applications. They have extremely high thermodynamic stability, have increased resistance to enzymatic degradation, and have been investigated as decoys for regulatory proteins. For potential nanotechnology applications, we have synthesized double stranded DNA catenanes by click ligation. Other researchers have studied covalently fixed multistranded DNA constructs including triplexes and quadruplexes.
Topics: Biology; Click Chemistry; DNA; Humans; Nanotechnology; RNA; Transcription, Genetic
PubMed: 22439702
DOI: 10.1021/ar200321n -
BioTechniques Jan 2007DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of...
DNA ligation is a routine laboratory practice, yet the yield of the desired product is often very low due to competing off-pathway reactions. The sensitivity of subsequent manipulations (e.g., selection via bacterial transformation) often obviates the need for a high yield of correctly ligated products. However the ability to perform high-yield, preparative-scale DNA ligations would benefit a number of downstream applications ranging from standard molecular cloning to biophysics and DNA computing. We describe here a ligation technique that specifically converts off-pathway ligation products back into substrate. We term this second-chance strategy enzymatic ligation assisted by nucleases (ELAN) and demonstrate the ordered assembly of four DNA fragments via simultaneous ligation and digestion in the presence of eight restriction enzymes. Use of ELAN increased the yield of the desired product by more than 30-fold.
Topics: DNA; DNA Restriction Enzymes; Deoxyribonucleases; Genetic Techniques
PubMed: 17269489
DOI: 10.2144/000112283 -
Genomics May 1989A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is...
A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is described. The product is increased by either linear or exponential amplification using sequential rounds of template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product, and an additional round of ligation is performed. After n rounds there is a (1 + x) X n-fold amplification of product, where x is the efficiency of the ligation reaction. Exponential amplification utilizes two pairs of oligonucleotides, one complementary to the upper strand and one to the lower strand of a target sequence. The products of the ligation reaction serve as templates for subsequent rounds of ligation. In this case there is (1 + x)(n-1)-fold amplification of product after n rounds. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates. At high template concentrations, the ligation reaction has an efficiency approaching 100%. In this report, we demonstrate the use of the ligation amplification reaction (LAR) to distinguish the normal from the sickle cell allele of the human beta-globin gene. We also report the use of LAR as a detection system for polymerase chain reaction-enriched DNA sequences.
Topics: Anemia, Sickle Cell; Base Sequence; DNA; DNA Ligases; DNA Mutational Analysis; Gene Amplification; Globins; Hemoglobin, Sickle; Humans; Oligonucleotide Probes; Templates, Genetic
PubMed: 2744765
DOI: 10.1016/0888-7543(89)90280-2 -
Angewandte Chemie (International Ed. in... Apr 2023Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA...
Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA nanostructures. However, it is challenging to prepare such ssDNA circles, particularly when the DNA size becomes very small (e.g. a 20 nucleotide (nt) long ssDNA circle). Often, such short ssDNA dominantly form concatemers (either linear or circular) due to intermolecular ligation, instead of forming monomeric ssDNA circles by intramolecular ligation. Herein, a simple method to overcome this problem by designing the complementary linker molecules is reported. It is demonstrated that ssDNA, as short as 16 nts, can be enzymatically ligated (by the commonly used T4 DNA ligase) into monomeric ssDNA circles at high concentration (100 μM) with high yield (97 %). This method does not require any special sequence, thus, it is expected to be generally applicable. The experimental protocol is identical to regular DNA ligation, thus, is expected to be user friendly for general chemists and biologists.
Topics: DNA, Single-Stranded; DNA; Nucleotides; Nanostructures; DNA Ligases; Nucleic Acid Amplification Techniques; DNA, Circular
PubMed: 36652628
DOI: 10.1002/anie.202218443 -
Angewandte Chemie (International Ed. in... Apr 2014Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer...
Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA.1 Key requirements for potential diagnostic tools not based on PCR include a fast templated chemical DNA ligation method that exhibits high pairing selectivity, and a sensitive detection method. Here we report on a solid-phase synthesis of oligonucleotides containing 5'- or 3'-mercapto-dideoxynucleotides and their chemical ligations, yielding 3'-5'-disulfide bonds as a replacement for 3'-5'-phosphodiester units. Employing a system designed for fluorescence monitoring, we demonstrate one of the fastest ligation reactions with half-lives on the order of seconds. The nontemplated ligation reaction is efficiently suppressed by the choice of DNA modification and the 3'-5' orientation of the activation site. The influence of temperature on the templated reaction is shown.
Topics: DNA; Disulfides; Molecular Structure; Oligonucleotides
PubMed: 24623660
DOI: 10.1002/anie.201310644 -
Analytica Chimica Acta Aug 2023Pathogen identification requires nucleic acid diagnosis with simple equipment and fast manipulation. Our work established an all-in-one strategy assay with excellent...
Pathogen identification requires nucleic acid diagnosis with simple equipment and fast manipulation. Our work established an all-in-one strategy assay with excellent sensitivity and high specificity, Transcription-Amplified Cas14a1-Activated Signal Biosensor (TACAS), for the fluorescence-based bacterial RNA detection. The DNA as a promoter probe and a reporter probe directly ligated via SplintR ligase once specifically hybridized to the single-stranded target RNA sequence, with the ligation product transcribed into Cas14a1 RNA activators by T7 RNA polymerase. This forming sustained isothermal one-pot ligation-transcription cascade produced RNA activators constantly and enabled Cas14a1/sgRNA complex to generate fluorescence signal, thus leading to a sensitive detection limit of 1.52 CFU mLE. coli within 2 h of incubation time. TACAS was applied in contrived E. coli infected fish and milk samples, and a significant signal differentiation between positive (infected) and negative (uninfected) samples was reached. Meanwhile, E. coli colonization and transmit time in vivo were explored and the TACAS assay promoted the understanding of the infection mechanisms of the E. coli infection, demonstrating an excellent detection capability.
Topics: Animals; Escherichia coli; DNA; Biosensing Techniques; RNA, Bacterial
PubMed: 37328250
DOI: 10.1016/j.aca.2023.341470