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ACS Synthetic Biology Sep 2023Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but...
Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.
Topics: High-Throughput Nucleotide Sequencing; DNA
PubMed: 37582217
DOI: 10.1021/acssynbio.3c00183 -
Nucleic Acids Research Sep 2000DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB...
DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA-3'-phosphotyrosyl-enzyme adduct and a free 5'-hydroxyl (5'-OH). Strand ligation results when the 5'-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3'-phospho-(para-nitrophenyl) oligonucleotides (3'-pNP DNAs), which mimic the natural 3'-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5'-OH strand on the 3'-pNP DNA (i.e., without a covalent protein-DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.
Topics: Arginine; Base Sequence; Binding Sites; Catalysis; DNA; DNA Topoisomerases, Type I; Esters; Integrases; Molecular Mimicry; Nitrophenols; Oligodeoxyribonucleotides; Organophosphorus Compounds; Substrate Specificity; Tyrosine; Vaccinia virus; Viral Proteins
PubMed: 10954601
DOI: 10.1093/nar/28.17.3323 -
Journal of the American Chemical Society Feb 2023Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising...
Functional nucleic acids (FNAs), such as DNAzymes and DNA aptamers, can be engineered into circular forms for improved performance. Circular FNAs are promising candidates for bioanalytical and biomedical applications due to their intriguing properties of enhanced biological stability and compatibility with rolling circle amplification. They are typically made from linear single-stranded (ss) DNA molecules via ligase-mediated ligation. However, it remains a great challenge to synthesize circular ssDNA molecules in high yield due to inherent side reactions where two or more of the same ssDNA molecules are ligated. Herein, we present a strategy to overcome this issue by first using in vitro selection to search from a random-sequence DNA library a ligatable DNA aptamer that binds a DNA ligase and then by engineering this aptamer into a general-purpose templating DNA scaffold to guide the ligase to execute selective intramolecular circularization. We demonstrate the broad utility of this approach via the creation of several species of circular DNA molecules, including a circular DNAzyme sensor for a bacterium and a circular DNA aptamer sensor for a protein target with excellent detection sensitivity and specificity.
Topics: DNA, Catalytic; Aptamers, Nucleotide; Ligases; Nucleic Acid Amplification Techniques; DNA; DNA, Circular; DNA, Single-Stranded; Biosensing Techniques
PubMed: 36657012
DOI: 10.1021/jacs.2c12666 -
Molecules (Basel, Switzerland) Aug 2020Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of...
Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5'-adenylated RNA (5'-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3'-5'-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5'-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.
Topics: Base Sequence; Catalysis; DNA; DNA, Catalytic; Kinetics; RNA; Substrate Specificity
PubMed: 32796587
DOI: 10.3390/molecules25163650 -
BioTechniques Sep 2017Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC...
Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.
Topics: Cloning, Molecular; DNA, Recombinant; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Genetic Vectors; Sequence Analysis, DNA; Temperature; Time Factors; Viral Proteins
PubMed: 28911316
DOI: 10.2144/000114588 -
Scientific Reports Feb 2020Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the...
Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and flexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 different adapters at low cost. Using this method, the flanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efficient and flexible method for identifying the flanking sequences in transgenic rice and Arabidopsis thaliana.
Topics: Arabidopsis; DNA, Bacterial; Mutagenesis, Insertional; Oryza; Plants, Genetically Modified; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 32103092
DOI: 10.1038/s41598-020-60411-w -
Scientific Reports Sep 2023DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this...
DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this work, we propose composite motifs, a framework that uses a mixture of prefabricated motifs as building blocks to reduce synthesis cost by scaling logical density. To write data, we introduce Bridge Oligonucleotide Assembly, an enzymatic ligation technique for synthesizing oligos based on composite motifs. To sequence data, we introduce Direct Oligonucleotide Sequencing, a nanopore-based technique to sequence short oligos, eliminating common preparatory steps like DNA assembly, amplification and end-prep. To decode data, we introduce Motif-Search, a novel consensus caller that provides accurate reconstruction despite synthesis and sequencing errors. Using the proposed methods, we present an end-to-end experiment where we store the text "HelloWorld" at a logical density of 84 bits/cycle (14-42× improvement over state-of-the-art).
Topics: DNA; Oligonucleotides; Consensus; Nanopores; Nutritional Status
PubMed: 37749195
DOI: 10.1038/s41598-023-43172-0 -
Nucleic Acids Research May 2017As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in...
As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.
Topics: CRISPR-Associated Proteins; DNA; Francisella; Taq Polymerase
PubMed: 28115632
DOI: 10.1093/nar/gkx018 -
Nature Communications Jul 2022DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) β during downstream steps of the base excision...
DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) β during downstream steps of the base excision repair (BER) pathway. However, how LIG1 discriminates against the mutagenic 3'-mismatches incorporated by polβ at atomic resolution remains undefined. Here, we determine the X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncover the ligase strategies that favor or deter the ligation of base substitution errors. Our structures reveal that the LIG1 active site can accommodate a G:T mismatch in the wobble conformation, where an adenylate (AMP) is transferred to the 5'-phosphate of a nick (DNA-AMP), while it stays in the LIG1-AMP intermediate during the initial step of the ligation reaction in the presence of an A:C mismatch at the 3'-strand. Moreover, we show mutagenic ligation and aberrant nick sealing of dG:T and dA:C mismatches, respectively. Finally, we demonstrate that AP-endonuclease 1 (APE1), as a compensatory proofreading enzyme, removes the mismatched bases and interacts with LIG1 at the final BER steps. Our overall findings provide the features of accurate versus mutagenic outcomes coordinated by a multiprotein complex including polβ, LIG1, and APE1 to maintain efficient repair.
Topics: Adenosine Monophosphate; DNA; DNA Repair; Mutagenesis; Mutagens
PubMed: 35790757
DOI: 10.1038/s41467-022-31585-w -
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