Chemical Synthesis Of DNA Research Articles

Watson-Crick Base Pairs with Protecting Groups: The 2-Amino Groups of Purine- and 7-Deazapurine-2,6-Diamine as Target Sites for DNA Functionalization by Selective Nucleobase Acylation.

The recognition of Watson-Crick base pairs carrying nucleobase protecting groups is reported as a new approach for DNA functionalization. The 2-amino groups of purine- and 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides served as molecular targets for this functionalization. The 2-amino group withstands oligonucleotide deprotection with ammonia, whereas all other protecting groups are released after chemical DNA synthesis. On this basis, a method was developed for the selective functionalization of oligonucleotides at the 2-position of purines and 7-deazapurines. Melting experiments and Tm values obtained from hybridization studies revealed that duplexes with protected (2-amino-dA) and (2-amino-7-deaza-dA)-dT base pairs are as stable as their nonprotected counterparts. Mismatch discrimination of protected purine- and 7-deazapurine-2,6-diamine DNA was superior to that of nonprotected DNA. Click functionalization in the minor groove of the DNA double helix became accessible via introduction of heptynoyl protecting groups bearing a terminal triple bond. Click reactions with pyrene azide validated the usability. DNA conjugates with bulky pyrene residues at the 2-position (minor groove) developed the same high stability as those functionalized at the 7-position (major groove). This demonstrates the potential of our new method using protected base pairs for DNA functionalization and paves the way for new DNA labeling strategies.

Assessment of enzymatically synthesized DNA for gene assembly.

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

Quantification of synthetic errors during chemical synthesis of DNA and its suppression by non-canonical nucleosides

Substitutions, insertions, and deletions derived from synthetic oligonucleotides are the hurdles for the synthesis of long DNA such as genomes. We quantified these synthetic errors by next-generation sequencing and revealed that the quality of the enzymatically amplified final combined product depends on the conditions of the preceding solid phase chemical synthesis, which generates the initial pre-amplified fragments. Among all possible substitutions, the G-to-A substitution was the most prominently observed substitution followed by G-to-T, C-to-T, T-to-C, and A-to-G substitutions. The observed error rate for G-to-A substitution was influenced by capping conditions, suggesting that the capping step played a major role in the generation of G-to-A substitution. Because substitutions observed in long DNA were derived from the generation of non-canonical nucleosides during chemical synthesis, non-canonical nucleosides resistant to side reactions could be used as error-proof nucleosides. As an example of such error-proof nucleosides, we evaluated 7-deaza-2´-deoxyguanosine and 8-aza-7-deaza-2´-deoxyguanosine and showed 50-fold decrease in the error rate of G-to-A substitution when phenoxyacetic anhydride was used as capping reagents. This result is the first example that improves the quality of synthesized sequences by using non-canonical nucleosides as error-proof nucleosides. Our results would contribute to the development of highly accurate template DNA synthesis technologies.

Enzymatic DNA synthesis gets a significant new player

The synthetic DNA maker Twist Bioscience announced at the J.P. Morgan Health Care Conference earlier this month that it is developing enzymatic DNA synthesis technology. The revelation—by a major player in chemical DNA synthesis—offers validation that the new technology is here to stay. “It doesn’t matter if chemical synthesis is better or enzymatic synthesis is better,” Twist CEO Emily Leproust said at the conference. “We have it, and we can continue to dominate the DNA synthesis market.” Twist shook up DNA synthesis about 7 years ago, when it introduced a method of conducting phosphoramidite chemistry on silicon chips rather than in traditional well plates. More recently, a group of start-ups has been trying to improve on the decades-old chemical process by using enzymes to make DNA. Those technologies are still in the earliest stages of commercialization. Companies working on synthetic biology, gene sequencing, pharmaceuticals, and next-generation data storage all need

Designer DNA biomolecules as a defined biomaterial for 3D bioprinting applications.

DNA has excellent features such as the presence of functional and targeted molecular recognition motifs, tailorability, multifunctionality, high-precision molecular self-assembly, hydrophilicity, and outstanding biocompatibility. Due to these remarkable features, DNA has emerged as a leading next-generation biomaterial of choice to make hydrogels by self-assembly. In recent times, novel routes for the chemical synthesis of DNA, advances in tailorable designs, and affordable production ways have made DNA as a building block material for various applications. These advanced features have made researchers continuously explore the interesting properties of pure and hybrid DNA for 3D bioprinting and other biomedical applications. This review article highlights the topical advancements in the use of DNA as an ideal bioink for the bioprinting of cell-laden three-dimensional tissue constructs for regenerative medicine applications. Various bioprinting techniques and emerging design approaches such as self-assembly, nucleotide sequence, enzymes, and production cost to use DNA as a bioink for bioprinting applications are described. In addition, various types and properties of DNA hydrogels such as stimuli responsiveness and mechanical properties are discussed. Further, recent progress in the applications of DNA in 3D bioprinting are emphasized. Finally, the current challenges and future perspectives of DNA hydrogels in 3D bioprinting and other biomedical applications are discussed.

Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity.

Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (SynViP). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See'm principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering.

Production of recombinant IGF1 and its action on neuroblastoma cells in vitro

This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein.
 To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography.
 The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons.
 Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

C-5 Propynyl Modifications Enhance the Mechanical Stability of DNA.

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or -medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base-pairing and easy integration during chemical DNA synthesis. Through single-molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C-5 propynyl modifications on the mechanical stability of double-stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non-modified DNA duplexes.

RNA dances to center stage.

RNA dances to center stage.

Template-directed chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages.

Up to now, the direct ligation of two DNA fragments with opposite directions to obtain 3′-3′ or 5′-5′ phosphate ester bonds is still challenging. The only way to obtain DNA oligonucleotides containing a 3′-3′ or 5′-5′ inversion of polarity sites is based on professional DNA chemical synthesis. Herein, we demonstrate a convenient template-directed chemical ligation that enables 3′-3′ and 5′-5′ linkages of two DNA oligonucleotides. This method is based on the assembly of two oligonucleotides on a template in opposite directions through forming antiparallel and parallel duplexes simultaneously, followed by coupling with N-Cyanoimidazole under mild condition. Moreover, on the basis of DNA oligonucleotides with 5′-5′ linkage obtained through our template-directed chemical ligation, we developed a new cDNA display technique for in vitro selection of functional polypeptides.

Synthesis of DNA/RNA and Their Analogs via Phosphoramidite and H-Phosphonate Chemistries

The chemical synthesis of DNA and RNA is universally carried out using nucleoside phosphoramidites or H-phosphonates as synthons. This review focuses on the phosphorus chemistry behind these synthons and how it has been developed to generate procedures whereby yields per condensation approach 100% with very few side products. Additionally the synthesis and properties of certain DNA and RNA analogs that are modified at phosphorus will also be discussed. These analogs include boranephosphonates, metallophosphonates, and alkylboranephosphines.

Inextricably bound : measurement and the bioeconomy

Synthetic biology has been described as the design and construction of biological devices and systems for useful purposes [1]. The synthesis of DNA is a critical part of this construction. Advanced measurements have been both enabling and motivating for advances in DNA synthesis chemistry. Building on decades of development of chemical synthesis of DNA [2] and the development of DNA microarrays [3], additional careful attention to minimizing rare side reactions and very small non-idealities in reaction yields has enabled unprecedented levels of synthesis perfection and throughput [4]. The industrialization of this advanced chemistry has been shown to serve as a robust and economical basis for highly sensitive and specific hybridization assays [5]. It has also been shown to serve as a robust and economical source of user defined DNA oligonucleotides of sufficient quality to be used for synthetic biology [6]. The availability of high quality DNA oligonucleotides, coupled with analogously industrialized processes for combining them into larger constructs, opens up the possibility of widespread adoption of synthetic biology methods. New measurement modalities are being developed as a consequence. These examples, along with others elaborated elsewhere in this journal, illustrate the close and sometimes unpredictable interplay amongst measurement, science, and biotechnology, and the foundational role of measurement in advancing the bio-economy.

Chemical Synthesis of DNA, RNA, and their Analogues

Chemical Synthesis of DNA, RNA, and their Analogues

Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine †

8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2′-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson–Crick pair.

Cloning Large Gene Clusters from E. coli Using in Vitro Single-Strand Overlapping Annealing

Despite recent advances in genomic sequencing and DNA chemical synthesis, construction of large gene clusters containing DNA fragments is still a difficult and expensive task. To tackle this problem, we developed a gene cluster extraction method based on in vitro single-strand overlapping annealing (SSOA). It starts with digesting the target gene cluster in an existing genome, followed by recovering digested chromosome fragments. Subsequently, the single-strand DNA overhangs formed from the digestion process would be specifically annealed and covalently joined together with a circular and a linear vector, respectively. The SSOA method was successfully applied to clone a 18 kb DNA fragment encoding NADH:ubiquinone oxidoreductase. Genomic DNA fragments of different sizes including 11.86, 18.33, 28.67, 34.56, and 55.99 kb were used to test the cloning efficiency. Combined with genetic information from KEGG and the KEIO strain collection, this method will be useful to clone any specific region of an E. coli genome at sizes less than ~28 kb. The method provides a cost-effective way for genome assembly, alternative to chemically synthesized gene clusters.

Foundations of organic chemistry: unity and diversity of structures, pathways, and reactions

Foundations of organic chemistry: unity and diversity of structures, pathways, and reactions

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

BackgroundRestriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective.ResultsAn enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb).ConclusionsThe long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

Streamlined Process for the Chemical Synthesis of RNA Using 2′-O-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase

An improved method for the chemical synthesis of RNA was developed utilizing a streamlined method for the preparation of phosphoramidite monomers and a single-step deprotection of the resulting oligoribonucleotide product using 1,2-diamines under anhydrous conditions. The process is compatible with most standard heterobase protection and employs a 2'-O-(1,1-dioxo-1λ(6)-thiomorpholine-4-carbothioate) as a unique 2'-hydroxyl protective group. Using this approach, it was demonstrated that the chemical synthesis of RNA can be as simple and robust as the chemical synthesis of DNA.

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.