Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures.
ABSTRACT: Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA that is compatible with polymerase enzymes.
Project description:We developed an efficient one-pot metal-free click polymerization procedure for the synthesis of 3,5-disubstituted polypyrazoles with high yields, high molecular weights, and narrow molecular weight distribution. The method involved two click reactions in a one-pot synthesis. The first reaction was the carbonyl chemistry of "non-aldol" type (condensation reaction of aldehydes with p-toluenesulfonylhydrazide), and the second was a click polymerization reaction of diazo compounds with alkynes. The reactions occurred sequentially by adding the reactants step by step. The diazo compound needed for the second click reaction was generated in situ by the first click reaction. The structures of the polypyrazoles were characterized by IR and 1H NMR analyses. And their thermal properties and solubility were also tested.
Project description:In this study, a simple one-pot method was proposed to synthesize water-soluble nucleoside-templated fluorescent silver nanoparticles (Ag NPs) and gold nanoparticles (Au NPs). The nucleoside-templated fluorescent Ag NPs and Au NPs were further characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and fluorescence spectroscopy (FLS). The effects of the molar ratio of reactants, reaction environment, and nucleotides on the synthesis of Ag NPs and Au NPs were also discussed. The results showed that nucleoside and ascorbic acid acted as a stabilizer and reductant, respectively, in the synthesis of Ag NPs and Au NPs, while citrate buffer acted as both a pH regulator and reductant. The synthesized nucleoside-templated fluorescent Ag NPs and Au NPs have good fluorescence stability and easy water solubility. In this study, a simple one-pot method was proposed to synthesize water-soluble nucleoside-templated fluorescent silver nanoparticles (Ag NPs) and gold nanoparticles (Au NPs).
Project description:The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards 'click' neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide-alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our 'click' neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.
Project description:A one-pot synthesis of triazolobenzodiazepine-containing polycyclic compounds is introduced. The reaction process involves a decarboxylative three-component [3 + 2] cycloaddition of nonstabilized azomethine ylides, N-propargylation, and intramolecular click reactions.
Project description:The construction of two libraries of triazole-containing isothiazolidine 1,1-dioxides is reported utilizing either a one-pot click/aza-Michael or click/OACC esterification protocol. One core dihydroisothiazole 1,1-dioxide scaffold was prepared rapidly on multigram scale via ring-closing metathesis (RCM) and was subjected to a one-pot multicomponent click/aza-Michael protocol with an array of amines and azides for the generation of a 180-member triazole-containing isothiazolidine 1,1-dioxide library. Alternatively, three daughter scaffolds were generated via the aza-Michael of three amino alcohols, followed by a one-pot, multicomponent click/esterification protocol utilizing a ring-opening metathesis polymerization (ROMP)-derived coupling reagent, oligomeric alkyl carbodiimide (OACC) to generate a 41-member library of triazole-containing isothiazole 1,1-dioxides.
Project description: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.
Project description:A facile, one-pot, and proficient method was developed for the production of various 2-arylaminobenzimidazoles. This methodology is based for the first time on a copper catalyst promoted domino C-N cross-coupling reaction for the generation of 2-arylaminobenzimidazoles. Mechanistic investigations revealed that the synthetic pathway involves a copper-based desulphurization/nucleophilic substitution and a subsequent domino intra and intermolecular C-N cross-coupling reactions. Some of the issues typically encountered during the synthesis of 2-arylaminobezimidazoles, including the use of expensive catalytic systems and the low reactivity of bromo precursors, were addressed using this newly developed copper-catalyzed method. The reaction procedure is simple, generally with excellent substrate tolerance, and provides good to high yields of the desired products.
Project description:We present a simple method called "ClickSeq" for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2',3'-dideoxynucleotides that randomly terminate DNA synthesis and release 3'-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are "click ligated" via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5'-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.
Project description:The one-pot synthesis of a target molecule in the same reaction vessel is widely considered to be an efficient approach in synthetic organic chemistry. In this review, the characteristics and limitations of various one-pot syntheses of biologically active molecules are explained, primarily involving organocatalytic methods as key tactics. Besides catalysis, the pot-economy concepts presented herein are also applicable to organometallic and organic reaction methods in general.