Synthesis Of DNA Oligonucleotides Research Articles

Silver(I)-mediated base pairing involving an S-glycosidic GNA nucleoside analogue

The 4S–Ag(I)–C base pair (4S, 3-((2-(methylthio)pyrimidin-4-yl)thio)propane-1,2-diol; C, deoxycytidine) represents the first metal-mediated base pair comprising an S-glycosidic nucleoside analogue. We report here the synthesis of the phosphoramidite suitable for the automated solid-phase synthesis of DNA oligonucleotides containing 4S and its silver(I)-binding ability. The DNA duplexes comprising a 4S:C mispair exhibit a large thermal stabilization upon the addition of one equivalent of silver ions, giving rise to the formation of the above-mentioned silver(I)-mediated base pair. By formally replacing the sulfur atom in the glycosidic bond by an oxygen atom, i.e., by applying 3-((2-(methylthio)pyrimidin-4-yl)oxy)propane-1,2-diol (4 O) as the artificial nucleoside analogue, the participation of this atom as a donor atom in silver(I)-mediated base pairing is shown to be neglectable. Supplemental data for this article is available online at

Synthesis of DNA and RNA Oligonucleotides Containing a Dual-Purpose Selenium-Modified Fluorescent Nucleoside Probe.

Development of efficient tools that would enable direct correlation of nucleic acid structure and recognition in solution and in solid state at atomic resolution is highly desired. In this context, we recently developed dual-purpose nucleoside probes made of a 5-selenophene-modified uracil core, which serves both as a conformation-sensitive fluorophore and as an X-ray crystallography phasing agent. In this article, we provide a detailed synthetic procedure to synthesize the phosphoramidites of 5-selenophene-modified 2'-deoxyuridine and 5-selenophene-modified uridine analogs. We also describe their site-specific incorporation into therapeutically relevant DNA and RNA oligonucleotide motifs by an automated solid support synthesis protocol. The dual-purpose and minimally invasive nature of the probes enables efficient analysis of the conformation and ligand binding abilities of bacterial decoding site RNA (A-site) and G-quadruplex structures of the human telomeric overhang in real time by fluorescence and in 3D by X-ray crystallography. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 5-selenophene-2'-deoxyuridine 2 and its phosphoramidite 5 Support Protocol 1: Synthesis of 2-(tri-n-butylstannyl) selenophene Support Protocol 2: Synthesis of 5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 Basic Protocol 2: Synthesis of 5-selenophene-modified uridine 7 and its phosphoramidite 11 Basic Protocol 3: Synthesis of DNA oligonucleotides containing 5-selenophene-modified 2'-deoxyuridine 2 Basic Protocol 4: Synthesis of an RNA oligonucleotide containing 5-selenophene-modified uridine 7.

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid·ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of β-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology.

The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read-write cost gap.

Analytical Devices Based on Direct Synthesis of DNA on Paper.

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

Synthesis of DNA oligonucleotides containing C5-ethynylbenzenesulfonamide-modified nucleotides (EBNA) by polymerases towards the construction of base functionalized nucleic acids

C5-Ethynylbenzenesulfonamide-modified nucleotide (EBNA) was investigated as substrate of various DNA polymerases. The experiments revealed that KOD, Phusion and Klenow DNA polymerases successfully accepted EBNA-T nucleotide as a substrate and yielded the fully extended DNA. KOD DNA polymerase was found to be the most efficient enzyme to furnish EBNA-T containing DNA in good yields. Phusion DNA polymerase efficiently amplified the template containing EBNA-T nucleotides by PCR.

ChemInform Abstract: Integrated Microfluidic Reactors

AbstractReview: 109 refs.

Parallel on-chip gene synthesis and application to optimization of protein expression

Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.

Optical tweezers directed one-bead one-sequence synthesis of oligonucleotides

An optical tweezers directed parallel DNA oligonucleotide synthesis methodology is described in which controlled pore glass (CPG) beads act as solid substrates in a two-stream microfluidic reactor. The reactor contains two parallel sets of physical confinement features that retain beads in the reagent stream for synthetic reaction but allow the beads to be optically trapped and transferred between the reagent and the inert streams for sequence programming. As a demonstration, we synthesized oligonucleotides of target sequence 25-nt, one deletion and one substitution using dimethoxytrityl (DMT) nucleoside phosphoramidite chemistry. In detecting single-nucleotide mismatches, fluorescence in situ hybridization of the bead-conjugated probes showed high specificity and signal-to-noise ratios. These preliminary results suggest further possibilities of creating a novel type of versatile, sensitive and multifunctional reconfigurable one-bead one-compound (OBOC) bead array.

Synthesis of DNA oligonucleotides in mesoporous silicon

AbstractIn‐situ oligonucleotide synthesis through an 8‐base sequence is demonstrated within 30 nm silicon mesopores utilizing sequential phosphoramidite reactions. Stepwise attachment of each base is confirmed by monitoring the porous silicon effective refractive index via a waveguide resonance angle. A linear increase in the effective refractive index is observed after the addition of each base, and the detection of oligonucleotides containing only a single thymine is possible. It is estimated that 50% of the available internal pore surface is occupied by the 8‐base oligonucleotides, which is a substantially greater coverage than obtained by using traditional methods of direct attachment of presynthesized DNA oligonucleotides. The greater surface coverage by DNA probes will enable more effective biosensing devices. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Thermolytic Release of Covalently Linked DNA Oligonucleotides and Their Conjugates from Controlled-Pore Glass at Near Neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.

Synthesis of Poly d(G-C) Oligonucleotides

Model sequences for evaluation of the GC dimer sequence repetition on synthesis success were prepared and analyzed by HPLC. Contiguous d(G-C) or d(C-G) sequences have a deleterious effect on DNA oligonucleotide synthesis. The critical number seems to be about 6 GCs in a row. If the GCs are separated by other nucleotides, the effect is not as severe.

Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2′-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2′-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.

Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides.

The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.

Conceptual "Heat-Driven" approach to the synthesis of DNA oligonucleotides on microarrays.

The discovery of deoxyribonucleoside cyclic N-acylphosphoramidites, a novel class of phosphoramidite monomers for solid-phase oligonucleotide synthesis, has led to the development of a number of phosphate protecting groups that can be cleaved from DNA oligonucleotides under thermolytic neutral conditions. These include the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, 3-(N-tert-butyl)carboxamido-1-propyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, and 4-methythiobutyl groups. When used for 5'-hydroxyl protection of nucleosides, the analogous 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group exhibited excellent thermolytic properties, which may permit an iterative "heat-driven" synthesis of DNA oligonucleotides on microarrays. In this regard, progress has been made toward the use of deoxyribonucleoside cyclic N-acylphosphoramidites in solid-phase oligonucleotide syntheses without nucleobase protection. Given that deoxyribonucleoside cyclic N-acylphosphoramidites produce oligonucleotides with heat-sensitive phosphate protecting groups, blocking the 5'-hydroxyl of these monomers with, for example, the thermolabile 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group may provide a convenient thermo-controlled method for the synthesis of oligonucleotides on microarrays.

3'-modified oligonucleotides by reverse DNA synthesis.

Reverse DNA oligonucleotide synthesis (i.e. from 5'-->3') is a strategy that has yet to be exploited fully. While utilized previously for the construction of alternating 3'-3'- and 5'-5'-linked antisense oligonucleotides, the use of nucleoside 5'-phosphoramidites has not generally been used for the elaboration of (modified) oligonucleotides. Presently, the potential of reverse oligonucleotide synthesis for the facile synthesis of 3'-modified DNAs is illustrated using a phosphoramidite derived from tyrosine. The derived oligonucleotide was shown to have chromatographic and electrophoretic properties identical with the modified oligonucleotide resulting from the proteinase K digestion of the vaccinia topoisomerase I-DNA covalent complex. The results confirm the nature of the structure previously assigned to this product, and establish the facility with which proteinase K is able to complete the digestion of the polypeptide backbone of the DNA oligonucleotide-linked topoisomerase I.

Synthesis of DNA oligonucleotides containing site-specifically incorporated O6-[4-oxo-4-(3-pyridyl)butyl]guanine and their reaction with O6-alkylguanine-DNA alkyltransferase.

DNA pyridyloxobutylation occurs during the metabolic activation of the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). This pathway contributes significantly to the carcinogenic and mutagenic activity of these nitrosamines. In general, the chemical structure of pyridyloxobutyl adducts are not well understood. Recently, an AGT reactive pyridyloxobutyl adduct was identified as O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). To better understand the importance of this adduct to the biological activity of pyridyloxobutylating agents, we developed a method for site-specifically incorporating O6-pobG into DNA oligonucleotides. They were synthesized using the phosphoramidite of the precursor 2'-deoxy-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guanosine. The dithiane group was oxidatively removed with N-chlorosuccinimide in a final postoligomerization reaction to generate the desired product. Human AGT with a polyhistidine tag was able to repair the O6-pobG-containing DNA oligonucleotide, generating unmodified oligonucleotide. These results are consistent with an alkyl group transfer mechanism for the repair of O6-pobG by AGT.

Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.

Helix Geometry and Hydration in A-DNA, B-DNA, and Z-DNA

The development of practical methods of large-scale, high-purity DNA oligonucleotide synthesis (Tan et al., this volume) has made it possible for the first time to carry out single-crystal X-ray structure analyses of short DNA molecules of preselected sequence. This has revolutionized our knowledge of the detailed molecular structure of DNA in much the same way that the perfection of multiple isomorphous replacement phase analysis did for proteins in the early 1960s. As listed in Table 1, at least three independent structure analyses are now available for each of the two families of DNA double helix known from fiber studies—A and B—plus a new and previously unsuspected family, left-handed Z-DNA. These analyses, of helices whose crystallographically unique units range from 3 bp to 12 bp, represent an advance beyond earlier studies of monomers and dimers (for review, see Rich et al. 1979), since they offer better approximations of the geometry that...