Automated DNA Synthesis Research Articles

Synthesis and antisense properties of 2'-O-(2S-methoxypropyl)-RNA-modified gapmer antisense oligonucleotides.

To ascertain whether increasing hydrophobicity can enhance the activity of second-generation antisense oligonucleotides (ASOs) in muscle, we investigated the antisense properties of 2'-O-(2S-methoxypropyl)-RNA (2S-MOP)-modified ASOs. Synthesis of the 2S-MOP 5-methyl uridine phosphoramidite was accomplished on a multi-gram scale by Lewis-acid-catalyzed ring opening of 5'-O-tert-butyldiphenylsilyl ether-protected 2,2'-anhydro-5-methyl uridine with 2S-methoxy-1-propanol. Synthesis of the 2S-MOP 5-methyl cytidine nucleoside from the corresponding 5-methyl uridine nucleoside was accomplished by formation and displacement of a 4-triazolide intermediate with aqueous ammonia. 2S-MOP-modified oligonucleotides were prepared on an automated DNA synthesizer and showed similar enhancements in duplex thermal stability as 2'-O-methoxyethyl RNA (MOE)-modified oligonucleotides. 2S-MOP-containing antisense oligonucleotides were evaluated in Balb-c mice and showed good activity for decreasing the expression levels of scavenger receptor B1 (Srb1) and phosphatase and tensin homologue (PTEN) mRNA in liver and muscle tissue.

Getting Things Backwards to Prevent Primer Dimers

This Commentary highlights the article by Satterfield that describes a new class of primer technology-cooperative primers, which prevent primer-dimer amplification.

Synthesis and Properties of 2′-O,4′-C-Ethyleneoxy Bridged 5-Methyluridine

2'-O,4'-C-Ethyleneoxy bridged 5-methyluridine (EoNA-T), possessing a seven-membered linkage and an anomeric 4'-carbon, was synthesized and introduced into oligonucleotides by using an automated DNA synthesizer. The EoNA-modified oligonucleotides significantly stabilized the duplexes with single-stranded RNA and triplexes with double-stranded DNA relative to the natural oligonucleotide and oligonucleotides modified by another seven-membered bridged 5-methyluridine, 2',4'-BNA(COC)-T. In addition, EoNA-T showed excellent nuclease resistance.

Triplex-forming ability of oligonucleotides containing 1-aryl-1,2,3-triazole nucleobases linked via a two atom-length spacer

Phosphoramidites containing 2-propynyloxy or 1-butyn-4-yl as nucleobase precursors were synthesized and introduced into oligonucleotides using an automated DNA synthesizer. Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition of the oligonucleotides with various azides gave the corresponding triazolylated oligonucleotides, triplex-forming ability of these synthetic oligonucleotides with double-stranded DNA targets was evaluated by UV melting experiments. It was found that nucleobases containing 2-(1-m-carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl units likely interacted with A of a TA base pair in a parallel triplex DNA.

Synthesis of 4-Aminophthalimide and 2,4-Diaminopyrimidine C-Nucleosides as Isosteric Fluorescent DNA Base Substitutes

The 4-aminophthalimide C-nucleoside 1 was designed as an isosteric DNA base surrogate, and a synthetic route to nucleoside 1 together with the 2,4-diaminopyrimidine-C-nucleoside 2 as a potential counterbase was worked out. The key steps in both synthetic routes represent a stereoselective Heck-type palladium-catalyzed cross-coupling with 2'-deoxyribofuranoside glycal followed by stereoselective reduction with NaBH(OAc)3. The nucleoside 1 shows a solvatofluorochromic behavior and significantly red-shifted fluorescence in solvents of high polarity and with hydrogen bonding capabilities. Both nucleosides 1 and 2 can be further processed to the corresponding phosphoramidite as DNA building blocks that allow incorporation of these chromophores as artificial DNA bases by automated DNA synthesis. The combination of the poor stacking properties of 1 and the hydrogen bonding interface at the phthalimide functionality that does not fit to any of natural DNA bases in the counterstrand yields destabilization of the duplex by 4-11 °C. The fluorescence of 1 in a representative double stranded DNA is characterized by a large Stokes' shift and a quantum yield of approximately 12%. These are remarkable optical properties considering the very small size of the chromophore and indicate a high potential of these nucleoside analogues for fluorescent DNA analytics and imaging.

Oligonucleotide Functionalization by a Novel Alkyne-Modified Nonnucleosidic Reagent Obtained by Versatile Building Block Chemistry

A convenient synthetic strategy has been designed to prepare an alkyne-modified synthon for automated DNA synthesis. It is based on the key O-DMTr-protected 4-(2-hydroxyethyl)morpholin-2,3-dione and building blocks obtained by its functionalization by various aliphatic amines. A respective nonnucleosidic phosphoramidite monomer containing a terminal alkyne in the side-chain was synthesized, and corresponding oligothymidylates incorporating the modification in various positions were prepared. The presence of the alkyne group was confirmed by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) between the functionalized oligonucleotide and an azide derivative of 7-nitro-2,1,3-benzoxadiazole.

Synthesis of Oligodeoxynucleotides with 5′‐Caps Binding RNA Targets

Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5'-O-terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of L-prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step-wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap-bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis.

Preparation of Azido Containing Oligonucleotides Through Diazo Transfer Reaction

The use of diazo transfer reagent, imidazole-1-sulfonyl azide hydrochloride (ISAHC), in the presence of Cu(2+) cation enables clean and efficient conversion of aminated oligodeoxyribonucleotides (ODNs) into their azido counterparts under mild conditions. ODNs bearing amino tether either at the 3', 5', or any internal position could be modified in this way, thus demonstrating the versatility of this reaction. The method also benefits from the commercial availability or easy access by routine automated DNA synthesis of amino-containing oligodeoxyribonucleotide starting material. Easy access to such azido-modified ODNs is of great interest for conjugation in particular through copper catalyzed 1,3-dipolar cycloaddition (CuAAC reaction).

Synthesis and Properties of Molecular Beacon DNA Probe Bearing Novel Silylated Pyrene Derivative

A novel stem-loop structured fluorescent oligoDNA probe (molecular beacon probe) bearing a silylated pyrene derivative at C-5 position of deoxyuridine has been synthesized. The fluorescently labeled modified nucleoside has been incorporated into two consecutive positions in the stem segment of the DNA using an automated DNA synthesizer. The resulting modified DNA exhibited an excimer fluorescent signal upon binding to the fully matched complementary DNA strand. The excimer emission was, however, effectively quenched while it stays alone or it hybridizes to a single base mismatched complementary target.

Quencher-free Linear Beacon Systems with Dual Fluorene-labeled Deoxyuridines

The identification of natural genetic variation in human populations is arguably one of the most important spin-offs of the effort toward the completion of the Human Genome Project. Single nucleotide polymorphisms (SNPs) are the most abundant natural genetic variations in the human genome. To satisfy the demand for molecular diagnostic assays for identifying SNPs, Tyagi and Kramer developed fluorogenic hairpin-shaped DNA probes, called “molecular beacons in 1996. Conventional molecular beacons are single-stranded oligodeoxynucleotides (ODNs) probes that exist in solution as stable stem-and-loop structures in which a fluorophore is covalently linked to one end and a quencher is covalently linked to the other end. The stem keeps the fluorophore and quencher of the molecular beacon in extremely close proximity to each other, when they are not bound to their targets; this causes the fluorescence of the fluorophore to be quenched via the fluorescence resonance energy transfer. By contrast, the conformational change induced by binding to a target nucleic acid causes the fluorophore and the quencher to move away from each other, thereby restoring the fluorescence. Therefore, the presence of the target nucleic acid is characterized by a sharp increase in fluorescence intensity. Recently, we reported that fluorescent hairpin oligonucleotides can function as molecular beacons even without the attachment of additional quencher moieties. Such probes can be called “quencher-free molecular beacons. A quencher-free molecular beacon consists of only a single fluorophore (2-ethynylfluorene-labeled deoxyuridine, U) positioned in the hairpin loop. When this probe undergoes DNA hybridization, it can discriminate between fully matched and single-base-mismatched sequences by exhibiting a strong dA-selective fluorescence. When the fluorene unit is intercalated within a DNA duplex as a result of mismatched base pairing, the fluorescence of the U unit is quenched as a result of a photoinduced charge transfer originating from interactions with neighboring nucleobases. In particular, the quencher-free molecular beacon probe having C-flanking bases (C-FBs) shows more efficient fluorescence ON/OFF behavior than do any other combinations of FBs. The only drawback of this quencher-free molecular beacon system is its poor detection sensitivity due to its relatively high intrinsic emission intensity. To solve this problem, we recently positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite the U unit to reduce the intrinsic fluorescence on hybridization with a probe. A short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective in reducing the intrinsic fluorescence of a linear beacon. In another effort to eliminate this drawback, we employed an ODN containing dual U units at specific distances (Figure 1). We observed that using this dual-labeled linear beacon system resulted in a clear improvement in the detection sensitivity for the perfectly matched target. The modified nucleotide U was synthesized through Sonogashira coupling of a 2-ethynylfluorene unit at the 5position of a 2-deoxyuridine base. Incorporations of the fluorene-labeled deoxyuridine U into the central position of the ODN 1 and of the two U units into the positions with one deoxycytosine spacer unit were effected using standard protocols of automated DNA synthesis (Figure 2). The fluorescence properties of fluorophore-containing DNA are strongly dependent on the electron injection and transfer properties of the FBs. In particular, fluorescent ODNs having C-FBs produce efficient fluorescence ON/OFF systems and have higher sensitivity as probes than do any other combinations of FBs. Therefore, we positioned two deoxy-

ChemInform Abstract: Design, Syntheses and Properties of Nucleic Acid Switch in Response to External Stimuli

AbstractReview: 52 refs.

New acid photogenerators based on thioxanthen-9-one sulfonium derivatives for detritylation in the oligonucleotide synthesis

Photochemical activity of a number of cationic thioxanthen-9-one derivatives for the formation of the trityl cation was studied, which resulted in the selection of compounds suitable for photodetritylation. 10-(4-Heptyloxyphenyl)-9-oxo-2-(N,N,N-triethylammonio)methyl-9H-thioxanthenium bis(hexafluorophosphate) and 2-methyl-, 2-(2-methyl-1-propanoyl-2-tosyl)-, 1-chloro-4-propoxy-, and 2,4-diethyl-10-(4-heptyloxyphenyl)-9-oxo-9H-thioxanthenium hexafluorophosphates were found to be photoactivators of detritylation of 5′-O-(4,4′-dimethoxytrityl)thymidine. The detritylation reaction is the most efficient in dichloromethane. 2,4-Diethyl-10-(4-heptyloxyphenyl)-9-oxo-9H-thioxanthenium hexafluorophosphate was used as a detritylation photoactivator in the oligonucleotide synthesis using an automated DNA synthesizer. The yield in the elongation step of the oligonucleotide chain was 98%.

Syntheses of DNA Duplexes That Contain a N4C‐Alkyl‐N4C Interstrand Cross‐Link

This unit describes a simple procedure for preparing short DNA duplexes that contain a single N(4)C-alkyl-N(4)C interstrand cross-link. The synthesis is carried out on an automated DNA synthesizer using standard phosphoramidite chemistry. The cross-link is introduced during the synthesis of the duplex. The method can be used to prepare milligram quantities of cross-linked duplexes suitable for physical studies and for the preparation of larger DNA molecules that can be used as substrates to study DNA repair in whole cell extracts and in living cells in culture.

Preparation of C5‐Functionalized Locked Nucleic Acids (LNAs)

Relative to conventional locked nucleic acids (LNAs), C5-functionalized LNAs with small entities at the C5-position display markedly higher (1) duplex thermostability with complementary single-stranded DNA/RNA targets, (2) target specificity, and (3) 3'-exonuclease stability. C5-Functionalized LNAs carrying a polarity-sensitive fluorophore enable sensitive and efficient discrimination of single nucleotide polymorphisms (SNPs) under non-stringent conditions. This unit describes protocols for chemical synthesis of an LNA uridine diol and corresponding C5-functionalized LNA uridine phosphoramidites. A procedure for incorporation of C5-functionalized LNA uridine phosphoramidites into oligodeoxyribonucleotides by automated DNA synthesis is also described.

Design, Syntheses and Properties of Nucleic Acid Switch in Response to External Stimuli

The control of molecular properties using external stimuli is an attractive research area that offers the potential for regulation of various biological phenomena. This review summarizes the concept, design, syntheses and properties of nucleic acid switches in response to external stimuli, namely, "external-stimuli-responsive bridged nucleic acids monomer". From (1)H-NMR experiments, every external-stimuli-responsive bridged nucleic acid monomer was found to have an N-conformation, while an S-conformation was predominantly observed after exposure to a proper stimulus. Each bridged nucleic acid monomer was effectively introduced into oligodeoxynucleotides using an automated DNA synthesizer. Moreover, oligonucleotides modified with these bridged nucleic acid monomer were changed in their hybridization property and tolerance to enzymatic digestion in response to each stimulus. These results clearly showed that external-stimuli-responsive bridged nucleic acids monomers should work as a nucleic acid switch, and have the potential for regulation of various biological phenomena.

Synthesis and Properties of Fluorescent Biological Molecules Labeled with Novel Silylated Perylene Derivative

We have synthesized novel perylene derivatives bearing a silyl function with modifiable functional groups as a feasible material for visualizing biological substances. The fluorescent perylene derivative was coupled to C-5 position of pyrimidine nucleotide and the nucleotide was subsequently incorporated to the middle position of oligoDNA using an automated DNA synthesizer. Also, the derivative was coupled with a cholesterol analog with different length of linking alkyl function. The resulting modified fluorescent oligoDNA exhibited a small change in its fluorescent signal upon hybridization with the complementary oligoDNA. While the cholesterol analogs bearing the silylated perylene exhibited marked fluorescent signal in living cells. The signal is stronger in the analog having longer linker function compared to the analog having shorter linker function. In each case, however, the signal was much prominent compared to that of dehydroergosterol (DHE), a fluorescent cholesterol analog widely used in biological study.

Metal–base pairing in DNA

The use of DNA as a molecular wire in nanoscale electronic architectures would greatly benefit from its capability of sequence-specific self-assembly. Although single electrons and positive charges have been shown to be transmitted by natural DNA over a distance of several base pairs, the high ohmic resistance of unmodified oligonucleotides imposes a serious obstacle. Exchanging some or all of the Watson–Crick base pairs in DNA by metal complexes may solve this problem and evolve DNA-like materials with superior conductivity for future nano-electronic applications. The so-called metal–base pairs are formed from suitable transition metal ions and ligand-like nucleosides which are introduced into both of the two pairing strands by automated DNA synthesis. This review illustrates the basic concepts of metal–base pairing and highlights recent developments in the field.

Native chemical ligation in the synthesis of internally modified oligonucleotide–peptide conjugates

Peptide-oligonucleotide conjugates have frequently been synthesized to improve cellular delivery of antisense or antigene compounds, to allow the immobilization of peptide and protein conjugates on DNA arrays, or to decorate nucleic acid architectures with peptide functions. In such applications, the site of conjugation is of little importance, and peptides have predominantly been appended to one of the terminal ends of the oligonucleotide by using an oxime-, thioether-, or disulfide-linkage or native chemical ligation. We, herein, demonstrate the first coupling of peptides to sequence internal sites. This attachment mode provides better control of the spatial arrangement of peptides presented by self-assembled nucleic acid scaffolds. Internal modification requires special phosphoramidite building blocks that can be used in automated DNA synthesis. For this purpose, Fmoc/StBu-protected cysteine was attached via an aminopropargyl linker to the C5-position of uridine. The rigid triple bond conferred a high reactivity in native chemical ligation reactions of 5-6mer peptide thioesters with up to 15 nucleotides long oligonucleotides. The desired peptide-oligonucleotide conjugates were obtained in high yields after purification. UV melt experiments revealed that the peptide modification does not hamper nucleic acid hybridization. This finding marked an important step in our research program devoted to studies of multivalent presentation of peptides via modular assembly of nucleic acid complexes.

A New Odorless Procedure for the Synthesis of 2′-Deoxy-6-Thioguanosine and Its Incorporation into Oligodeoxynucleotides

6-S-[2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxy-6-thiogua nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl 3-mercapto-propionate. The phosphoramidite precursor derived from 2 was successfully applied to an automated DNA synthesizer to produce 2′-deoxy-6-thioguanosine containing ODN. The results showed that 2-ethylhexyl 3-mercaptopropionate is useful as an odor less reagent and also as an S-protecting group of 2′-deoxy-6-thioguanosine.

Efficient Guest Inclusion by β-Cyclodextrin Attached to the Ends of DNA Oligomers upon Hybridization to Various DNA Conjugates

Two types of 5'-β-CyD-DNA conjugates were synthesized using two different strategies and were hybridized with cDNA conjugates bearing various possible guest compounds at the 3' ends. One of the β-CyD conjugates was synthesized on the basis of solid-phase postmodification of DNA with a monoamino-β-CyD derivative on a synthesis column for automated DNA synthesis. The other β-CyD conjugate was synthesized by the solution-phase coupling of DNA with a monomercapto-β-CyD derivative using a heterobifunctional cross-coupling reagent. When these 5'-β-CyD-DNA conjugates were hybridized with cDNA conjugates bearing 1-adamantaneacetic acid at the 3' ends, significant stabilization of the duplexes was observed as compared with the control duplex without β-CyD. Duplexes of 5'-β-CyD-DNA conjugates and complementary 3'-dansyl-glycine-DNA conjugates were also moderately stabilized. Thermodynamic measurements revealed that the host-guest inclusion interactions between β-CyD and 1-adamantaneacetic acid or dansyl-glycine are roughly as strong as those found in bulk solution even if they are tethered to the ends of the DNA.