DNA Synthesizer Research Articles

Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the intercalators at the junction point.

3'-3'-Linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point were synthesized on a DNA synthesizer using a controlled pore glass (CPG), which has pentaerythritol carrying the intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer. The inhibitory activity of the 3'-3'-linked ODNs against the cleavage of the target DNA by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer and the 3'-3'-linked ODN without the intercalator.

Synthesis and characterization of oligodeoxyribonucleotides carrying arrays of intercalating groups.

A novel strategy for attaching the functional groups to oligodeoxyribonucleotides (ODNs) is described. A thymidine carrying anthraquinone derivative at the N3 position (AnT) was synthesized and introduced into ODNs using the phosphoramidite method on a DNA synthesizer. Py-Pu-Py type triplex formation of ODNs was enhanced by attaching AnT to the 5'-end of the third strand.

Reusable solid-phase supports for oligonucleotides and antisense therapeuticsElectronic supplementary information (ESI) available: CGE data for octathymidine phosphodiester sequences on supports P1–5, P7, P9, and P11–13 PAGE data for phosphorothioate products, dimethoxytrityl analysis and UV measurements on supports P1–7, P9 and P15, step listing for ABI394 DNA synthesizer and CGE and HPLC data for phosporothioate syntheses on support P3. See http://www.rsc.org/suppdata/p1/b1/b105089n/

A general method for oligonucleotide synthesis on reusable solid-phase supports has been developed which will significantly lower the cost of large-scale synthesis. It consists of five steps: 1, nucleoside attachment to an hydroxy derivatized support through a Q-linker (hydroquinone-O,O′-diacetic acid) linker arm; 2, chloro-acetylation of unreacted surface groups; 3, conventional phosphodiester or phosphorothioate oligonucleotide synthesis; 4, cleavage from the support with aqueous or anhydrous ammonia; and 5, support regeneration with methanolic potassium carbonate. The recycling process is fast, fully automatable, and does not require removal of the support from the synthesis column. Fifteen solid-phase supports were evaluated with glycerol-CPG providing the best results. Consecutive syntheses of ISIS 2302, a dGCCCAAGCTGGCATCCGTCA phosphorothioate sequence, on the same synthesis column were performed. A glycerol-CPG synthesis column was satisfactorily used six consecutive times when NH4OH was the cleavage reagent. However, anhydrous NH3 allowed twelve consecutive syntheses without any deterioration in support loading, product quality, or amount of product produced. An improved method for preparing the essential nucleoside-3′-hemiesters of the Q-Linker and an unexpectedly slower rate of cleavage for phosphorothioate DNA vs. phosphodiester DNA are also described.

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca(2+)-bound protein exists as a dimer at protein concentrations lower than 150 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca(2+)-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.

Nucleosides and nucleotides. 208. Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point.

The synthesis of 3'-3'-linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3'-3'-linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3'-3'-linked ODN 15 without the intercalator.

Characterization of oligodeoxyribonucleotide synthesis on glass plates.

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5'-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using (32)P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5'-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.

SYNTHESES OF DNA DUPLEXES CONTAINING A C‒C INTERSTRAND CROSS-LINK

Short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link were prepared on controlled pore glass supports using a DNA synthesizer. The C‒C cross-link was introduced via a convertible nucleoside on the support or by using a protected C‒C cross-link phosphoramidite. An orthogonal protection scheme allowed selective chain growth in either a 3′ → 5′ or 5′ → 3′ direction. The cross-linked duplexes were purified by HPLC and characterized by MALDI-TOF mass spectrometry and/or by enzymatic digestion.

Synthesis and duplex stability of oligonucleotides containing 7-vinyl-7-deazaguanine as a strong electron-donating nucleobase

We synthesized 7-vinyl-7-deazaguanine ( vzG), an N7-modified guanine base, which has a smaller oxidation potential than G and 7-deazaG. The incorporation of vzG into oligonucleotides was achieved by phosphoramidite chemistry on a DNA synthesizer. The T m of the duplex containing vzG was higher than that of the corresponding duplex containing G or 7-deazaG. The stabilization of the duplex containing vzG is due to the favorable enthalpy change by increased π-stacking between the vinyl group and the 5′-flanking base.

Industrial-strength profiling: Rosetta Inpharmatics, Inc.

Rosetta’s approach is, at its most basic, an argument for big biology. “Absolutely that trend is going to happen,” says Friend. “Once you have a critical mass of data you can say a lot more about the biology. But unlike physics, a single lab can focus in on a very narrow area and still make a contribution.”Rosetta, of course, plans to be more ambitious. Most of its test case experiments have been in yeast, but the company has been busy working with mammalian cells. The results of that work will be along shortly. “Stay tuned,” says Boguski, “for some landmark scientific publications in mammalian biology.”

Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding.

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.

Synthesis and properties of a new type DNA dendrimer.

We have designed a new type of a DNA dendrimer which has rigid branched structure. The branching molecule was prepared from 1,3,5-tribromobenzene. The dendrimer unit, in which three oligonucleotide-chains, two molecules of T15 and one molecule of A15, linked to the branching molecule, was synthesized by an automated DNA synthesizer. The properties of the dendrimer unit and dendrimer formation by inter-molecular association of T15 and A15 chains of the dendrimer unit will be presented.

Nucleosides and Nucleotides. Part 206: Introduction of Lipophilic Groups into 4′α- C-(2-Aminoethyl)thymidine-Containing Phosphodiester Oligodeoxynucleotides and Thermal Stabilities of the Duplexes

Synthesis and properties of the 4′α- C-(2-aminoethyl)thymidine ( 1)-containing oligodeoxynucleotides (ODNs), which have lipophilic groups such as palmitic acid, oleic acid, and cholesterol at the terminal of the aminoethyl linker of the compound 1, are described. The ODNs were synthesized in a DNA synthesizer by using controlled pore glasses (CPGs) having the 4′α- C-[ N-(palmitoyl), N-(oleoyl), and N-(cholesteryloxycarbonyl)aminoethyl]thymidine analogs ( 9a, b, and c). The stability of the duplexes formed by these ODNs and a complementary DNA 15 or RNA 16 was studied by thermal denaturation. It was found that the bulky functional group such as cholesterol thermally destabilizes the DNA/DNA duplexes, but that such thermal destabilization can be offset by the effects of the aminoethyl linker of 1. Furthermore, these lipophilic groups do not largely influence the thermal stability of the DNA/RNA duplexes.

Regulation of Human COL2A1 Gene Expression in Chondrocytes: IDENTIFICATION OF C-Krox-RESPONSIVE ELEMENTS AND MODULATION BY PHENOTYPE ALTERATION

Regulation of Human COL2A1 Gene Expression in Chondrocytes: IDENTIFICATION OF C-Krox-RESPONSIVE ELEMENTS AND MODULATION BY PHENOTYPE ALTERATION

Branched oligonucleotides containing bicyclic nucleotides as branching points and DNA or LNA as triplex forming branch

Various Y-shaped branched oligonucleotides containing a 2′- O,3′- C-ethylene linked or 2′- O,4′- C-methylene linked bicyclic nucleotide as branching point were synthesized on an automated DNA synthesizer. Thermal denaturation experiments at 260 and 284 nm showed increased thermal stabilities of complexes formed between these Y-shaped oligonucleotides and complementary DNA compared with those formed with the corresponding linear reference. The most significant effect was observed when LNA (locked nucleic acid) monomers were used in the triplex forming branch.

High-Loading Supports for Oligonucleotide Synthesis

b ), and A simple protocol for the preparation of high-loading supports, suitable for large-scale synthesis of oligonucleotides, has been developed. The method involves the use of inexpensive reagents and is amenable to large-scale preparation of supports. The derivatized supports were successfully employed in an automated DNA synthesizer without any difficulty. The quality of the synthesized oligonucleotides was found to be comparable to that of the corresponding oligomers prepared with commercially available standard supports. Introduction. - Synthetic oligonucleotides and their modified analogs have become vital tools for various biological studies. They are finding widespread use in the development of oligonucleotide-based therapeutic and diagnostic tools (1 - 4). Gen- erally, oligonucleotides, in the size range of 15 - 30 residues, are required in abundance, but in tiny quantities for studies like DNA sequencing, etc. However, in case of developing DNA based diagnostics and therapeutics (for use as antisense oligomers), relatively larger quantities of short oligomers are required. To synthesize these molecules in large quantities, the present day methodologies (5 - 7) have proven to be somewhat expensive. Hence, there is a need to develop economically viable routes or protocols for the synthesis of oligonucleotides to meet this exponentially growing demand for these molecules. In the last several years, tremendous improvements have been reported in the area of oligonucleotide synthesis (8) (9). The introduction of new sets of labile protecting groups, fast coupling reagents, and improved polymer supports have revolutionized the entire field so that even a non-chemist can synthesize these molecules quite easily. Since most of the syntheses are currently carried out on solid supports, which play a significant role in deciding the cost of these molecules, there is a need to develop supports having a relatively higher density of the functional groups, which could ultimately reduce the cost of these molecules without compromising the quality of the final product. During the last few years, however, although several polymer supports have been developed and tested, LCAA-CPG (long-chain alkyl- amine/controlled-pore glass) is still the standard polymer support (10) for routine synthesis of oligonucleotides. Recently, we published an express protocol for the functionalization of polymer supports useful for oligonucleotide synthesis (12), in which we reported the loading of LCAA-CPG in the range of 45 - 50 mmol/g of support.

Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection

Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.

Essential Role of Human Leukocyte Antigen-encoded Proteasome Subunits in NF-κB Activation and Prevention of Tumor Necrosis Factor-α-induced Apoptosis

The multisubunit proteasome complex is the principal mediator of nonlysosomal protein degradation. The proteasome subunit varies minimally between cells with the exception of LMP2, LMP7, and LMP10 subunits in rodent and human cells. LMP2 and LMP7 subunits are encoded by the human lymphocyte antigen region, and they optimize proteolytic mediated antigen presentation. The proteasome is also important for the function of transcription factor nuclear factor-kappaB (NF-kappaB). It is required for NF-kappaB subunits p50 and p52 generation and catalyzes degradation of phosphorylated IkappaBalpha. These proteasome-mediated reactions have now been shown to be defective in T2 cells, a human lymphocyte cell line that lacks both LMP2 and LMP7. Although T2 cells contain normal expression of p100 and p105, the abundance of p50 and p52 was greatly reduced. Tumor necrosis factor-alpha (TNF-alpha) induced normal phosphorylation of IkappaBalpha but failed to induce degradation of phosphorylated IkappaBalpha. Both DNA binding assays and luciferase assays revealed that TNF-alpha-induced NF-kappaB activation is defective in T2 cells. Unlike parental cells, T2 cells were susceptible to TNF-alpha-induced apoptosis. These data indicate human leukocyte antigen-linked proteasome subunits are essential for NF-kappaB activation and protection of cells from TNF-alpha-induced apoptosis.

Syntheses and properties of conformationally restrained nucleosides and oligonucleotides analogues

This review summarizes our efficient syntheses of novel bicyclic nucleoside analogues, 3'-O,4'-C-methyleneribonucleosides (1) (4-BC type nucleoside analogue), 2'-O,4'-C-methyleneribonucleosides (2) (5-BC), 3'-amino-3'-deoxy-3'-N,4'-C-methyleneribonucleosides (3) (aza 4-BC), and 3'-azido- and 3'-amino-3'-deoxy-2'-O,4'-C-methyleneribonucleosides (4, 5) (aza 5-BC). From 1H-NMR and X-ray crystallographic analyses, the 4-BC and aza 4-BC type nucleoside analogues (1, 3) were found to have a S-conformation predominantly, while the conformations of 5-BC and aza 5-BC type nucleoside analogues (2, 4, 5) were exclusively locked in N-form. The 4-BC and 5-BC type nucleoside analogues (1, 2) were effectively introduced into oligonucleotides using a DNA synthesizer. Furthermore, unprecedented hybridizing ability towards complementary RNA and DNA, RNA selectivity, potent triplex forming ability, and sufficient enzymatic stability of these modified oligonucleotides were also confirmed. These results should reveal a promising route to the development of antisense/antigene methodology.

A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development.

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4'-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide. Stepwise addition of the linker phosphoramidite graduated at 1% (relative to the total sites available) per step at 50 degrees C resulted in an optimal yield of immobilized oligonucleotides at a density of 2.24 x 10(10) strands/mm2. This observed loading maximum lies well below the theoretical maximum loading owing to nonspecific adsorption of HEG on the glass and subsequent blocking of reactive sites. Surface loadings as high as 3.73 x 10(10)/mm2 and of excellent sequence quality were achieved with a reverse amidite protocol. The support was first modified into a 2-cyanoethyl-N,N-diisopropylphosphoramidite analog followed by coupling with 4,4'-dimethoxytrityl-protected HEG. This protocol is conveniently available when using a conventional DNA synthesizer. The reverse amidite protocol allowed for control of the surface loading at values suitable for subsequent analytical applications that make use of immobilized oligonucleotides as probes for selective hybridization of sample nucleic acids of unknown sequence and concentration.

Synthesis and Duplex Stabilization of Oligonucleotides Consisting of Isonucleosides

Novel oligonucleotide analogues built from isonucleosides were synthesized by the phosphoramidite approach on an automated DNA synthesizer. The phosphoramidite building blocks were synthesized by phosphitylation of the corresponding protected isonucleosides. The oligonucleotide analogues C ‐ G containing the isonucleoside 1 ‐ 3 were studied with respect to their hybridization properties and enzymatic stability. The oligomers bearing an isonucleoside at the end of the strands all proved stable towards snake-venom phosphodiesterase, but only the oligomers D ‐ G exhibit acceptable duplex stability when hybridized with complementary d(A14).