Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state

Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8(+) T cell response, but the development of similar reagents for detection of CD4(+) T cells based on class II MHC proteins has been more difficult. We evaluated fluorescent streptavidin-based oligomers of HLA-DR1 for use as reagents to analyze Ag-specific human CD4(+) T cells. Staining was blocked at low temperatures and by drugs that disrupt microfilament formation and endocytosis. Cell-associated MHC oligomers were resistant to a surface stripping protocol and were observed by microscopy in intracellular compartments. This behavior indicates that detection of CD4(+) T cells using class II MHC oligomers can depend on an active cellular process in which T cells cluster and/or endocytose their Ag receptors. T cells of identical specificity but in different activation states varied greatly in their ability to be detected by class II MHC oligomers.     

Synthesis and hybridization properties of oligodeoxynucleotides containing 3'-deoxy-3'-C-methyleneuridine

3'-Deoxy-3'-C-methyleneuridine nucleoside 1 has been incorporated into oligodeoxynucleotides. Relative to the unmodified references, oligomers containing nucleoside 1 displayed reduced binding affinities towards complementary DNA and RNA with a tendency towards RNA-selective hybridization.     

Selective O-phosphitilation with nucleoside phosphoramidite reagents.

In contrast to tetrazole, pyridine hydrochloride/imidazole converts nucleoside phosphoramidites to intermediates that show a high preference for phosphitilating hydroxyl groups relative to nucleoside amino groups. Use of this activating agent and incorporation of a pyridine hydrochloride/aniline wash step in the synthetic cycles permit synthesis of mixed base twenty-mer oligonucleotides from nucleoside reagents containing unprotected amino groups. This approach should be useful for the synthesis of oligonucleotide analogues containing substituents sensitive to reagents used in conventional deblocking steps. Pyridine hydrochloride itself is an effective reagent for activating nucleoside methylphosphonoamidites and ribonucleoside phosphoramidites, as well as deoxyribonucleoside phosphoramidites, when high O/N selectivety is not needed.     

A facile synthesis of DNA oligomers with modified backbones via the phosphoramidite method without nucleoside base protection.

A convenient synthesis of DNA oligomers with modified back bones including phosphorothioates and phosphotriesters has been developed on the basis of the phosphoramidite strategy without nucleoside base protection. In this synthesis, a new, convenient method for the preparation of N-free-5'-O-dimethoxytrityl-2'-deoxyribonucleoside by the direct O-selective dimethoxytritylation of the parent nucleosides has also been disclosed.     

The Degradation of dG Phosphoramidites in Solution

The reaction of 2′-deoxynucleoside phosphoramidites with water is an important degradation reaction that limits the lifetimes of reagents used for chemical deoxyoligonucleotide synthesis. The hydrolysis of nucleoside phosphoramidites in solution has therefore been investigated. The degree of degradation depends not only on the presence of water but also on the specific nucleoside, 2′-deoxyguanosine (dG) being especially susceptible. Additionally, the nature of the group protecting the exocyclic amine on the nucleoside base strongly influences the rate of hydrolysis. For dG, the degradation is second order in phosphoramidite concentration, indicating autocatalysis of the hydrolysis reaction. Comparison of the degradation rates of dG phosphoramidites with different protecting groups as well as with phosphoramidites containing bases that are structurally similar to dG affords clues to the nature of how dG catalyzes its own destruction and indicates a direct correlation between ease of protecting group removal and propensity to undergo autocatalytic degradation.     

Synthesis and Hybridization Properties of Oligodeoxynucleotides Containing 3′-Deoxy-3′-C-methyleneuridine

Abstract

3′-Deoxy-3′-C-methyleneuridine nucleoside 1 ¹ has been incorporated into oligodeoxynucleotides. Relative to the unmodified references, oligomers containing nucleoside 1 displayed reduced binding affinities towards complementary DNA and RNA with a tendency towards RNA-selective hybridization.     

Unexpected results and recourse in process optimization of nucleoside 3'-O-succinates

An improved and scalable protocol for the synthesis of 3'-O-succinates of nucleosides has been developed using succinic anhydride. As a result, formation of unwanted dimer has been completely eliminated and use of toxic and smelly reagents have been avoided during synthesis of nucleoside succinates. All succinates were isolated in pure state without silica gel column chromatography.     

Selectivity in subunit composition of Ena/VASP tetramers

The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The vertebrate members of the Ena/VASP family, VASP, Mena and EVL, have many overlapping properties and expression patterns, but functional and regulatory differences between paralogues have been observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP activity. While it has been assumed that family members can form mixed oligomers, this possibility has not been investigated systematically. Using cells expressing controlled combinations of VASP, Mena and EVL, we evaluated the composition of Ena/VASP oligomers and found that VASP forms oligomers without apparent bias with itself, Mena or EVL. However, Mena and EVL showed only weak hetero-oligomerization, suggesting specificity in the association of Ena/VASP family members. Co-expression of VASP increased the ability of Mena and EVL to form mixed oligomers. Additionally, we found that the tetramerization domain (TD) at the C-termini of Ena/VASP proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD with a synthetic tetramerizing coiled coil sequence supports homo-oligomerization and normal VASP subcellular localization.     

Effect of structural variations in cholesteryl-conjugated oligonucleotides on inhibitory activity toward HIV-1.

A number of oligonucleotide analogues containing internucleoside phosphorothioate linkages and a covalently attached cholesteryl residue was synthesized and tested for activity against HIV-1 in cultures of Molt3 cells. Structural features important for high antiviral activity are the presence of a cholesteryl moiety, a run of terminal phosphorothioate groups, and the presence of nucleoside residues. An increase in length of the tether between cholesteryl and phosphorus from six to 14 atoms has no significant effect on antiviral activity, and up to one-half of the internucleoside links in a cholesteryl-conjugated phosphorothioate oligomer and one-third of the internucleoside links in a nonconjugated phosphorothioate can be replaced with phosphodiester links without much change in antiviral activity. However, replacement of nucleoside units in the oligomers by a simple analogue (-OCH2CH2CH2O-) yields inactive or very weakly active compounds, even in the presence of a cholesteryl group. Dose-response patterns for assays in which cholesteryl-conjugated oligomers are added to test cells either simultaneously or subsequently to viral infection are similar for homooligomer derivatives and for oligomers containing "antisense" sequences, suggesting a similarity in mode of action for the two classes of oligomers in this system.     

Synthesis of 2’-aminometylmorpholino nucleoside analogues containing the 4’-carboxymethyl linker group

A simple and efficient method has been developed for the preparation of 2′-aminomethylmorpholino-4′-carboxymethyl nucleoside analogues and their 2′-N-Boc-modified derivatives as synthons for obtaining oligomers by peptide synthesis methods.     

Multiple oligodeoxyribonucleotide syntheseson a reusable solid-phase CPG support via the hydroquinone-O,O'-diacetic acid (Q-Linker) linker arm

A strategy for oligodeoxyribonucleotide synthesis on a reusable CPG solid-phase support, derivatized with hydroxyl groups instead of amino groups, has been developed. Ester linkages, through a base labile hydroquinone- O, O '-diacetic acid ( Q-Linker ) linker arm, were used to couple the first nucleoside to the hydroxyl groups on the support. This coupling was rapidly accomplished (10 min) using O -benzotriazol-1-yl- N, N, N ', N '-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole as the activating reagents. Oligodeoxyribonucleotide synthesis was performed using existing procedures and reagents, except a more labile capping reagent, such as chloro-acetic anhydride, methoxyacetic anhydride or t-butylphenoxyacetic anhydride, was used instead of acetic anhydride. After each oligodeoxyribonucleotide synthesis, the product was cleaved from the support with ammonium hydroxide (3 min) and deprotected as usual. Residual linker arms or capping groups were removed by treatment with ammonium hydroxide/methylamine reagent and the regenerated support was capable of reuse. Up to six different oligodeoxyribonucleotide syntheses or up to 25 cycles of nucleoside derivatization and cleavage were consecutively performed on the reusable support. This method may provide a significant cost advantage over conventional single-use solid supports currently used for the manufacture of antisense oligodeoxyribonucleotides.     

10 The montmorillonite-catalyzed synthesis of RNA dimer

A synthesis has been developed, providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5′-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately, 30% of the imidazolide is converted into each 2′-5′ dimer and 3′-5′ dimer with the rest hydrolyzed to the 5′-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5′-monophosphates. Both homo- and heterochiral combinations of reagents have been tried. The montmorillonite-catalyzed oligomerization of 5′-activated nucleotides leads to oligomers up to 50 residues in length (Huang & Ferris, 2007) using the excellent catalyst Volclay®. However, all oligomers must necessarily begin as dimers, so we considered it important to study in detail the formation of these products under prebiotic conditions. Then, a meaningful comparison could be drawn between our syntheses and the formation of long oligomers that is part of our studies of the origins of life. In the synthesis of trimers from these dimers, we looked for alternative synthetic methods via a 5′-phosphate dimer with activated nucleotides as well as 5′-hydroxy nucleotide dimers with the same reactant. The method has shown promise in targeting trimer synthesis and the procedure lends itself to the development of combinatorial libraries. The use of enzymatic hydrolysis has played a crucial role in this work, facilitating product identity across the spectrum of products prepared. The yields of the corresponding homochiral and heterochiral dimers from A and U will require careful modeling of the reactants in their interactions with both the clay and one another to locate the source of the similarities and differences. The lack of reactivity of arabino- and xylo-nucleosides also poses interesting structural, modeling, and origins of life issues. Results with clays that catalyze long oligomer formation only poorly reveal that they too catalyze these dimer syntheses, albeit less well than Volclay.^®      

UNEXPECTED RESULTS AND RECOURSE IN PROCESS OPTIMIZATION OF NUCLEOSIDE 3′-O-SUCCINATES

An improved and scalable protocol for the synthesis of 3′-O-succinates of nucleosides has been developed using succinic anhydride. As a result, formation of unwanted dimer has been completely eliminated and use of toxic and smelly reagents have been avoided during synthesis of nucleoside succinates. All succinates were isolated in pure state without silica gel column chromatography.     

Synthesis and fluorescent properties of 5-(1-pyrenylethynyl)-2'- deoxyuridine-containing oligodeoxynucleotides

Novel reagents for the fluorescent labeling of oligo- and polynucleotides have been prepared: 5-(1-pyrenylethynyl)-2'-deoxyuridine 3'-phosphoramidite and a solid support carrying this nucleoside. Oligonucleotides containing one or several modified units have been synthesized, and the fluorescence of these probes has been shown to change upon hybridization with the complementary sequence.     

Oligonucleotides with 2,6-Diaminopurine Base Replacing for Adenine: Synthesis and Properties

Abstract

Synthesis of three nucleoside building blocks with a benzoyl protected diaminopurine (DAP) base and their incorporation at different positions of DNA, RNA and hexitol oligomers (HNA) have been accomplished. DNA hairpins with a DAP substituted for one (or more) adenine in the loop structure were not found to be more stable. But the stability of RNA-hexitol as well as hexitol-hexitol duplexes improved when the adenine base was replaced with DAP.     

Efficient cross-linking to cytidine by functional nucleobases capable of in situ activation.

We have previously demonstrated that the ODNs with 2-amino-6-(2-phenylsulfoxyethyl)purine nucleoside derivative were capable of efficient interstrand cross-linking with cytidine selectively. In this new strategy, less reactive precursor was auto-activated within a duplex to generate 2-amino-6-vinylpurine derivative. However, it turned out that 2-amino-6-(2-phenylsulfinyl)-ethylpurine nucleoside was not applicable as the precursor for the synthesis of DNA oligomers with G-rich sequences. In this report, 2-amino-6-(2-methylsulfinylethyl)purine nucleoside has been proven to be more suitable as a precursor for DNA synthesis. In addition, the ODNs incorporating either 2-amino-6-(2-phenylsulfoxy ethyl)purine or 2-amino-6-vinylpurine showed high reactivity toward the cytidine at the target site but quite less reactivity was observed for it at non-target site, demonstrating high site-selectivity.     

Phosphine derivatives for selective phosphorylation of nucleoside and their application to oligonucleotide chemistry

Some new phosphorylating reagents have been developed. They were classified into two types; one reacts selectively to the cis-glycol of ribonucleoside and the other has the selective reactivity to the primary alcohol of nucleoside. The application of these selective phosphorylation reactions to the synthesis of oligonucleotides is described.     

Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

Guanine modification during chemical DNA synthesis.

Base modification during solid-phase phosphoramidite synthesis of oligodeoxynucleotides has been investigated. We have discovered chemical modification that converts dG and dG-containing oligomers to a fluorescent form. This modification has been linked to N,N-dimethylaminopyridine (DMAP), an acylation catalyst, which can displace phosphate triester adducts at the 6-position of guanine. Further, we have found that this fluorescent intermediate can be converted in ammonium hydroxide solution to 2,6 diaminopurine deoxyribonucleoside (2,6 DAP), a potentially mutagenic nucleoside analog. We have shown that N-methylimidazole (NMI) in place of DMAP eliminates the fluorescent species and reduces 2,6 DAP contamination.