A comparative study of supports for the synthesis of oligonucleotides without using ammonia

A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2-nitrobenzyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9-fluorenylmethyl supports.We are grateful to CICYT (PB92-0043) and E.E.C.C. Biomedicine and Health Programme (BMH1-CT93-1669) for financial support. We thank Drs. Matthias Mann, Gitte Neubauer, Matthias Wilm (EMBL) and Irene Fernández (University of Barcelona) for obtaining mass spectra.Peer reviewe

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides

A novel approach for the preparation of oligonucleotides carrying amino groups at the 3'-end is described. Several CPG supports having aminoalkyl groups and 3'-amino-2',3'-dideoxynucleosides linked through base-labile carbamate linkages such as 2-(2- nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl were prepared using two different strategies. These supports are compatible to the standard solid phase phosphite-triester methodology and yield oligonucleotides containing amino groups at the 3'-end. Several properties of the 3'-amino oligonucleotides, such as nuclease resistance, hybridization, and preparation of oligonucleotide conjugates are discussed.Financial support from CICYT (PB92-0043) and E.E.C.C. Biomedicine and Health Programme (BMH1-CT93-1669) is gratefully acknowledged. We thank Drs P. Herdewijn, A. van Aerschot, T. Saison- Behmoaras, and W. Pfleiderer for their helpful suggestions. We are grateful to Marten Wiersma for his technical assistance.Peer reviewe

Synthesis of oligoribonucleotides containing 4-thiouridine using the convertible nucleoside approach and the 1-(2-fluorophenyl)-4-methoxypiperidin-4- yl group

Oligoribonucleotides containing 4-thiouridine were prepared using the Fpmp group for protection of the 2′-OH. Two uridine derivatives with the 1,2,4-triazolyl and the 2-nitrophenyl groups at position 4 were used to obtain 4-thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.We thank Drs. Matthias Mann, Gitte Neubauer, Matthias Wilm and Ole Jensen for obtaining mass spectra. Anna Aviñó was recipient of a EMBO short-term fellowship. This work was supported by the Dirección General de Investigación Científica y Técnica (grant BQU2003- 00397), and the Generalitat de Catalunya (2001-SGR-0049).Peer reviewe

Inhibition of Hha I DNA (cytosine-c5) methyltransferase by oligodeoxyribonucleotides containing 5-Aza-2′-deoxycytidine: Examination of the intertwined roles of co-factor, target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between Hha I methyltransferase (M.Hha I) and oligo-deoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.Hha I. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.Hha I that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.Hha I.Partial support for this work was provided by the DAMD Breast Cancer Program (DAMD 17-98-1-8215) to JKC and fellowship support from the Graduate College at UNMC to ASB. We are also greatful to Drs. X. Cheng and S. Kumar for their generous gifts of purified M.HhaI.Peer reviewe

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2′-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.a European Molecular Biology Laboratory, D-69117 Heidelberg, Germany b Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 69198-4525, United States c UNMC/Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 69198-4525, United States d Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, United States e Institut de Biología Molecular de Barcelona, CSIC, Jordi Girona 18-26, E-08034 Barcelona, SpainN

Parallel-stranded hairpins containing 8-aminopurines. Novel efficient probes for triple-helix formation

We describe novel oligomers with a greater propensity to form triplexes than oligomers containing only natural bases. They consist of a polypyrimidine sequence linked head-to-head with a polypurine sequence carrying one or several 8-aminoadenine or 8-aminoguanines. The presence of 8-aminopurines also stabilised the parallel-stranded duplex structure. © 2001 Elsevier Science Ltd.The authors thank the Dirección General de Investigación Cientı́fica y Técnica (projects PB98-1222, PM99-0046 and BQU2000-0649), the Generalitat de Catalunya (project 2000-SGR-0018) and CyGene, Inc. for financial supportPeer Reviewe

DNA-triplex stabilizing properties of 8-aminoguanine

A DNA-triplex stabilizing purine (8-aminoguanine) is designed based on molecular modeling and synthesized. The substitution of guanine by 8-aminoguanine largely stabilizes the triplex both at neutral and acidic pH, as suggested by molecular dynamics and thermodynamic integration calculations, and demonstrated by melting experiments. NMR experiments confirm the triplex-stabilizing properties of 8-aminoguanine and demonstrate that few changes are found in the structure of the triplex due to the presence of this modified base

Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence