Modification of guanine bases by nucleoside phosphoramidite reagents during the solid phase synthesis of oligonucleotides

Pon, Richard T.; Damha, Masad J.; Ogilvie, Kelvin K.

doi:10.1093/nar/13.18.6447

Cited by 57 publications

(15 citation statements)

References 31 publications

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“…Because the unprotected 0-4 group of the uracil ring is reactive toward nucleophilic displacement resulting in chain branching during synthesis, we used the p-nitrophenylethyl group to protect the 0-4 atom of 5-fluorodeoxyuridine (16,17 Step 1. 5-Dimethoxytrityl-5-fluorodeoxyuridine: 5-Fluorodeoxyuridine (42 mmol, 9.74 g) was suspended in 100 ml of dry pyridine in a 200-ml round-bottom flask attached to a drying tube.…”

Section: Methodsmentioning

confidence: 99%

5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.

Habener

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic complementary oligonucleotides are useful hybridization probes for the detection of mRNAs and genes encoding proteins for which only a partial amino acid sequence is known. Usually this involves the synthesis of mixtures of oligonucleotides complementary to all possible bases in degenerate positions of codons. As an alternative we have prepared and characterized a series of unique oligonucleotides containing a pyrimidine analog, 5-fluorodeoxyuridine (F). Thermodynamic parameters and the melting temperatures of hybrid duplexes containing A-F and G-F base pairs showed that they are considerably more stable than duplexes containing APT and GOT base pairs. The stability of a duplex decreased linearly with the number ofmismatches introduced at positions at least a codon apart. A 5-fluorodeoxyuridine-substituted oligonucleotide cDNA detects rat liver pyruvate carboxylase mRNA on a RNA gel blot with a dissociation temperature only 10'C below the measured melting temperature in solution. We suggest that the complexity of oligonudeotide cDNAs used for screening gene libraries can be reduced by the design of single hybridization probes containing the substituted bases-5-fluorodeoxyuridine to pair with adenosine or guanosine, guanosmne to pair with cytidine or thymidine, and deoxyinosine to pair with adenosine or cytidine at positions of codon degeneracy-and still retain near-maximum stability of hybrid duplexes.Recombinant DNA technology allows the isolation of genes encoding specific proteins. When the partial amino acid sequence of the protein is known, an approach to isolation of the genes is the synthesis of mixtures of 8-64 oligonucleotides of 14-17 bases that are complementary in sequence to all possible codons predicted for the corresponding amino acid sequence (1-3)J A difficulty in these syntheses, however, is that coupling efficiencies of the nucleotides at each step are not uniform resulting in unequal representations of the oligonucleotides in the mixture; certain of the sequences may be practically absent (4). In general the successful detection of the correct sequence has been possible only when it is present in the screened library at a relatively high abundance (>0.1-1% of sequences). A complex mixture of oligonucleotide probes may lead to spurious hybridization to incorrect sequences, particularly in attempts to identify a correct single-copy sequence in mammalian gene libraries with a complexity approaching 109 base pair.To circumvent these problems, base analogs have been designed that can potentially base pair with any of the four natural bases (adenosine, thymidine, guanosine, and cytidine) in DNA. For example, deoxyinosine (5), 2-amino-2'-deoxyadenosine (4), and phenyl derivatives (5) have been used to prepare unique probes at positions of base degeneracy, but base pairing is significantly less stable than the normal Watson-Crick base pairing (6). Similarly, deoxyguanosine has been used in positions opposite to cytidine or thymidine ambiguities because the GOT wobble base pair is be...

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Section: Methodsmentioning

confidence: 99%

5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.

Habener

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…[80] Deblocking of the 5 -OH group was accomplished with 80% acetic acid. [81] The product was purified with two-dimensional preparative thin layer chromatography using 20% methanol in both directions.…”

Section: Phosphatemethylated D(t P A)mentioning

confidence: 99%

Dna Systems for B-Z Transition and Their Significance as Epigenetic Model: The Fundamental Role of the Methyl Group

Buck¹

2011

Nucleosides, Nucleotides and Nucleic Acids

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Epigenetic systems involved in the dynamics of gene expression, which are fundamental to cell determination and function without alteration in DNA sequences, are based on methylation of the N-terminal tails of lysine residues and DNA methylation. We demonstrate the vital importance for genetic transfer by different (hydrogen) networks, suggesting a complex interaction between the two epigenetic modifications. In other words, the methylation of local lysines can prescribe C(P)G methylation, which requires that methylation of histones and DNA are cooperative in carrying out an epigenetic instruction for integrating gene-silencing networks. To give a bio-organic description of the epigenetic coherence between histone and base methylation, we used the well-known B- into Z-DNA dynamics in combination with the unique properties of phosphatemethylated DNA on different levels of chemistry.

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“…(b) Ac½O/N-MeIn capping: 'cap to column' step, 20 sec followed by a 'wait' step, 30 sec (this extra step was added by editing the standard cycle program). This prolonged capping treatment efficiently reverses guanine modification which takes place during the amidite coupling step (32,33). (c) Oxidation: 'iodine to column' step, 30 sec, followed by a 'wait' step, 20 sec.…”

Section: Reagentsmentioning

confidence: 99%

“…HPLC grade solvents, pyridine, dichloromethane, and acetonitrile were purchased from BDH and Caledon (Toronto).Derivatization of controlled pore glass supportsLong chain alkylamine controlled-pore glass (LCAA-CPG, 500 A, Pierce Rockford, IL, or CPG, Inc., N.J.) was derivatized with 5'-O-MMT-uridine, 5'-O-DMT-deoxythymidine, 5'-O-MMT-N6-Bz-adenosine, and 5 '-O-MMT-N4-Bz-cytidine following minor modifications of the procedures described by Damha et al (30) (for ribonucleoside bound CPG) and Pon et al (31) (for deoxythymidine bound CPG). Final nucleosides loadings were in the range of[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] .tmol/g and are given inTable 2. A typical preparation of 5'-MMT-uridine bound LC-AA-CPG follows: LCAA-CPG (5 g) was slowly stirred or shaken in a solution of 3 % trichloroacetic acid in dichloroethane at room c Analysis of a Nuclease P1 digest of bRNA F on a 24% polyacrylarnide/7 M urea gel.…”

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confidence: 99%

Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates

et al. 1992

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The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo.

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Modification of guanine bases by nucleoside phosphoramidite reagents during the solid phase synthesis of oligonucleotides

Cited by 57 publications

References 31 publications

5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.

5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.

Dna Systems for B-Z Transition and Their Significance as Epigenetic Model: The Fundamental Role of the Methyl Group

Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates

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