Synthesis and cleavage of oligodeoxynucleotides containing a 5- hydroxyuracil residue at a defined site

June Fujimoto, Linh Tran, Lawrence Sowers

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous Ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amine groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by β-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.

Original languageEnglish (US)
Pages (from-to)1254-1258
Number of pages5
JournalChemical Research in Toxicology
Volume10
Issue number11
DOIs
StatePublished - 1997
Externally publishedYes

Fingerprint

Oligodeoxyribonucleotides
Uracil-DNA Glycosidase
Oligonucleotides
DNA
Ammonia
Hydrolysis
Acetylation
Oxidation
Uracil
Cytosine
High performance liquid chromatography
5-hydroxyuracil
Acidity
Hydroxyl Radical
Amines
Protons
Digestion
Substitution reactions
High Pressure Liquid Chromatography
Derivatives

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Synthesis and cleavage of oligodeoxynucleotides containing a 5- hydroxyuracil residue at a defined site. / Fujimoto, June; Tran, Linh; Sowers, Lawrence.

In: Chemical Research in Toxicology, Vol. 10, No. 11, 1997, p. 1254-1258.

Research output: Contribution to journalArticle

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