Synthesis, Structure, and Deoxyribonucleic Acid Sequencing with a Universal Nucleoside: 1-(2’-Deoxy-β-D-ribofuranosyl)-3-nitropyrrole

Donald E. Bergstrom, Peiming Zhang, Pascal H. Toma, Philip C. Andrews, Ruthann Nichols

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


A nucleoside analogue, 1-(2’-deoxy-β-D-ribofuranosyl)-3-nitropyrrole (4) was designed to function as a universal replacement for any of the natural nucleosides in DNA sequences. Compound 4 was synthesized by the reaction of 3-nitropyrrole with sodium hydride and 1-chloro-2-deoxy-3,5-di-O-toluoyl-D-erythropentofuranose, and the structure was confirmed by X-ray diffraction. Nucleoside 4 was transformed to 1-(2’-deoxy-5’-dimethoxytrityl-β-D-ribofuranosyl)-3-nitropyrrole-3’-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (6) for incorporation into oligonucleotides by conventional synthesis protocols. Analogues of the oligonucleotide, 5’-d(CGT AAT CAG AAA ACA AT)-3’ with nucleoside 4 replacing the natural nucleosides in up to 9 positions were constructed and tested as primers for dideoxy sequencing. Sequencing studies show that a substantial number of nucleotides can be replaced by 4 without loss of primer specificity. Sequencing primer 4 with substitutions of 4 at the third position in each of four codons gave a sequencing ladder comparable to primer 1, the exact match, while a 256-fold degenerate oligonucleotide mixture (primer 2) gave an unreadable sequencing ladder. Primers containing two or more mismatches gave indecipherable results. A unique property of 4 was its ability to replace long strings of contiguous nucleosides and still yield functional sequencing primers. Sequences with three (primer 8), six (primer 9), and nine (primer 10) 4 substitutions all gave readable sequencing ladders. Optical thermal profiles obtained for the oligonucleotide pairs 5’-d(C2T5XT5G2)-3’ and 5’-d(C2A5YA5G2)-3’ (X, Y = A, C, G, T, and 4) fit the normal sigmoidal pattern observed for the DNA duplex to single strand transition. The melting temperatures (Tm) of the oligonucleotides containing X-4 base pairs (X = A, C, G or T, Y = 4) all fell within a 3 °C range of one another. However, the Tm’s were significantly lower than the corresponding sequences containing only A-T and G-C base pairs. The ability of 4 to associate by stacking with a natural nucleoside was confirmed by constructing the dimer d(Ap4) and determining the CD spectrum.

Original languageEnglish (US)
Pages (from-to)1201-1209
Number of pages9
JournalJournal of the American Chemical Society
Issue number4
StatePublished - 1995
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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