SPECIFIC RECOGNITION OF CG BASE-PAIRS BY 2-DEOXYNEBULARINE WITHIN THE PURINEA.PURINE.PYRIMIDINE TRIPLE-HELIX MOTIF

The sequence-specific recognition of double-helical DNA by oligodeoxyribonucleotide-directed triple-helix formation is limited mostly to purine tracts. Within the geometric constraints of the phosphate-deoxyribose position of a purine.purine.pyrimidine triple-helical structure, model building studies suggested that the deoxyribonucleoside 2'-deoxynebularine (dN) might form one specific hydrogen bond with cytosine (C) or adenine (A) of Watson-Crick cytosine-guanine (CG) or adenine-thymine (AT) base pairs. 2-Deoxynebularine (dN) was incorporated by automated methods into purine-rich oligodeoxyribonucleotides. From affinity cleavage analysis, the stabilities of base triplets within a purine.purine.pyrimidine (Pu.Pu.Py) triple helix were found to decrease in the order N.CG is similar to N.AT >> N.GC is similar to N.TA (pH 7.4, 37-degrees-C). Oligodeoxyribonucleotides containing two N residues were shown to bind specifically within plasmid DNA a single 15 base pair site of the human immunodeficiency virus genome containing two CG base pairs within a purine tract. This binding event occurs under physiologically relevant pH and temperature (pH 7.4, 37-degrees-C) and demonstrates the utility of the new base. Quantitative affinity cleavage titration reveals that, in the particular sequence studied, an N.CG base triplet interaction results in a stabilization of the local triple-helical structure by 1 kcal.mol-1 (10 mM NaCl, 1 mM spermine tetrahydrochloride, 50 mM Tris-acetate, pH 7.4, 4-degrees-C) compared to an A.CG base triplet mismatch.