Specific Recognition of CG Base Pairs by 2-Deoxynebularine Within the Purine-Purine-Pyrimidine Triple-Helix Motif.

摘要

The sequence-specific recognition of double-helical DNA by purine-rich oligodeoxyribonucleotide-directed triple-helix formation is limited 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 order N.CG approx. N.AT >>N.GC approx. N.TA (PH 7.4, 37 deg 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 deg 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-I (10 mM NaCl, 1 mM spermine tetrahydrochloride, 50 mM Tris-acetate, pH 7.4, 4 deg C) compared to an A.CG base triplet mismatch.