Stilz H U, Dervan P B
Beckman Institute, California Institute of Technology, Pasadena 91125.
Biochemistry. 1993 Mar 9;32(9):2177-85. doi: 10.1021/bi00060a008.
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 approximately N.AT >> N.GC approximately 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.
通过寡脱氧核糖核苷酸定向三链体形成对双螺旋DNA进行序列特异性识别主要限于嘌呤序列。在嘌呤 - 嘌呤 - 嘧啶三链体结构的磷酸 - 脱氧核糖位置的几何限制范围内，模型构建研究表明，脱氧核糖核苷2'-脱氧新制癌菌素（dN）可能与沃森 - 克里克胞嘧啶 - 鸟嘌呤（CG）或腺嘌呤 - 胸腺嘧啶（AT）碱基对中的胞嘧啶（C）或腺嘌呤（A）形成一个特定的氢键。通过自动化方法将2-脱氧新制癌菌素（dN）掺入富含嘌呤的寡脱氧核糖核苷酸中。从亲和切割分析发现，嘌呤 - 嘌呤 - 嘧啶（Pu.Pu.Py）三链体中碱基三联体的稳定性按以下顺序降低：N.CG≈N.AT >> N.GC≈N.TA（pH 7.4，37℃）。含有两个N残基的寡脱氧核糖核苷酸被证明能在质粒DNA内特异性结合人免疫缺陷病毒基因组的一个单一15碱基对位点，该位点在嘌呤序列内含有两个CG碱基对。这种结合事件发生在生理相关的pH和温度条件下（pH 7.4，37℃），并证明了这种新碱基的实用性。定量亲和切割滴定表明，在所研究的特定序列中，与A.CG碱基三联体错配相比，N.CG碱基三联体相互作用导致局部三链体结构在1 kcal·mol-1下稳定（10 mM NaCl，1 mM四盐酸精胺，50 mM Tris - 乙酸盐，pH 7.4，4℃）。
