Gargallo R, Vives M, Tauler R, Eritja R
Department of Analytical Chemistry, University of Barcelona, E-08028 Barcelona, Spain.
Biophys J. 2001 Nov;81(5):2886-96. doi: 10.1016/S0006-3495(01)75929-2.
2-Aminopurine (P) is a mutagen causing A.T to G.C transitions in prokaryotic systems. To study the base-pairing schemes between P and cytosine (C) or thymine (T), two self-complementary dodecamers containing P paired with either C or T were synthesized, and their protonation equilibria were studied by acid-base titrations and melting experiments. The mismatches were incorporated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C or T. Spectroscopic data obtained from molecular absorption, circular dichroism (CD), and molecular fluorescence spectroscopy were analyzed by a factor-analysis-based method, multivariate curve resolution based on the alternating least squares optimization procedure (MCR-ALS). This procedure allows determination of the number of acid-base species or conformations present in an acid-base or melting experiment and the resolution of the concentration profiles and pure spectra for each of them. Acid-base experiments have shown that at pH 7, 150 mM ionic strength, and 37 degrees C, both C and P are deprotonated. At pH near 4, the majority of species shows C protonated and P deprotonated. Finally, at pH values near 3, the majority of species shows both protonated C and P. These results are in agreement with NMR studies showing a wobble geometry for the P x C base pair and a Watson-Crick geometry for the P x T base pair at neutral pH. Melting experiments were carried out to confirm the proposed acid-base distribution profile. For the sequence including the P x T mismatch, only one transition was observed at neutral pH. However, for the sequence including the P x C mismatch, two transitions were detected by CD but only one by molecular absorption. This behavior agrees with that observed by other authors for oligonucleotides of similar sequence and suggests the following sequence of conformational changes during melting: duplex --> hairpin --> random coil.
2-氨基嘌呤（P）是一种诱变剂，在原核系统中可导致A.T到G.C的转换。为了研究P与胞嘧啶（C）或胸腺嘧啶（T）之间的碱基配对方案，合成了两个包含与C或T配对的P的自互补十二聚体，并通过酸碱滴定和熔解实验研究了它们的质子化平衡。错配被引入自互补序列d（CGCPCCGGXGCG）中，其中X为C或T。通过基于因子分析的方法，即基于交替最小二乘优化程序的多元曲线分辨（MCR-ALS），对从分子吸收、圆二色性（CD）和分子荧光光谱获得的光谱数据进行了分析。该程序允许确定酸碱或熔解实验中存在的酸碱物种或构象的数量，并解析它们各自的浓度分布和纯光谱。酸碱实验表明，在pH 7、150 mM离子强度和37℃下，C和P均去质子化。在pH接近4时，大多数物种显示C质子化而P去质子化。最后，在pH值接近3时，大多数物种显示C和P均质子化。这些结果与核磁共振研究一致，该研究表明在中性pH下，P x C碱基对呈摆动几何形状，P x T碱基对呈沃森-克里克几何形状。进行熔解实验以确认所提出的酸碱分布概况。对于包含P x T错配的序列，在中性pH下仅观察到一个转变。然而，对于包含P x C错配的序列，通过CD检测到两个转变，但通过分子吸收仅检测到一个转变。这种行为与其他作者对类似序列的寡核苷酸观察到的行为一致，并表明熔解过程中构象变化的顺序如下：双链体→发夹→无规卷曲。
