Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine

2-Aminopurine (P) is a mutagen causing A(.)T G(.)C transitions in prokaryot ic systems. To study the base-pairing schemes between P and cytosine (C) or thymine M, two self-complementary dodecamers containing P paired with eith er C or T were synthesized, and their protonation equilibria were studied b y acid-base titrations and melting experiments. The mismatches were incorpo rated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C o r T. Spectroscopic data obtained from molecular absorption, circular dichro ism (CD), and molecular fluorescence spectroscopy were analyzed by a factor -analysis-based method, multivariate curve resolution based on the alternat ing least squares optimization procedure (MCR-ALS). This procedure allows d etermination 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 sho wn that at pH 7, 150 mM ionic strength, and 37 degreesC, both C and P are d eprotonated. 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 sh owing a wobble geometry for the P-C base pair and a Watson-Crick geometry f or the P-T base pair at neutral pH. Melting experiments were carried out to confirm the proposed acid-base distribution profile. For the sequence incl uding the P-T mismatch, only one transition was observed at neutral pH. How ever, for the sequence including the P-C mismatch, two transitions were det ected by CD but only one by molecular absorption. This behavior agrees with that observed by other authors for oligonucleoticles of similar sequence a nd suggests the following sequence of conformational changes during melting : duplex --> hairpin --> random coil.