Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine.
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.
Gargallo
R
R
Department of Analytical Chemistry, University of Barcelona, E-08028 Barcelona, Spain. raimon@apolo.qui.ub.es
Vives
M
M
Tauler
R
R
Eritja
R
R
D015075:2-Aminopurine / Q000737:chemistry*; D020137:Base Pair Mismatch / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics; D002942:Circular Dichroism; D003596:Cytosine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016018:Least-Squares Analysis; D008956:Models, Chemical; D015999:Multivariate Analysis; D016296:Mutagenesis / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics*; D011522:Protons; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry
D009838:Oligodeoxyribonucleotides; D011522:Protons; D015075:2-Aminopurine; D003596:Cytosine; D013941:Thymine