M. Vives

Study of the intercalation equilibrium between the polynucleotide poly(adenylic)-poly(uridylic) acid and the ethidium bromide dye by means of Multivariate Curve Resolution and the multivariate extension of the continuous variation and mole ratio methods

The intercalation equilibrium between the polynucleotide poly(adenylic)-poly(uridylic) acid and the dye ethidium bromide was studied using molecular absorption spectrophotometry, spectrofluorimetry, and circular dichroism. The presence of an intercalation complex, its pure spectrum, and its concentration profile were clearly deduced using Multivariate Curve Resolution and a multivariate extension of the classical continuous variation and mole-ratio methods. The value of the polynucleotide/dye ratio (r(poly/dye)) in the intercalation complex and of its apparent formation log constant were estimated to be 4 and 6.2, respectively. The proposed multivariate methodology allowed an accurate qualitative and quantitative description of the intercalation process.

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.

Resolution of temperature dependent conformational multiequilibria processes

Abstract Multivariate Curve Resolution (MCR) is applied to the study of temperature dependent conformational multiequilibria evolving processes. Experimental data sets are obtained by UV spectral monitoring of the melting behavior of the heteropolynucleotide poly(adenylic) acid–poly(uridylic) acid (poly(A)–poly(U)) and of the hompolynucleotides poly(adenylic) (poly(A)) acid and poly(urydilic) acid (poly(U)), i.e., recording UV spectra at different temperatures during the melting process of these polynucleotides. Separate study of every individual melting experiment by MCR did not give a satisfactory resolution of the heteropolynucleotide poly(A)–poly(U) melting process because of unresolved rotational ambiguities and rank deficiency problems. Conversely, the simultaneous MCR analysis of the melting process of poly(A)–poly(U) heteropolynucleotide together with the separate melting processes of the poly(A) and poly(U) homopolynucleotides, allowed the resolution of the species profiles and the elimination of the rank deficiency problems present in the individual analysis of the melting behavior of poly(A)–poly(U).

Study of the interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)Pt(II) complex with poly(I), poly(C) and poly(I)·poly(C)

The interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)platinum(II) (Ptpyrr) with the polynucleotides poly(I), poly(C) and poly(I) x poly(C) acids was studied by circular dichroism, molecular fluorescence and (1)H NMR spectroscopies. Multivariate Curve Resolution, a factor analysis method, was applied for the analysis and interpretation of spectroscopic data obtained in mole ratio and kinetics studies. This procedure allows the determination of the number of different interaction complexes present during the experiments and the resolution of both concentration profiles and pure spectra for all of them. Two different interaction complexes were observed at the experimental conditions studied. The first one, at low Ptpyrr:polynucleotide ratio (r(Ptpyrr:poly)) values, corresponds to the interaction of Ptpyrr with hypoxanthine bases in the poly(I) moiety. This interaction leads to the destabilization and dissociation of the double-stranded conformation. The second complex was observed at higher r(Ptpyrr:poly) values and corresponds to the interaction of Ptpyrr to cytosine bases in poly(C) moiety. The formation of both complexes showed that the interaction of Ptpyrr with hypoxanthine bases occurred at the first stages of the reaction and with cytosine bases at longer reaction times. The results obtained show the utility of the Multivariate Curve Resolution approach for the analysis of data obtained by monitoring spectroscopically the interaction equilibria of platinum compounds with nucleic acids.

Study of the interaction of a cis-dichloroaminopyrrolidine Pt(II) complex and the polynucleotide poly(I)–poly(C) acid by means of 1H-NMR and multivariate curve resolution

Multivariate curve resolution is proposed for the analysis of spectroscopic data obtained from kinetic studies on the interaction of compounds analogous to cis-platinum (cisplatin) and nucleic acids. In particular, the interaction between the cis-dichloro-(1,2-diethyl-3-aminopyrrolidine) Pt(II) compound (Ptpyrr) and the polynucleotides poly(inosinic) (poly(I)), poly(cytidylic) (poly(C)) and poly(I)-poly(C) acids were monitored by 1 H-NMR spectroscopy. Multivariate curve resolution was applied for the first time to analyze 1 H-NMR spectroscopic data, which allowed to determine the number of interaction complexes and the recovery of both kinetic concentration profiles and pure NMR spectra for all the detected species. For poly(I) and poly(C), two complexes were detected upon interaction with Ptpyrr, which corresponded to the mono-adduct and to the bis-adduct complex formation. For poly(I)-poly(C), three components were detected, which corresponded to: (a) the initial non-platinated double-stranded polynucleotide, (b) to a mixture of non-platinated single-stranded poly(C) and platinated poly(I), and (c) to a mixture of poly(C) and poly(I), both platinated. Multivariate curve resolution chemometric procedure based on an alternating least-squares optimization (MCR-ALS) is, thus, proposed as a useful tool for the recovery of analytical information from 1 H-NMR data.

Study of the influence of metal ions on tRNAPhe thermal unfolding equilibria by UV spectroscopy and multivariate curve resolution

The influence of metal ions (Na(+), Mg(2+) and Cd(2+)) on the thermal unfolding of phenylalanine transfer ribonucleic acid (tRNA(Phe)) was studied by UV spectroscopy-monitored melting experiments. Absorbance data were obtained during the unfolding process in the range 220-340 nm and later analyzed by a multivariate curve resolution approach (MCR-ALS) based on factor analysis. This procedure determines the number of spectroscopically distinct conformations present during the unfolding process and reveals their concentration profiles and pure spectra, without any initial assumption having to be made about the number of steps in the unfolding pathway. From the concentration profiles and pure spectra, information such as T(m) values can be recovered. The results were compared with those obtained previously in spectroscopic and calorimetric unfolding experiments, showing that the multivariate approach recovers information that complements that obtained in traditional spectroscopic melting experiments.