Spectroscopic study of the interaction of actinomycin D with oligonucleotides carrying the central base sequences -XGCY- and -XGGCCY- using multivariate methods

10 pages, 5 figures, 1 table.-- PMID: 17123067 [PubMed].-- Published online Nov 23, 2006.Supporting information (4 pages, 3 suppl. figures) available at: http://www.springerlink.com/content/870650451902431q/MediaObjects/216_2006_946_MOESM1_ESM.docThe interactions of actinomycin D (ACTD) with the oligonucleotides 5′-CAAAGCTTTG-3′, 5′-CATGGC CATG-3′ and 5′-TATGGCCATA-3′ were investigated by means of acid–base titrations and mole-ratio and melting experiments monitored by molecular absorption and circular dichroism (CD) spectroscopies. For each experiment, CD and molecular absorption spectra were recorded at each point in the experiment, and later analyzed via appropriate multivariate data analysis methods. The study of the interactions between these oligonucleotides and ACTD at 25°C showed the formation of an interaction complex with a stoichiometry of 1:1 (ACTD:duplex) and values for the log(formation constant) of 5.1 ± 0.3, 6.4 ± 0.2, and 5.6 ± 0.2, respectively. An additional interaction complex at higher temperatures was also detected, which might be related to the single-stranded forms of the oligonucleotides.We acknowledge two grants from the Spanish Ministerio de Educación y Ciencia (projects BFU2004-02048/BMC and BQU2003-0191).Peer reviewe

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

11 pages, 9 figures, 1 scheme, 1 table.-- PMID: 11606299 [PubMed].-- PMCID: PMC1301753.2-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.This study was supported by the Ministerio de Educacio´n y Cultura (grant DGICYT PB96–0377) and the Generalitat de Catalunya (grant 1999SGR-00048). M. V. also thanks the University of Barcelona for a Ph.D. grant.Peer reviewe

Synthesis, stability, and protonation studies of a self-complementary dodecamer containing the modified nucleoside 2'-deoxyzebularine

17 pages, 6 figures, 1 scheme.-- PMID: 14691937 [PubMed].-- Available online Nov 3, 2003.The nucleoside 2'-deoxyzebularine (K) was incorporated into the self-complementary dodecamer 5-CGTACGKGTACG-3 by solid-phase 2-cyanoethylphosphoramidite chemistry using dimethoxytrityl (DMT) as the 5-hydroxyl protecting group. Standard synthesis cycles using trichloroacetic acid and short ammonia treatment (50ºC for 30 min) were found to be the optimal conditions to obtain the desired dodecamer with minimum acid and basic degradation of the acid- and base-sensitive 2-pyrimidinone residue. The protonation equilibria of the K nucleoside and of the dodecamer at 37ºC were studied by means of spectroscopically monitored titrations. For the K nucleoside, a pKa value of 3.13 0.09 was obtained. For the dodecamer, four acid-base species were found in the pH range 2-12, with pKa values of 9.60 0.07, 4.46 0.16, and 2.87 0.19. Melting experiments were carried out to confirm the proposed acid-base concentration profiles. Finally, kinetic experiments were also carried out at several pH values to evaluate the stability of the K nucleoside and of the dodecamer. An increased stability was shown by the K nucleoside when incorporated into the dodecamer. Multivariate methods based on both hard- and soft-modeling were applied for the analysis of spectroscopic data, allowing the estimation of concentration profiles and pure spectra.Ministerio de Educación y Cultura; Grant Number: MCYT BQ42000-0788, Generalitat de Catalunya; Grant Number: 2001SGR00056.Peer reviewe

Multivariate resolution of NMR labile signals by means of hard- and soft-modelling methods

12 pages, 5 figures, 1 table.-- Printed version published Aug 25, 2003.-- Papers presented at the 8th International Conference on Chemometrics and Analytical Chemistry (Seattle, Washington, United States, Sep 22-26, 2002).One of the difficulties frequently encountered when studying acid–base equilibria with NMR spectroscopy is the labile behaviour of the measured signal, which hinders the application of bilinear multivariate data analysis methods. In this work, a mathematical transformation is proposed for the conversion of NMR labile signals to inert signals, which make possible the application of multivariate data analysis methods, based on bilinear data models. The procedure has been applied to the analysis of NMR data corresponding to the acid–base equilibria of nucleotides dCMP and dGMP. Both hard-modelling (EQUISPEC) and soft-modelling (MCR-ALS) approaches have been applied for the analysis and resolution of transformed bilinear NMR data matrices.This research was supported by the Spanish MCYT (BQU2000-0788) and by the Generalitat de Catalunya (2001SGR0056).Peer reviewe