Spectrometric Study of the Oligodeoxyribonucleotide Protonation in Aqueous Solution

UV absorption spectra and circular dichroism spectra of aqueous solutions of cytosinand thymine-containing single-stranded Oligodeoxyribonucleotide 5'-CCTTTCCTTTTCCTTTCC-3'(ckit4) were measured at various рН in the range 3.3–8.9. The chemometric analysis of the multiinstrumental data matrix was carried out. The diagrams of relative contents of complex forms of the DNA molecule absorbing in the studied wavelength range (220–320 nm) were constructed by the ALS–MCR soft simulation procedure without initial postulation of their chemical compositions. The model of equilibrium complex formation describing observed changes in the spectra depending on the solution acidity was developed on the basis of the matrix method. Intrinsic protonation constants of the oligonucleotide ckit4 were calculated. The formation of intramolecular complexes between cytosine С.С bases in the studied DNA molecule is of a cooperative nature, and their subsequent protonation is an anticooperative process. DOI: 10.1134/S1070363210030205 Cooperativity of the ligand binding by a central molecule of a complex-forming agent plays a defining role in a number of biologically important processes [1]. A number of mathematical models have been developed to describe such interactions with infinite polymers [2–6]. The matrix method [7] has been offered for the calculation of equilibrium constants of the ligand cooperative binding by homogeneous oligomers. Alongside with so-called “rigid” simulation methods for the description of conformation and acid– base conversions of biopolymers, chemometric methods of the “soft” simulation [8–11] are widely used. The chemometric analysis is integrated in the MCR–ALS (Multivariate Curve ResolutionAlternating Least Squares) procedure [12]. To examine the possibility of simulating a cooperative complex formation, in the present work we have considered a specific example of folding a single-stranded oligomer with the formation of an intramolecular structure in the course of the cytosincontaining DNA protonation. It is known that cytosine-rich DNA chains in weakly acid or even in neutral aqueous solutions can form tetraplexes, which bear the name imotif [13–17]. The formation of the i-motif structure by the oligodeoxyribonucleotide 5'-CCTTTCCTTTTCCTTTCC-3' (ckit4), which was selected as the object of study in the present work, has been shown earlier by the NMR method [18]. It is supposed that in the case of ckit4 this structure consists of 4 locks organized from 2 parallel duplexes fastened in the antiparallel orientation. The structure is stabilized due to formation of intramolecular complexes between protonated and free cytosine bases С.С (see scheme). Synthetic oligomers of definite length and sequence have recently found a wide application as models of the polymeric DNA and RNA [19]. The oligonucleotides forming i-motif structures have a transcriptional activity in some genes and also find application in nanotechnologies [20–23]. Though i-motif structures are preferably formed in weakly acid solutions, there are proves of their existence in vivo. It is supposed that i-motif structures are formed in human chromosomes and in promoting locales of some genes [24]. The present work was fulfilled with the aim of developing a procedure for joint application of ALS– MCR and matrix simulation methods, taking, as a case in point, the study of the mechanism of protonation of a singlestranded DNA oligomer in an aqueous solution on the basis of spectrometric titration data. Expansion of multivariable dependences by means of the MCR–ALS procedure [12]. The use of the chemometric analysis allows spectrometric data tj be described using so-called “soft model.” The soft simulation of equilibrum interconversions consists in constructing diagrams of concentration profiles of abstract spectral forms participating in interconversions in a system. These profiles can be used to build the model of the system having a physical sense, so-called “rigid model.” Parameters of a rigid model (equilibrium constants) in the case of a complex formation with non-polymeric ligands can be calculated from diagrams constructed with the use of evolving factor analysis [25–27]. It has been shown that in the case of a single-stranded polymer [28, 29] parameters of equilibrium reactions in a system with a concentration selectivity can be calculated immediately from the diagram constructed by means of ALS– MCR. In the absence of selectivity, the parameters of a complex formation involving a polydentate molecule are calculated from diagrams ALS–MCR with a constant error [30]. Calculation of intrinsic constants of the oligonucleotide protonation by a matrix method. In the present work the modified matrix model [7] was used to calculate equilibrium protonation constants of the oligomer under study. The basic allowance of the matrix model consists in the fact that the central molecule contains vacancies in fixed positions, which can add ligands. In the case under consideration the process is in essence the adsorption of protons by a DNA molecule caused by the presence of specific functional groups. In a neutral medium the molecule has the conformation of a stretched helix, and when рН decreases, the molecule is rolled up due to formation of intramolecular hydrogen bonds between protonated and neutral cytosine bases (see scheme). Thus an intramolecular complex is formed, in which half of the bound bases is capable to add protons, but constants of their protonation differ from the corresponding constants for the free bases in a neutral medium. According to the matrix method, the model of the complex formation with an oligomer, capable of essential variation of its molecular structure depending on the complex formation degree, can be presented as follows. Let complexes [PnHn] (n = 0, 1, 2, ..., N) are formed in equilibrium conditions by the addition of ligands (in our case of protons) to vacancies of a polynucleotide molecule (Pn). The whole set of complexes is present irrespective of concentrations of components, and the relative oligomer distribution between forms is defined by the stability of corresponding compounds. The set of constants describing stability of each configuration of [PnHn] complexes can be presented in the form of the product of equilibrium constants of ligand addition to separate vacancies (βki) [Eq. (1)]. (1) To calculate βki, we shall introduce a matrix of configurations M(2, N). A matrix row Mk reproduces one of possible configurations of the complex with a sequence of zeros in positions with free vacancies, and with a sequence of unities in positions with vacancies occupied by ligands. The expression for the equilibrium constant of a ligand addition to a particular vacancy looks like Eq. (2).