Influence of Magnetic Microparticles Isolation on Adenine Homonucleotides Structure

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Materials 2014, 7, ; doi: /ma Article OPEN ACCESS materials ISSN Influence of Magnetic Microparticles Isolation on Adenine Homonucleotides
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Materials 2014, 7, ; doi: /ma Article OPEN ACCESS materials ISSN Influence of Magnetic Microparticles Isolation on Adenine Homonucleotides Structure Monika Kremplova 1, Dana Fialova 1, Lukas Nejdl 1, David Hynek 1,2, Libuse Trnkova 1,2,3, Jaromir Hubalek 1,2, Rene Kizek 1,2 and Vojtech Adam 1,2, * Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno CZ , Czech Republic; s: (M.K.); (D.F.); (L.N.); (D.H.); (L.T.); (J.H.); (R.K.) Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, Brno CZ , Czech Republic Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno CZ , Czech Republic * Author to whom correspondence should be addressed; Tel.: ; Fax: Received: 17 November 2013; in revised form: 16 December 2013 / Accepted: 17 February 2014 / Published: 25 February 2014 Abstract: The electroactivity of purine and pyrimidine bases is the most important property of nucleic acids that is very useful for determining oligonucleotides using square wave voltammetry. This study was focused on the electrochemical behavior of adenine-containing oligonucleotides before and after their isolation using paramagnetic particles. Two peaks were detected peak A related to the reduction of adenine base and another peak B involved in the interactions between individual adenine strands and contributes to the formation of various spatial structures. The influence of the number of adenine bases in the strand in the isolation process using paramagnetic particles was investigated too. Keywords: adenine; adenine interaction; magnetic beads; square wave voltammetry; aptamer; biosensor; nanobiotechnology Materials 2014, Introduction It is well known that a DNA molecule is composed of nucleotides, the basic building blocks of DNA. The nucleotide consists of 2-deoxy-β-D-ribose, phosphate group and purine (adenine, guanine) or pyrimidine (cytosine, thymine) base [1]. For the study of nucleic acids, various instrumental methods such as ultraviolet-visible (UV/Vis) spectrometry, electrophoretic methods, polymerase chain reaction (PCR) and circular dichroism are used [2 6]. Besides these methods, electrochemical methods are also possible to use [7]. Electroactivity of nucleic acids bases on mercury electrodes is one of the most sensitive ones. Palecek was the first who used modern oscillographic polarography for successful detection of redox DNA signals [8 11]. Since then, great progress and development has been made in the electrochemistry of nucleic acids on various electrodes [7,12,13]. The attention is paid to various electrochemical methods using a mercury electrode as a working one including linear sweep and cyclic polarography/voltammetry (elimination polarography/voltammetry), differential pulse polarography/voltammetry, square wave polarography/voltammetry, alternating currents (AC) polarography/voltammetry, and chronopotentiometry for analysis of DNA [7,14]. The connection of adsorptive transfer stripping technique with the above-mentioned methods is very promising tool for studying nucleic acids [14]. Square wave voltammetry (SWV) is one of the most sensitive electrochemical methods for determination of oligonucleotides (ODNs) [15 17]. SWV is generally the best choice among all pulse methods, because it offers background suppression combined with the effectiveness of differential pulse voltammetry (DPV), slightly greater sensitivity than that of DPV, much faster scan times, and applicability to a wider range of electrode materials and systems. The most reproducible behavior and lowest detection limits are generally found on mercury surfaces [18]. From the point of view of DNA electroanalysis, this method belongs to the most sensitive label free ones with the lowest limits of detection [19 24]. Aptamers represent one of the specific parts of the whole wide oligonucleotide group. They are defined as molecules of ribonucleic (RNA) and single-strand (ss) deoxyribonucleic (ssdna) acids or peptides that can bind to targets with high affinity and specificity due to their specific three-dimensional structure [25]. Especially RNA and ssdna aptamers can differ from each other in the sequence and the folding pattern, although they bind to the same target [26]. Applications of aptamers in the area of biosensing have been widely developed during last decade. Aptamers have been studied as a biomaterial in numerous investigations concerning their use as diagnostic and therapeutic tools, biosensing probe, and in the development of new drugs, mainly drug delivery systems [25,27,28]. A phenol-chloroform extraction is considered a standard method for nucleic acid isolation, but this method needs special laboratory equipment, and it is also time-consuming. For these reasons, the research has been focused on other alternative methods for biomolecules isolation [29 31]. One possible way is the isolation of nucleic acids using paramagnetic and/or superparamagnetic particles (MPs) [32,33]. Paramagnetic particles are the particles with size ranges from nm to mm, responding to an external magnetic field and facilitating the binding of bioactive molecules due to their modified surface by biocomponents [34 36]. The main advantages of MPs are as follows: easy to use, fast sample preparation without centrifugation and dialysis. Physicochemical properties of MPs are very important for their biological applications. The most commonly used MPs in biosensor applications are Materials 2014, composed of ferrous oxide or ferric oxide [37]. Nanoparticles of ferric oxide can provide adequate surface for binding biomolecules. There are two methods of surface modifications of MPs. The first method is based on the electric envelope layer, which ensures the electrostatic adsorption of biomolecules [38]. The second method of surface treatment of paramagnetic particles is based upon biomolecules anchored on the particle, which are able to bind the target biomolecule specifically [23,39]. The isolation of adenine containing nucleotides is based on this principle, because oligo (deoxythymine) 25 is anchored on the surface of MPs and can be hybridized by molecules of adenine-containing nucleotides [12]. The aim of our study was to investigate the electrochemical behavior of adenine-containing nucleotides on the surface of a mercury electrode and their behavior after separation using paramagnetic particles. The description of this phenomenon could be useful for understanding of some aspects of isolation processes using magnetic materials. 2. Results and Discussion 2.1. Square Wave Voltammetry of Adenine-Containing Oligonucleotides As it was mentioned above, square wave voltammetry is one of the most frequently used and sensitive electrochemical methods for determination of DNA [22,40,41]. For oligonucleotides, containing different bases, both CA and G peaks can be measured using this method. Current peaks of guanine can be obtained on mercury or amalgam electrodes by re-oxidation of the product of guanine reduction, 7,8-dihydroguanine [42]. Inset of Figure 1A shows typical voltammograms of oligonucleotide that contains adenine bases. Homonucleotide da 15 was selected as an example. Two peaks were recorded: the first one at potential 1.37 V, well-known and often described as peak A (related to adenine reduction on HMDE) and the second one recorded at potential 1.10 V, described as peak B in this study. In Figure 1A, it is also shown that the potentials of both the peaks are measured within the concentration range from 0.09 to 50 µg/ml of da 15. For peak A, there are no changes in the peak potential with the increasing concentration of the adenine oligonucleotide da 15. On the contrary, the potentials of peak B are shifted to more positive values with increasing concentration of da 15. The absolute potential difference, determined in the concentration range from 0.09 to 50 µg/ml of da 15, was V. Palecek defined and described the peak II SW, which has high similarity (in accordance with the obtained voltammograms) with our peak B, only in a native double-stranded DNA structure [24,43]. In our samples, only adenine single-stranded oligonucleotides were occurring. Due to this fact, we assumed the detection of the peak B as a result of formation of more complex structures of the adenine chains. The interactions in the nucleosides structures, in general, can be considered as electrostatic interactions. These interactions lead to the formation of planar base-pair structures as those of the Watson-Crick (WC) type [44]. One of them, the formation of the trans-configuration of adenine-adenine pairs, is expected, when the phosphodiester bonds have the same orientation. In addition, the non-covalent interactions that stabilize both DNA and RNA can be divided into three groups as follows: hydrogen bonding, base-stacking, and electrostatic effect of the strands [44]. The base-stacking interaction is also recognized as crucial to stabilize the structure of nucleic acids. It Materials 2014, is particularly important to note that the planar structures are generally more stable than stacked structures. These effects have been shown in numerous studies of behavior of the adenine bases and there are several possibilities of formation of complexes [45 51], mainly possible hydrogen bonds in A A structures [52,53]. Figure 1. (A) Dependence of position of peak A and B of adenine-containing nucleotide da15. Red points indicate potential values of peak A, green points indicate potential values of peak B, determined by square wave voltammetry with following parameters: start potential 0.1 V, end potential 1.6 V, deposition potential 0.1 V, accumulation time 720 s, equilibration time 5 s, voltage step V, amplitude 0.02 V, frequency 280 Hz (sweep rate V/s). In inset: typical SW voltammograms of adenine-containing nucleotide da15 at various concentrations; (B) Dependence of relative height and potential of peak A and B on the applied deposition potential for da15 at concentration 25 µg/ml; (C) Dependence of a relative height and potential of peak A and B on the accumulation time for da15 at concentration 25 µg/ml; (D) Influence of applied frequency on intensity of peak A and B for da15 at concentration 25 µg/ml. Two different frequencies were applied, 100 (blue points) and 280 Hz (red points). According to the angle between the planes of two bases, five structural types of pairing of bases can be distinguished: (1) planar, H-bonded; (2) non-planar, H-bonded; (3) T-shaped; (4) planar stacked; and (5) non-planar stacked [54]. The planar base-pair structure for dimers of bare adenine has been observed and assigned to that having two moieties that are doubly hydrogen bonded [53]. Plutzer et al. [53] identified various conformations of adenine pair by IR-UV resonance spectroscopy. Some of the isomers are stabilized by the inter-fragment interactions similar to the H-bonding. Five of Materials 2014, them have hydrogens of NH 2 of the one fragment pointing towards nitrogen atoms of the other fragment. However, the study of structures revealed that these bonds are relatively long and, consequently, are weaker than regular hydrogen bonds [51]. A symmetric H-bonded dimer, labelled as AA-HB1, is the most stable adenine dimer [51,52,54]. The stacked structures, stabilized dimers of adenine nucleosides, have been widely investigated and related to the various energy changes by some authors [50,51]. The results demonstrate that the dimer possesses a stacked structure being stabilized by the formation of hydrogen-bonding network involving the two sugar groups. In many presented variations of adenine and thymine dimers, the adenine dimer AA-ST1 was suggested as the most stable stacked configuration [51]. Dependence of Measured Signals on Different SWV Parameters All measurements were carried out using square wave voltammetry. To use the most sensitive conditions for analysis of the compounds of interest, we optimized some parameters. Deposition potential was the first optimized parameter (Figure 1B). Five different values of potential, at which the effect of sample deposition on the mercury drop on the electrochemical signal was investigated, were selected. The highest and best A signal was detected using the deposition potential of 0.1 V. This potential was used in all further measurements. Other selected deposition potentials showed lower signals or potential shift at different concentrations of the sample occurred. On the other hand, the peak B had the best signal at the potential of 0.3 V. The difference between both the best accumulation potentials indicates the different nature of both signals. Because of the fact that the peak A has been well described and known, we decided to realize next measurements with the accumulation potential of 0.1 V. The second factor in support of this decision was that the SWV scans were performed from 0 to 1.6 V and in such case the accumulation at 0.1 V causes no problems, but only lower signal. The effect of accumulation time of monomeric adenine deoxyribonucleotides on the surface of mercury electrode was the second studied parameter. Figure 1C clearly shows the increasing trend of the electrochemical signal depending on the increasing time of the accumulation. The tested time interval was from 60 to 720 s. The accumulation time of 720 s provided the highest signal and therefore was chosen as the best time of accumulation. The potentials of the signals during the varying accumulation time showed no significant changes. The frequency is a very important parameter in determination of DNA by the square wave voltammetry. Figure 1D shows the effect of frequency on the size and quality of both A and B peaks. Two values of frequency, 100 and 280 Hz, were used in this experiment. The intensity of signal was measured within the concentration range from da to 50 µg/ml. The electrochemical signal at the frequency of 280 Hz was two times higher in comparison to that measured at the frequency of 100 Hz for both the peaks. Therefore, the frequency 280 Hz was used for all further measurements Influence of Number of Adenine Bases on the Electrochemical Signal Thirteen oligonucleotides differing in the number of adenine bases in their sequence were used in this study, oligonucleotides da 2 da 10, then da 15, da 20, da 25, and da 30. For each ODN, calibration curves were determined within the concentration range from 50 ng/ml to 25 μg/ml. The applied Materials 2014, amount of ODN, quantitatively determined using UV/Vis spectrometry [20,55 57], (data is not shown) served as a control. The influence of ODN concentration on peak A and B height was determined. The reduction signal of adenine (peak A) changed linearly with increasing ODN concentration and peak B had quadratic concentration dependences. The slopes of the calibration curves related to peak A depending on the length of the oligonucleotide chain are shown in Figure 2A. From oligonucleotide da 2 to da 4, we can assume that the slope of the calibration curve of peak A have similar values with a slightly declining trend. Generally, the slopes of the calibration curves gradually decreases with the increasing number of adenine bases in the oligonucleotide chain, thus the sensitivity of the method for this determination is limited. Figure 2. (A) Slopes of linear concentration dependencies obtained from evaluation of intensity of peak A within the concentration range from 50 ng/ml to 25 µg/ml of adenine nucleotide; (B) Calculated quadratic dependencies based on the measured data obtained for the peak B within the concentration range from 50 ng/ml to 25 µg/ml of adenine nucleotide; (C) Determined concentrations of adenine nucleotides related to the local maxima/minima of calculated quadratic dependencies for peak B; not restricted to model concentration interval of adenine nucleotide; (D) Dependence of quadratic coefficient calculated from quadratic dependencies on various number of adenine bases in strand; (E) Dependence of linear coefficient of calculated quadratic dependencies on the various number of adenine bases in strand. Calibration curves were also evaluated for the peak B. Figure 2B shows the calculated quadratic calibration curves. The quadratic regression equations were based on the calibration set of data of the individual oligonucleotides with confidence interval at least Generally, the increasing length of the adenine oligonucleotide strand causes reduction of the maximum electrochemical signal and its Materials 2014, shift to the lower intensity. Nevertheless, for the set of da 2 da 7 the shift of the maximum is not relevant and only intensity of the signal decreases. This phenomenon could be explained by the modification of the surface of the electrode by adenine nucleotides. The longer strands (da 7 da 30 ) cause decrease of the intensity of the signal of peak B, which indicates isolation character of longer strands towards forming bonds between individual adenine strands. For short the adenine strands, there is a prerequisite for the pairing of adenine bases between two strands. The electron transport through the strands is a complex process and the transmission onto the surface of the electrode is not fully understood. For the longer adenine strands, there is apparently no connection between adenine bases in two strands, but the adenine-adenine connection can be formed in one strand. From da 8 there is a probability of the intra-molecular binding of adenines due to the conformational freedom in individual adenine strands and thus the electrochemical signal of peak B is lowered. Peak B, as it has been assumed and confirmed by Palecek s experiments [24,43], is connected with the formation of binding between various strands. The shape of the obtained curves changed dramatically for the borderline between da 20 and longer oligonucleotides. The successive lengths of the oligonucleotide strand (da 25 and da 30 ) present only minimal changes in the intensity of the peak B with the increasing concentration of da. This effect could be caused by the forming of relatively stable spatial intra-conformation of individual strands that is stabilized by A A bonds. The formation of inter-strand binding is, in this case, unlikely and unexpected due to high conformational freedom in the individual adenine strands. Such structures could easily cover the surface of the electrode and thus eliminate the response to the concentration changes. For the model of quadratic equations, the concentrations related to the local maxima/minima were calculated (Figure 2C). This calculated concentration is necessary to understand as an optimal amount of individual da for the most creation of space structures based on adenine interactions. As it is obvious from the figure, da 2 da 7 does not change the calculated concentration maxima. It means that the same concentration of da (variable in the length of chain) induces the maximum of peak B measured (variable in the peak intensity), it means maximal spatial arrangement of da molecules. From eight adenines in the strand (da 8 ), the concentration maxima increase rapidly up to twenty adenines, where the breakpoint of the shape of the calculated curves is present. Further, the local minimum is presented for da 25 and da 30. The length of 20 adenines is critical in the obtained curve shape too. As it is indicated in Figure 2B, the shape of the curve is changed with the increasing number of adenines in the strand. The length of twenty adenines is the borderline between the negative and positive quadratic parts of the calculated curves (Figure 2D) and thus limits the shape of the obtained curves. The linear parts of the calculated dependencies (Figure 2B) are shown in Figure 2E. These dependencies directly reflect the changes in the length of the strand. As it was mentioned above, the shape of the calculated curves changes with the increasi
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