FTIR SPECTROSCOPIC STUDY OF POLYETHYLENE GLYCOL AND CALF THYMUS DNA COMPLEX IN DIFFERENT INCUBATION TIME Amal Muftah1, Ali M Bentaleb2, Nagib A Elmarzugi3, Terin Adali2
1National Medical Research Center, 2Near East University,Cyprus , 3IBD-UTM , Malaysia & BTRC/Faculty of Pharmacy Tripoli, Libya
0.30 1330.007 0.003 0.25
1085.139 10.187 945.869 0.917
1294.514 0.011 1250.648 0.298
2291.772 0.053
PEG 400 only
1096.046 22.308
885.917 0.092
1455.945 0.657
0.10
0.25 1455. 336 2.101
0.05
0.20 1717.906 -0.021 2865.949 14.439 0.10
944.770 1.402
836.833 0.109
1786.832 0.462
2929.309 -0.866 3207.006 -0.000 1621.185 -0.029
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1349.426 0.536
3749.394 4.416
2879.722 0.023
DNA-PEG 1:1 48H
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1793.530 1.014
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885.957 1. 528
1717.164 0.247 1541.082 0.073
1978.431 0.062 Absorbance
0.00
-0.05
-0.15
2158.643 0.713
1086.162 9.579 1456.794 0.522
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DNA+PEG 1:1 T=0
1350.356 0.392 945.988 0.918
-0.25
-0.10
1250.584 0.299 1631. 318 -0.083
-0.15
885.924 0.083
2933.166 -0.996 2878.106 0.014
-0.30
1248.744 0. 517
1295.240 0.304
3275.316 -0.138
-0.20
3710.172 0.049 3191.578 0.001
836.399 0.398
-0.35 1293.715 0.018
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1612.181 -0.000
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2030.656 0.162
-0.45 -0.50
1457.220 0.609 1252.160 0.002 1975.721 0.163 1606.395 -0.002 1350.733 0.534 1699.439 0.228
2879.246 -0.044
DNA+PEG 1:1 T=1
2915.453 -0.756
-0.55
2158.673 1.582
1088.713 11.976 946.690 1.174 886.419 0.108
2568.641 0.067 3528.914 -0.000
3210.862 -0.000
837.155 0.276
-0.60 1296.022 0.220
-0.65
1541.200 0.138
Figure 1 FTIR total spectra of polyethylene glycol 400 3800
Characterization of Pure ctDNA Figures 2 shows the FTIR finger print spectrum of ctDNA without the addition of PEG. As can be seen, ctDNA possess characteristic peaks at ( 958, 1017, 1086, 1219, 1313, 1398, 1541, and 1651 cm-1). Ring vibrations of nitrogenous bases (C=O, C=N stretching), PO2 stretching vibrations (symmetric and asymmetric) and deoxyribose stretching of DNA backbone are limited in the spectral region 2000- 750 cm-1. Therefore, this particular region is of interest here in this study. The vibrational bands of DNA at 1716, 1651, 1611, and 1491 cm-1 are assigned to guanine (G), thymine (T), adenine (A) and cytosine (C) nitrogenous bases, respectively . Bands at 1219 and 1086 cm-1 are related to phosphate asymmetric and symmetric vibrations respectively. These are the major bands of pure DNA which are monitored during this study. Changes in these bands (shifting and intensity) upon incubation time were more further studied.
1637.167 0.000
0.0015 0.0010 2229.136 0.011 0.0005
1611.202 -0.001
1651.838 0.111 2015.024 0.007
1541.314 -0.001 A bsorbance
771.299 0.001
1086.208 0.013
958.962 -0.000
1219.010 0.011
0.0000 1716.490 -0.001
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Figure 3 FTIR total spectra of PEG 400 and ctDNA at different incubation time PEG and PO2 Binding Poly ethylene glycol-PO2 interaction was evident from an increase in the absorption and shifting of the PO2 antisymmetric band at 1219 cm-1 in the spectra of the PEGctDNA complexes. The PO2 band at 1219 cm-1 shifted toward higher frequencies after zero time to 1250 cm-1 , and to 1252 cm-1 after 1 hour, and return to 1950 cm-1 after 48 hours. While the band at 1086 cm-1 related to the phosphate symmetric stretching vibration was exhibited a minor absorption changes and shifting to a lower frequency at 1085 after 48 hours, higher frequency at 1088 after 1 hour, and with no shifting changes after zero time upon DNA complexation. PEG and Base Pairs Binding. Some evidences for PEG-base binding observed from the spectral changes for free ctDNA upon PEG complexation. After zero time a minor shifting of the bands at 1716 cm-1(guanine) toward higher wave number 1717 cm-1 , and to 1699 cm-1 after 1 hour, and to 1786 cm-1 after 48 hours. For cytosine at 1491 cm-1, it has been seen that there is high band shift toward lower wave number after zero time (1456 cm-1) , after 1 hour (1457 cm-1), and after 48 hours 1455 cm-1. Over all these changes could be attributed to direct PEG binding to the base pairs of the ctDNA.
DNA only
0.0020 0.005 2187.132
1491.414 -0.001
-0.0005 -0.0010 -0.0015 -0.0020 -0.0025 -0.0030
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Figure 2 FTIR finger print of ctDNA 2100- 700 cm-1
The IR spectral features, and major peaks for PEG 400 figure .1 , ctDNA figure print figure 2 , and PEG-ctDNA complexes samples at different incubation times fig 3 are presented below. Characterization of pure polyethylene Glycol using FTIR As demonstrated in spectrum analysis of pure PEG 400 figure 4.1, which showed many characteristic peaks at (885, 944, 1096, 1146, 1287, 1296,1349, 1556, 1772, 2710, 2886, and 3711 cm-1 ). The strong absorptions of PEG are assigned to the -CH2CH2- stretching around 2886 and 3749 cm-1 which demonstrates the presence of saturated carbons (CH2CH2)n-
1350.297 0.022 1349.749 0.022
0.15
The current study aim is to assess the effect of incubation time, determine mode of action, and binding site interaction between PEGctDNA, and optimization of ctDNA-PEG complexation using FourierTransform Infra-Red (FTIR) spectroscopy.
Complex samples PEG/ctDNA for FTIR were prepared by rapid addition of Equal volumes of ctDNA to PEG solution and kept for three incubation times ( Zero, 1hr, 48 hrs ) to allowing reaction. The composition of complexes is defined by the complex charge ratio, which is the ratio of PEG (positive charge) to DNA negative charge. To improve the stability of the complexes, samples were prepared in buffered purified water, The plots of wave number and absorption of ctDNA in plane vibration related to A-T, G-C base pairs and PO2 stretching vibration versus PEG were obtained after peak normalization.
1350.222 0.392
0.20
Absorbance
Interactions of DNA with various molecules are interesting because of its importance as biochemical tools for many biomedical applications, such as visualization of DNA , DNA hybridization , DNA biosensors , action mechanisms and determination of some DNA targeted drugs, and developing gene and drug delivery systems Therefore, understanding DNA interaction patterns based on the study of molecules that bind to DNA is one of the most important parameters, which can speed up the drug discovery and development processes Several synthetic polymers play a major role as biomaterial and vehicles for many drug delivery systems, and selecting molecules that bind genomic DNA to form a complex is a central requirement for drug delivery system development, necessitating new invitro methods for rapid and low-cost assessment of the binding affinity and location of molecule along DNA molecules. Many applications of DNA-polymer complex have already been demonstrated and characterized. Among synthetic polymers, polyethylene glycol (PEG) show potential applications in different biotechnical, industrial, and clinical applications including biosensors , gene and drug delivery system development due to their solubility, non toxicity and biocompatibility . Therefore PEG is a highly investigated polymer for modification of biological macromolecules and surfaces for many pharmaceutical formulation and biotechnical applications as well . Detailed structural analysis of PEG complexes with calf thymus DNA are not well known.
Effect Of Incubation Time on FTIR spectra of PEG-ctDNA To determine effect of incubation time on the chemical binding between PEG and ctDNA, FTIR Background analysis for pure PEG 400, and pure ctDNA are previously collected. Spectral analysis comparing of PEG-ctDNA complex samples are also obtained at zero time, after 1hour, and after 48 hours Figure 4.3. As can been seen in spectral analysis of PEG/ctDNA after zero time, one hour, and 48 hours at 1:1 ratio in buffer pH 7.4 shown new absorption peaks at 2878 and 2933 cm-1 , 2875 and 2933 cm-1 after 1 hour, and 2879- 2929 cm-1 after 48 hours. These peaks corresponding to some of the characteristic peaks of PEG 400. because these peaks were not present in the pure PEG sample, and the only difference between is the addition of PEG to ctDNA, which attributed effect the incubation time on the chemical binding of PEG to the ctDNA.
Understanding the forces and the mode involved in the binding of certain molecules to DNA is of prime importance. In this study and based on the FTIR spectra we conclude the following: . The FTIR spectroscopic and structural analysis results was indicate that PEG binds with DNA, by weak to moderate complex formation with both hydrophilic and hydrophobic contacts through DNA base pair, with little binding preference towards phosphate backbone of DNA helix. . Binding reaction of PEG and ctDNA proceeds rapidly at room temperature and complexation formation vary by time.) . The mode of interaction between PEG and ctDNA was via outside groove binding or electrostatic binding modes.. The study shown the significant effect of incubation time in PEGctDNA complex formation. . FTIR technique in this experiment was shown rapid, good stability, and a valuable tool for studying the biological properties of PEG with ctDNA.
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