Spectroscopic Studies on the Interaction between Polyamido-amine Dendrimers and DNA

Polyamide-amine type (PAMAM) dendrimers are a class of three-dimensional, highly ordered, novel polymers that can be designed and strictly controlled at the molecular level when compared to traditional polymers. The size, shape, structure, and terminal functional groups of the product are generally highly symmetrical and monodisperse, and thus have a wide range of potential uses. In recent years, there have been many reports on the application of PAMAM dendrimers to gene transfer vectors. Under normal physiological conditions, the amino-terminated PAMAM dendrimer will have a positive charge due to protonation of the amino group, which can interact with negatively charged DNA molecules to form a nano-sized polyelectrolyte complex. Compounds that are tightly bound by electrostatic forces can protect DNA outside the cell and inside the cell, preventing it from being degraded by enzymes, and the complex can easily penetrate the cell membrane into the cell, thus achieving the purpose of transferring the gene into the cell. . Therefore, by studying the interaction between PAMAM dendrimer and DNA, it will provide important theoretical basis for improving the transfer efficiency of these polymer gene vectors and designing and synthesizing novel gene vectors.

Using ethidium bromide (EB) as a probe, the PAMAM dendrimer and the sperm DNA were studied under normal physiological conditions by UV-Vis absorption spectroscopy and fluorescence spectroscopy combined with infrared microscopy and circular dichroism. Interaction, from the molecular level, discussed the interaction mechanism between PAMAM dendrimer and fish sperm DNA, and proposed its possible mode of action.

1 Experiment (i) Main reagents and instruments. Fish sperm DNA (biochemical reagent, Sigma, purity 60 nm/4280 nm>1.9, the bath concentration was determined with the absorbance at 260 nm, the molar absorption coefficient s = 6600 cm was stored at 4C in advance); ethidium bromide EB (biochemical reagent) (Fluka); 5th generation (G5) PAMAM dendrimer with ethylenediamine as the core, laboratory-made; experimental water for the second deionized water, other reagents are of analytical grade. PB-10 precision pH meter (Sartorius, Germany); FTS6000 Fourier infrared spectrometer (Bio-Rad, USA); Sakamoto ASCO-715 circular dichroism analyzer; CARYEclipse UV spectrophotometer (Varian, USA); CARYEclipse Fluorometer (Varian, USA). The solution used in the test experiment was the synthesis of G5 PAMAM dendrimer with pH of ethylenediamine as the core. In accordance with the method, ethylenediamine is used as the core, and Michael is added to excess methyl propionate in methanol to obtain a semi-generational product with ester end groups. After purification, the ester is esterified with a large excess of ethylenediamine. The aminolysis reaction yields a whole generation of dendritic molecules with terminal amino groups, whose structural formulae are as follows. Repeat this operation to obtain G5PAMAM dendrimer.

PAMAM dendrimers were prepared as solutions by dissolving different terminal amino groups in PBS.

UV-visible absorption spectroscopy analysis. The respective absorption spectra were measured at room temperature in the wavelength range of G5 nm to which different terminal amino group concentrations were added to a PBS solution containing 20 [mu]amol/LEB and 80 [mu]mol/L of protamine DNA.

A solution of pmol/L herring sperm DNA in PBS was added to different concentrations of terminal amino groups in G5PAMAM solution, and the respective fluorescence spectra were measured at room temperature. The excitation wavelength is 535 nm and the emission wavelength is 595 nm. The 80 pmol/L fish sperm DNA solution is mixed with different terminal amino group concentrations of G5PAMAM, spotted on a cesium fluoride wafer, and the solvent is vacuumed to form a film on the infrared spectrometer. Infrared spectroscopy before and after binding of the sperm DNA and G5 PAMAM dendrimer was measured by infrared microscopy.

(Vi) Circular dichroism (CD) analysis. The final concentration of immobilized sperm DNA was 80 pmol/L. G5 PAMAM dendrimer solution with different terminal amino groups was added and its CD spectrum was measured at room temperature at 220320 nm and CD spectra of heat-denatured fish sperm DNA were performed. Compare.

2 Results and discussion 2.1 UV-Visible absorption spectroscopy to study the interaction of PAMAM dendrimer with DNA Curve 1 is the UV-visible absorption spectrum of EB in PBS solution. When the fish sperm DNA was added, the absorption intensity of EB decreased, and the maximum absorption wavelength shifted red (line 2). This is due to the insertion of the planar phenanthroline ring of EB into the base pairs of the DNA stack. When the G5PAMAM dendrimer was gradually added to the DNA/EB solution, the absorption intensity of EB began to increase gradually, and the maximum absorption wavelength gradually returned to the original position (line 36). This phenomenon indicates that the PAMAM dendrimer can interact with DNA binds to each other and can disrupt the insertion of EB and DNA, replacing the EB from the DNA molecule.

2.2 Fluorescence Spectrometry Study of PAMAM dendrimer interaction with DNA The fluorescence intensity of EB itself is very weak, but can be inserted in parallel between the base pairs in the DNA double helix structure, so that the fluorescence intensity is greatly enhanced. If the compound interacts with DNA and the EB is displaced from the DNA molecule, the fluorescence intensity will be significantly reduced. Therefore, EB can be used as a probe for studying the interaction pattern of the compound and DNA. It is the relative fluorescence intensity map of fish sperm DNA/EB system in the presence of G5PAMAM dendrimer solution with different terminal amino groups concentration. It can be seen that with the addition of G5PAMAM, the fluorescence intensity of DNA/EB system gradually decreases, which is when G5 PAMAM When the terminal amino group concentration increased from 30 pmol/L to 80 pmol/L, the relative fluorescence intensity of the DNA/EB system had a significant change, from the initial 87% to 35%, while continuing to increase the concentration of G5PAMAM, The decrease in relative fluorescence intensity of the DNA/EB system is no longer evident. This phenomenon shows that when the PAMAM dendrimer is added to the DNA/EB system, the PAMAM molecule can interact strongly with the DNA. May change the configuration of the DNA molecule, which greatly reduces the degree of binding of EB to DNA, leading to fluorescence quenching. Moreover, the trend of the middle curve also shows that the beginning of this interaction will increase with the increase of PAMAM concentration, and when the concentration of the terminal amino group of G5 PAMAM and the concentration of DNA reaches a certain proportion, the curve will no longer fall in different terminal amino groups. The relative fluorescence intensity change of EB-fish sperm DNA system in the presence of G5PAMAM solution is obvious in PBS solution, which indicates that the interaction between the two will reach the saturation value. Under our experimental conditions, we found that when the ratio of the two concentrations>2:1, the interaction force between G5PAMAM and DNA can reach saturation.

The characteristics of the EB-DNA binding reaction can be described using the classical Scatchard equation: the number of sites for binding, and Keb is the binding constant of DNA and EB that are inherent to each site.

Under our experimental conditions, when the presence of PAMAM dendrimers in the system competes with EBs for DNA binding, the binding properties of EBs to DNA can be described by the derivation of the Scatchard equation: when the concentration of PAMAM in the system is 0, it corresponds to that of Keb. equal.

The data measured by fluorescence and absorption spectra were plotted against rEB by rEB/CEB. We can obtain Scatchard plots of DNA/EB systems in the presence of different concentrations of G5PAMAM. The slope and intercept of each line can be obtained separately from n. Values, the results are listed in Table 1. The results of the synthesis and Table 1 we can find, compared with the pure DNA/EB system (-1), with the G5PAMAM's force, the mixed DNA/EB system (-24) The Kobs and n values ​​were reduced, which indicates that the Scatchard diagram system interacts with the EB-fish sperm DNA system when the different terminal amino group concentrations of G5PAMAM solution are added to the DNA/EB system. G5PAMAM and EB with different terminal amino groups concentration - Apparent binding constants and number of sites of interactions between the sperm DNA system a) After PBS solution of dendrimers, the strong interaction between PAMAM molecules and DNA inhibits the binding of EB to DNA, and the Ks of the mixed system The changes in the values ​​of n and n also indicate that the PAMAM molecule is not a simple electrostatic or intercalation, but rather a complex interaction with DNA molecules. In addition, from Table 1, we can also find that when the ratio of the concentration of amino group at the terminal amino group of G5 PAMAM to the DNA concentration is greater than or equal to 2:1, the value of n and the value almost no longer change, indicating that the interaction between G5 PAMAM and DNA has reached a saturated concentration value. This is also consistent with the results of previous fluorescence quenching experiments.

We transform Equation (3) into the data in Table 1 and the previously measured cp value into Equation (4) and then plot it. After linear regression analysis, we get the formula: 1 Condition-0.057cp+2.55x10-5 ( r = 0.998, n = 3). From this we can calculate the binding of G5 PAMAM to DNA in a PBS system at pH 7.4. 2.3 Infrared Microscopy Techniques The interaction of PAMAM dendrimers with DNA is the result of infrared microscopy of phosphate and bases in G5 PAMAM molecules. The result of the pair. In addition, we also found that in the IR spectrum of the mixed system, the characteristic absorption peak of the B configuration DNA at 1013 cm1 disappeared before, indicating that the interaction between G5PAMAM and the sperm DNA resulted in the secondary structure of DNA. Variety.

2.4 Circular dichroism study of PAMAM dendrimer interaction with DNA Protein, nucleic acid and other biological macromolecules are mostly asymmetric molecules, with optical activity, can be determined by circular dichroism and its secondary structure in the solution state . It is the CD spectrum of the mixed system after the fish sperm DNA was treated with heat denaturation and before the addition of G5 PAMAM solution with different amino group concentrations. The IR spectrum (700~2000cm) before and after the interaction with the fish sperm DNA, (a) is the IR spectrum of the fish sperm DNA, the main features are: 1692cm1 is the guanine or pyrimidine base in the DNA molecule heterocyclic ring C== The absorption peaks of the O stretching vibrations, 1228 and 1060 cm1, are the absorption peaks of the anti-symmetrical stretching and the symmetrical stretching vibration of the phosphate in the DNA molecule, respectively. On the other hand, 1013cm1 is the absorption peak of C=N stretching vibration in the ribo unit of DNA molecule, and it is also the characteristic absorption peak of B-shaped DNA. (b) is the IR spectrum of G5PAMAM. Its main features are: absorption peak of C==O stretching vibration of PAMAM dendrimer amide part at 1627cm1, NH bending vibration and CN stretching of PAMAM dendrimer amide part at 1550cm1. The combined absorption peaks of the vibrations, while the 1464 cm1 is the absorption peak of CH bending vibration in the PAMAM dendrimer CH2 chain segment. From the IR spectrum of G5.0PAMAM and DNA mixed system ((c)), we can find that after adding the PAMAM molecule, the three characteristic absorption peaks representing the phosphate and the base in the DNA have shifted to varying degrees. In the previous 1060, cm1, and the intensity of each absorption peak has also changed, the characteristic absorption peaks representing the PAMAM molecules have also undergone similar changes, respectively from the previous 1627, cm1, these results indicate that the PAMAM dendrimer There is an interaction with the DNA molecule, in which the terminal amino group charged positively by protonation in the PAMAM molecule can be combined with the negatively charged phosphate by the charge in the normal physiological environment, and the amide moiety in the PAMAM molecule skeleton. The C==O and CN groups may also interact with the corresponding groups on the nitrogen-containing base pair in the DNA molecule, so this interaction is the interaction of the PAMAM molecule with both the DNA G5PAMAM and the fish sperm DNA. The CD spectrum shows that the CD spectrum of the normal spermatozoa DNA in the free state (curve 1) has a positive absorption peak at 276 nm corresponding to the stacked structure of the nucleic acid bases and a negative at 248 nm. The absorption peak corresponds to the double helix structure of the nucleic acid, which is the CD profile of a typical B configuration DNA, while the positive absorption peak at 276 nm of the CD spectrum (curve 2) of the fish sperm DNA after heat denaturation is unchanged. Absorption peaks in the negative direction are lost due to the double helix structure of the nucleic acid. When the concentration of 40pmol/LG5PAMAM dendrimer was added to the solution of fish sperm DNA, the CD spectrum of the mixed system showed a decrease in the amplitude of the positive and negative absorption peaks and a red shift in the peak position, respectively, from the previous 276 and 248 sites. At 283 and 252 nm (curve 3), this indicates that the sperm DNA starts to change from the B configuration to the C configuration. As the concentration of PAMAM increases (curve 4, 5), the positive and negative peak positions of the CD spectrum of the mixed system will The faint red shift continues to occur and the amplitude continues to decrease, but at high concentrations of PAMAM, the spectral lines begin to become blurred. This may be due to the fact that the PAMAM molecules form a tightly-structured complex with the DNA, making the DNA molecules around Increased environmental hydrophobicity. Moreover, we also found that the CD spectrum of the mixed system differs from the CD spectrum of the denatured fish sperm DNA in that each of its spectral lines has an absorption peak in the negative direction, which indicates the mutuality between the PAMAM dendrimer and the sperm DNA. The effect does not denature the DNA.

3 Conclusions In this paper, ethidium bromide (EB) was used as a probe to study the interaction between PAMAM dendrimer and fish sperm DNA under normal physiological conditions through various spectroscopic methods. UV-visible absorption and fluorescence were observed. Spectral test results show that there is a strong interaction between the PAMAM dendrimer and the fish sperm DNA, and through the Scatchard model analysis of this interaction, we also get the binding constant of the two. Infrared microscopy and circular dichroism test results indicate that this strong interaction is the result of the interaction of the PAMAM dendrimer with the phosphate and base pairs of the DNA molecule, and this effect leads to secondary DNA levels. The structure changes. These conclusions are of great significance at the molecular level to explore the interaction mechanism between these novel dendrimers and DNA.