salmon sperm DNA

Accepted Manuscript Comparison on the interaction of Al sperm DNA

3+ / nano-Al13 with calf thymus DNA /salmon

Fei Ma, Yue Ma, Changwen Du, Xiaodi Yang, Renfang Shen PII:

S0022-2860(15)30126-5

DOI:

10.1016/j.molstruc.2015.07.015

Reference:

MOLSTR 21670

To appear in:

Journal of Molecular Structure

Received Date: 13 February 2015 Revised Date:

7 July 2015

Accepted Date: 7 July 2015

Please cite this article as: F. Ma, Y. Ma , C. Du , X. Yang , R. Shen, Comparison on the interaction of 3+ Al / nano-Al13 with calf thymus DNA /salmon sperm DNA, Journal of Molecular Structure (2015), doi: 10.1016/j.molstruc.2015.07.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Comparison on the interaction of Al3+/ nano-Al13 with calf thymus DNA

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/salmon sperm DNA

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Fei Ma a, Yue Ma b, Changwen Du a, Xiaodi Yang b,*, Renfang Shen a, *

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a

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China

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b

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School of Geograph Science, Nanjing Normal University, Nanjing 210026, P.R. China

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Jiangsu Key Laboratory of New Power Batteries, School of Chemistry and Materials Science, and

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Abstract: The conformation change, binding mode and binding site between

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Al3+/nano-Al13 and calf thymus DNA/salmon sperm DNA were investigated by UV-Vis

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absorption, FTIR spectra, Raman spectroscopy and CD spectra, as well as melting curves

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measurement. The UV-vis spectra and circular dichroism spectra results suggested that

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the phosphate group structure was changed when Al3+ interacted with DNA, while the

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double-helix was distorted when nano-Al13 interacted with DNA. The FTIR and Raman

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spectroscopy revealed that the binding sites were Al3+…PO2, Al3+…N7/guanine

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PO2…Al13…N7-C8/guanine with calf thymus DNA, and Al3+…N3-O2/cytosine,

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Al3+…N7-C8/guanine,

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salmon sperm DNA, respectively. The electrostatic binding was existed between Al3+ and

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DNA, and the electrostatic binding and complexing were found between nano-Al13 and

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DNA.

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Keywords: Nano-Al13, Al3+, DNA, conformation, binding mode, binding site.

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PO2…Al13…N1/adenine

with

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PO2…Al13…N7-C8/guanine,

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Introduction:

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Aluminum is one of the most abundant metal exist in the Earth’s crust. Acidic

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precipitation processes account for most of the redistribution of aluminum released from

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the soil to the environment. Foods additives, pharmaceutical preparation, and special

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industry productions cause large increasement in the amount of aluminum existing [1].

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Previous studies have demonstrated that when present at a high level, the aqua aluminum

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ion and its hydrolytic species in the environment were responsible for the toxic effects on

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humans, animals and plants due to ion-toxicity stress [2-6]. Aluminum implicates in

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disturbances of cerebral function and it has been identified as a major risk factor in

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dialysis-induced encephalopathy, microcytic anaemia, and osteomalacia [7-9]. Exposure

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of Aluminum is known to activate apoptotic cascades, provoke cell cycle arrest, and

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interfere with cell signaling pathways [10].

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The common exist ion Al3+, which was abbreviated from the octahedral hexahydrate

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form of Al (H2O)63+, is believed to be the most toxic form below pH 4.5. Al3+ can interact

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with biological systems and interfere with biological functions. It has been linked to

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many diseases such as dialysis dementia and microcytic anemia without iron deficiency

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[11]. The potential effects of nanoparticles have also raised considerable concerns on

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toxicity. The Keggin-type Al13 polycation polymer (AlO4Al12 (OH)24(H2O)

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called nano-Al13) is an effective coagulation agent with rapid aggregation rate over a

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relatively wide range of pH, which has been widely applied in water treatment. Nano-Al13

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is also used as pillaring agents, antacids, antiperspirants and surface active agents [12].

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Nano-Al13 is a major species of aluminium under physiological conditions, and the

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process of Al(OH)3 formation requires the presence of nano-Al13 as a precursor [13].

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Various studies of nano-Al13 have been conducted in the field of environmental chemistry

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and the activity of proteins, nucleic acid and enzymes at a molecular level. Al3+ could

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disrupt biological membranes by interacting with the membrane phospholipids and

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damages DNA directly [14]. Since nano-Al13 exists with metastable state, it could be

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found in the tea root, stem and leaf of Camellia sinensis [15]. The reports found that

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nano-Al13 showed 10-fold toxicity to plant root relative to the monomeric Al3+ species

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and comparable toxicity to algae as the monomeric Al3+ species [16, 17].

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7+

, also

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Chemical interactions between ions and specific binding sites on DNA are

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meaningful. Na+, K+, Mg2+, and Ca2+ ions are important neurotransmitters; Zn2+ ions

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through the zinc finger play an important role in the regulation of DNA transcription and

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replication. Positively charged metal cations are Lewis acids and they can interact either

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with donor/acceptor groups on nucleobases (ring-nitrogen atoms and exocyclic keto

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groups) or with negatively charged phosphate groups. As we known, Al3+ is not regarded

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as an essential nutrient in plant and animal life, but it can promote plant growth and

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induce other desirable effects at low concentration though it is just passing through in

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vivo rather. Our lab’s previous studies have reported the effects of different species of

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aluminum on the malate dehydrogenase activity, glutamate dehydrogenase activity,

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glutathione reductase activity, reduced glutathione and hemoglobin [18-21], and the

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results indicated that nano-Al13 displayed stronger toxicity for the larger surface of

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nano-Al13 cluster formed in aqueous solution. Thus, it is valuable to study the effects of

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Al3+ and nano-Al13 to DNA. The purposes of the current work are i) to find the

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conformation change, bind mode and binding site of Al3+/nano-Al13 on calf thymus DNA

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/salmon sperm DNA; ii) to make comparisons between them. In order to establish the

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binding mode, multiple analytical methods were employed for characterization, including

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UV-vis, and CD spectroscopy and melting curves experiment in solution. The FTIR

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spectroscopy and Raman spectroscopy were used to detected binding site. The conclusion

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could be of useful in elucidating the nature of its interaction in the processes of biological

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relevance.

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2 Materials and Methods

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2.1 Materials

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Calf thymus DNA (ct-DNA) and salmon sperm (ss-DNA) were purchased from

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Sigma Biological Co., and used as received, which was used without further purification

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and dissolved in doubly distilled water at a final concentration of 4×10-5 mol L-1 and

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stored at 0 - 4 °C. The purity of DNA was checked on UV-vis spectrophotometry by 3

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monitoring the ratio of A260/A280 = 1.84 (ct-DNA) and 1.80 (ss-DNA). The concentration

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of DNA was determined by the absorption at 260 nm (ε=6600 L mol-1 cm-1). Nano-polynuclear aluminum sulfate (nano-Al13) was synthesized by our previous

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reported method in laboratory [22] and the NMR spectrum was examined. Aluminum

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chloride solution (0.25 mol L-1, 25 mL) was heated at 70 °C using a thermostat. The

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NaOH solution (0.25 mol L-1, 60 mL) was slowly added to the above solution with

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controlling of velocity accurately with 2 mL/min and the mixture were kept stirring, and

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the adding process lasted about 30 minutes. The obtained solution was incubated at room

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temperature for 24 h, and then Na2SO4 solution (0.1 mol L-1, 62.5 mL) was added and the

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kept reacting about 10 min, then the solution was filtrated and aged for 48 h. The crystal

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obtained from centrifugalization was washed twice with distilled water and 70% ethanol

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solution, respectively, finally air-dried and stored in desiccator for future use. The Al

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stock solution was prepared by dissolving high purity (99.99 %) metallic Al powder in

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hydrochloric acid to prevent hydrolysis of Al3+ ion. Tirs-HCl buffer solution was

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prepared by dissolving appropriate amount of Tirs aminomethane (hydroxymethyl), and

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adjusting pH by concentrated hydrochloric acid to weak acidic condition (pH 6.5). All

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reagents were of analytical grade and they were dissolved with deionized Milli-Q water.

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2.2 Method

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2.2.1 UV-vis spectra

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UV-vis measurements of mixture solution were made on Varian Cary 5000

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spectrophotometer at room temperature from 200 nm - 340 nm. The UV-vis titrations

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were performed by successive addition of Al3+ and nano-Al13 (0 to 250 µL per 50µL) to 3

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mL DNA solution in 1 cm cuvette. The mixture solution were kept stirring for about 10 s

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for each addition before scanning, respectively. The concentration of DNA solution was

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4×10-5 mol L-1 and the finally concentration of Al species added in the DNA solution was

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0, 1.3, 2.6, 3.9, 5.2, 6.5×10-5 mol/L, respectively.

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2.2.2 FTIR spectroscopy In order to get solid samples for FTIR measurements, the mixture solution

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(cDNA=4×10-3 mol L-1, cAl=3.9×10-3 mol L-1) 10 mL in culture dish was dried at room

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temperature (25 °C) in vacuum drying oven, and then the solid sample were collected for

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FTIR spectroscopy investigation. The spectra were recorded on a FTIR Niocolet Fourier

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Transform detector and KBr beam splitter, using AgBr windows. Interferograms were

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accumulated over the spectral range 4000 to 400 cm-1 with a nominal resolution of 2 cm-1

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and a minimum of 100 scans. Each set of infrared spectra was taken three times.

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2.2.3 Raman spectroscopy

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The solid samples were also studied by Raman spectroscopy. Raman spectra were

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collected on a Jobin Yvon LABRAM HR800 spectrometer. The spectra were excited with

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the 514.5 nm line of an argon laser with 163 radiant powers from Spectra-Physics using

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approximately 100 mV at the samples. Each spectrum displayed in the figures

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represented the average of at least 140 exposures of 15 s each. Each set of Raman spectra

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was taken three times.

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2.2.4 Circular dichroism measurements

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Circular dichroism (CD) measurements of mixture solution were obtained with on

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an Applied Photophysics Chriascan circular dichroism spectrometer at room temperature.

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The DNA solution of 3 mL in 1 cm cuvette was titrated with Al3+ and nano-Al13 solution,

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and the reaction time was about 10s. The concentration of DNA solution 4×10-5 mol L-1

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and the finally concentration of Al species added in DNA solution was 4×10-5 mol/L. The

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buffer solution signals of CD spectra were also investigated for error subtraction. The

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spectra were scanned with 1 nm spectral bandwidth and 0.5 nm step resolution from 220

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nm - 320 nm. Each spectrum was accumulated twice and the obtained results expressed

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as millidegrees (mdeg).

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2.2.5 The melting curves experiments The melting experiments were performed on Varian Cary 5000 spectrophotometer in

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conjunction with a thermostat cell compartment. The temperatures of DNA, Al3+-DNA

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and Al13-DNA were determined by monitoring the absorbance at 260 nm of the samples

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as a function of temperature ranged from 25 to 90 °C with probes and increased at a

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heating rate of 5 °C min-1 (Thermal software).

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2.2.6 Statistical analysis

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All data were obtained in triplicate experiments, the mean values, standard deviations

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and statistical difference were estimated utilizing analysis of variance (ANOVA). The

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t-test was made to compare the mean values.

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2.3 Theory and calculation

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2.3.1 The melting temperature

The melting temperature Tm was obtained from the mid-point of the hyperchromic

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transition. The following equation was employed to describe the absorbance versus

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temperature (Tm).

f ss =

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A − A0 Af − A0

(1)

Where A0 was the absorbance at initial temperature (25 °C) and Af was the

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absorbance at final temperature (90 °C). A was the absorbance at Tm. Moreover, Tm was

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calculated when fss=0.5 [23, 24].

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3. Results and Discussion

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3.1 Conformation change and Binding Mode

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3.1.1 UV-vis Absorption

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The effect of addition amount of Al species on DNA were showed in Fig. 1. The

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UV-vis spectra at 258 nm were the characteristic absorption band of DNA. Generally

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speaking, there exist two effects called "hyperchromic effect" and "hypochromic effect"

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on DNA. Hyperchromism effect results from the damage of DNA double-helix structure.

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Hypochromism effect results from the contraction of DNA in the helix axis, as well as the

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conformation change [25-29].

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Fig. 1

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When the Al3+ solution was gradually added into the ct-DNA and ss-DNA,

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respectively, the absorption at 258 nm both decreased (Fig. 1 a1 and b1). The decreasing

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of absorption revealed hypochromic effect, which suggested that Al3+ led to the

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contraction of DNA in the helix axis, and it was influenced by the conformation change

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of phosphate group of DNA backbone (base pairs of nucleic acid). With an increasing

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concentration of nano-Al13, the absorption both increased (Fig. 1 a2 and b2). The

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hyperchromism effect indicated that nano-Al13 was interacted with the base nitrogen rings

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of DNA and double-helix structure, causing a little distortion of the DNA backbone.

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3.1.2 CD spectra

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The CD spectrum is a vigorous technique to follow the DNA interaction via

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investigating the changes in the DNA morphology for the sensitivity signal to subtle

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variation of DNA achiral conformation [30, 31]. In particular, B-DNA shows two

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conservation bands at 278 nm (due to base stacking) and 246 nm (due to the helicity, in

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right handed B form) in the UV region. Both the bands are quite sensitive to the

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interaction with other molecules [32-34]. The base stacking interaction would enhance

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the intensity of DNA, while groove binding and electrostatic interaction shows less or no

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perturbation on the base stacking and helical band [35]. Moreover, when DNA base are

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disrupt and base stacking are weaker, the intensities of CD bands shows decrease.

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Fig. 2

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Fig. 2 showed the CD spectra of DNA in the absence and in the presence of Al3+ and

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nano-Al13. CD spectrum of DNA itself has characteristics of B-DNA conformation. Al3+

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and nano-Al13 were not optically active and therefore they showed no absorption in CD

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spectra in the region. The spectra of ct-DNA and ss-DNA were different. With addition of

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Al3+, there was a decrease in the intensity of positive band. The results indicated that Al3+

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could lead to base stacking of ct-DNA. Moreover, electrostatic interaction was occurred

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on helical band. The spectra of ct-DNA showed decrease both in positive band and

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negative band after interacting with nano-Al13, indicating that nano-Al13 influenced the

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base stacking and the helicity of ct-DNA.

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The spectra of ss-DNA and Al3+ mixture showed decrement in positive band and

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negative band, which reflected the base stacking and the helicity of DNA. With addition

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of nano-Al13, the ss-DNA spectra increased in the positive band. Hegde et al. [36]

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reported that the increasing of positive band was induced by the conformational transition

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of B-DNA to A-DNA. The increase of CD band was associated with each base transition,

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which occurred due to the coulombic interaction and also close contact between

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hydrophobic of nucleotides and small molecules [21, 37].

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3.1.3 The melting curves experiments

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When in the condition of pH or heat, the double helical structure of DNA would

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undergo a transition into a randomly single-stranded form at the melting temperature (Tm).

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It has been known that double stranded DNA gradually dissociate to single strands with

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increasing solution temperature and result in hyperchromic affect [38]. Tm is strictly

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related to the stability of double helix, and the interaction of the molecules with DNA

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may alter the Tm by stabilizing or destabilizing the final complex. Interaction of additive

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with DNA can influence the melting temperature. Moreover, it is possible to obtain

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information on the strength of the interaction. Namely, small molecules that go into the 8

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double helix and bind to base pairs can increase Tm [39] (5-8 °C) and the double helix

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structure is still stable. Electrostatic binding to DNA stabilized the DNA double helix

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structure and would cause lower increase Tm [40, 41]. The values of Tm of ct-DNA,

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Al3+-ct-DNA,

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calculated by equation (1) were showed in Fig. 3 and Table 1.

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Fig. 3

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Table 1

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Al3+-ss-DNA and

nano-Al13-ss-DNA

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ss-DNA,

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nano-Al13-ct-DNA,

In the presence of Al3+, the Tm of ct-DNA and ss-DNA both increased less than 5 °C.

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But for nano-Al13-ss-DNA system, the changes were more than 5 °C. The results revealed

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that the modes of Al3+-ct-DNA and Al3+-ss-DNA were electrostatic binding. The reason

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that Al13 exhibited a stronger harmful effect to DNA may be on account of the larger

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surface effects of Al13 cluster formed in aqueous solution. The model built by Cai et al

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[21] showed the backbone structure of the aluminum clusters, from which the conclusion

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were that Al3+ clusters could exert strong effect to biomolecules. The increased active

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sites on the surface made it possible to develop more significant interactions between

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ligands and cluster surface. Of course, there were 13 aluminum atoms in one nano-Al13

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molecular which was 13-fold than free Al3+ and theoretically displays greater effect at the

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same concentration. However, nano-Al13 as an independent species was rather than 13

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aluminum atoms to discuss the inhibitory ability. As a matter of fact, the interaction

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between nano-Al13 and biomolecules should not to be regarded as the sample plus of 13

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free Al3+. In addition, because of the seven positive charges on nano-Al13, there also

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existed electrostatic binding [42].

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3.2 Binding Site

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3.2.1 FTIR spectroscopy 9

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FTIR techniques are widely used to determine biomolecular structures. Evidence for

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binding to A-T, G-C or phosphate comes from spectral changes in DNA in plan vibrations

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at 1710-1717 cm-1 (G), 1662 cm-1 (T), 1609 cm-1 (A), 1492 cm-1 (C), 1222 cm-1 (PO2

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asymmetric) and 1088 cm-1 (PO2 symmetric), 964 cm-1 (deoxyribose C-C), 893 cm-1 and

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860 cm-1 (sugar-phosphate stretching), 836 cm-1 (phosphodiester mode) (Table 2) [43-46].

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Fig. 4

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Table 2

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The addition of Al3+ and nano-Al13 resulted in the enhancement of intensity of

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ct-DNA, respectively. However, the intensity changes of ss-DNA have opposite effects

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(Fig. 4). Generally, cation bind to DNA bases and the phosphate group causes major

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increase of intensity and shifting of these vibrations, but the loss of intensity has been

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attributed to DNA aggregation, condensation or helix stabilization. There are two main

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binding: Al-phosphate binding and Al-base binding. Previous literatures claimed that the

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intensity ratio variations were used to determine the metal ion binds to DNA bases and

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the backbone phosphate group. The metal-phosphate binding was reflected by the

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alterations of the relative intensities of the symmetric and asymmetric vibrations of the

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backbone PO2 group, associated with the ratio νs/νas (1088/1222 cm-1) change in

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phosphate interaction in cation-DNA complexes [47-49]. The intensity ratio variation of

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symmetric and asymmetric PO2 bands at 1088/1222 cm-1 changed from 1.19 (ss-DNA) to

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1.29 (ss-DNA-Al3+) and 1.23 (ss-DNA-Al13), indicating the Al-PO2 interaction. For

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ct-DNA, the ratios of νs and νas were 1.42 (ct-DNA), 1.44 (ct-DNA-Al3+) and 1.51

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(ct-DNA-Al13), suggesting the Al-PO2 interaction. The guanine band at 1700 cm-1 gained

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intensity and shifted to 1697 cm-1, thymine band at 1651cm-1 gained intensity and shifted

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to 1646 cm-1-1647cm-1, and adenine band at gained intensity and appeared at 1606 cm-1

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to 1609 cm-1 for ct-DNA. The shifting and increasing observed for the guanine, thymine

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and adenine vibrations were due to Al-base binding via guanine and adenine N7 atoms

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and thymine O2 in the major and minor grooves of DNA duplex. Thus, there were formed

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of -PO2…Al13…N7 (G) ct-DNA complex, and Al3+…N7 (G) ct-DNA complex [50, 51].

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From above information, it was found that the Al-binding of the peptides were existed

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when they at the N-terminus. This was rather surprising as Al3+ [52, 53], was known to

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have a low affinity for amines, and the N-terminal group was therefore not expected to be

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a strong binding site. Tamas Kiss et al [54] have reported that in the interactions of Al3+

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with oligopeptides, the Al3+-binding capabilities of the peptides were stronger when they

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had a free Pro-NH function at the N-terminus, and coordination would much more likely

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through the N donor group. However, the reductions of intensity for the bands at 1696

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cm-1, 1650 cm-1 to 1608 cm-1 were due to ss-DNA aggregation and condensation, which

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would be discussed further. Moreover, the sugar-phosphate bands at 836 cm-1 and 893

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cm-1 exhibited no major shifting in the spectra, suggesting the B-family structure of

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DNA.

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3.2.2 Raman spectroscopy

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Raman spectroscopy has been exploited previously as a conformational probe of

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genomic DNA complexes. Fig. 5 showed that ss-DNA and ct-DNA displayed similar B

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conformations, as evidenced by Raman markers of C2-endo/anti G (680 cm-1), A (727

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cm-1), and T (748 cm-1) deoxynucleosides and by gauche- (g-) conformations of

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phosphodiester torsions α and ζ (780 cm-1) [55, 56]. Raman bands and assignments were

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summarized in Table 3. The integrated intensity of 1576 cm-1 band was known to be

22

largely invariant to transformations between different double-helical forms DNA and to

23

changes in solvent environment that do not lead to DNA denaturation [57-59]. The

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intensity change in the 1576 cm-1 region, accompanied by a slight shift, was also

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consistent with binding at either the N7 or N1 position, although the latter was probably

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not involved, being engaged in intersrand hydrogen bonding and therefore protected

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against metal attack in duplex DNA. The band of 782 cm-1 reflected the average

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backbone torsion angles, α (O3-P-O5-C5), β (P-O5-C5-C4) and γ (O5-C5-C4-C3), from

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the g-/t/g+ range to the t/t/t range. The PO2- maker band at 1092 cm-1 was used for

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intensity normalizations in studies of DNA denaturation [60-63]. The changes of

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wavelength and intensity reflected the changes of local electrostatic environment of the

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DNA phosphates. There were pronounced changes after interacted with Al13 and Al3+.

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They could be explained in part by a hypochromic effect associated with increased

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base-pair stacking interactions. The intensity changes with Al13 were caused by a much

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more direct type of interaction (covalent binding) with one particular oxygen atom of the

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phosphate group, gave a -PO2…Al13…N7 type of complex. For ss-DNA, besides changes

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of wavelength and intensity, there was broader band in centered 1094 cm-1. The intensity

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changes would be caused by interaction with one particular oxygen atom of the phosphate

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group; and the shape changes such as broader band would be caused by N3-O2 of

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cytosine，the structure changes influenced the conformation of the phosphate group. It

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was resulted from disruption of the hydrogen bonds between cytosine and guanine. The

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loss in intensity of the band at 1487 cm-1, which was predominantly due to a guanine

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vibrational mode with a strong N7-C8 stretching. Thus, the band at 1480 cm-1 (ct-DNA)

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and 1847 cm-1 (ss-DNA) represented the state of hydrogen bonding at guanine N7 sites.

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The band of 1660 cm-1 (ct-DNA) and 1668 cm-1 (ss-DNA) coincided with thymine that

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was diagnostic of the state of hydrogen bonding of the thymine C4=O site [64, 65]. The

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decreasing intensity noted that the interactions of nano-Al13, Al3+ and DNA altered the

21

hydrogen bonding of the thymine C4=O site. For the bands of 1306 cm-1/1305 cm-1, 1337

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cm-1/1338 cm-1, 1369 cm-1/1374 cm-1 (ct-DNA/ss-DNA) were responsive the base pairs

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[66, 67].

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Fig. 5

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Table 3

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4. Conclusions

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Table 4

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In this study, the effects of Al3+/nano-Al13 on calf thymus DNA/salmon sperm DNA

5

were comprehensive investigated. Table 4 showed the conformation change, binding

6

mode and binding site of them. In aqueous solution, the UV-vis spectra displayed that

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Al3+ led a slight hypochromic effect on two types of DNA, which suggested that Al3+

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could bind to phosphate group of DNA in solution. With adding of nano-Al13 into DNA

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solution, hyperchromism was observed. The results indicated that nano-Al13 could bind to

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the base nitrogen rings of DNA, and the double-helix structure of DNA was damaged.

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The CD spectra suggested that electrostatic interaction was occurred between Al3+ and

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ct-DNA, and nano-Al13 influenced the base stacking and the helicity of ct-DNA. The

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change of base stacking and the helicity was occurred between ss-DNA and Al3+. The

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interaction of ss-DNA and nano-Al13 was associated with each base transition, which was

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due to the coulombic interaction and close contact between hydrophobic of nucleotides

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and small molecules. The melting curves experiments displayed the helicity and base

17

stacking conformation changes on DNA. In solid state condition, FTIR spectroscopy and

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Raman spectroscopy results suggested that the binding sites were Al3+…PO2,

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Al3+…N7/guanine PO2…Al13…N7-C8 /guanine on ct-DNA, while binding sites were

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Al3+…N3-O2/cytosine,

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PO2…Al13…N1/adenine on ss-DNA, respectively.

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Al3+…N7-C8/guanine,

PO2…Al13…N7-C8/guanine,

AC C

22

RI PT

4

23

Acknowledgments

24

The project is supported by the National Natural Science Foundations of China (No.

25

41401256), the national key Basic Research Program of China (2014CB441000) and the

26

open fund of the State Key Laboratory of Soil and Sustainable Agriculture (No.

27

0812201216).

13

ACCEPTED MANUSCRIPT

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17

ACCEPTED MANUSCRIPT 3+

Table 1 The melting temperature of Al

Samples

ct-DNA

and Al13 interact with ct-DNA and ss-DNA.

Al3+-ct-DNA Al13-ct-DNA

Al3+-ss-DNA

Al13-ss-DNA

53.1

76.8

ss-DNA 59.4

63.8

74.3

49.5

AC C

EP

TE D

M AN U

SC

RI PT

Tm / °C

ACCEPTED MANUSCRIPT Table 3 The main assignments peaks of DNA Wavenumber cm-1

Assignments Thymine

1609

Adenine

1492

Cytosine

RI PT

1662

PO2 asymmetric

1088

PO2 symmetric

964

deoxyribose C-C

893, 860

phosphate stretching

AC C

EP

TE D

M AN U

SC

1222

ACCEPTED MANUSCRIPT Table 3 The characteristic Raman bands of DNA, Al3+-DNA and Al13-DNA Wavenumber / cm-1 Assignments

ct-DNA

ct-DNA +Al3+

ct-DNA +Al13

ss-DNA

ss-DNA +Al3+

679

675

673

668

668

—

—

680

675

676

729

729

729

731

734

—

—

748

754

—

782

782

782

787

789

—

—

802

800

844

845

844

830

—

872

878

881

888

884

916

912

920

915

—

966

963

969

965

—

1005

1005

1006

1010

1009

1022

T

1055

1060

1060

1057

1067

1056

ν (CO)

1092

1091

1090

1092

1094

1094

ν (PO2-)

1174

1176

1177

—

—

—

—

—

—

1184

1183

1183

dC,dT

1242

1243

—

—

1306

1306

1338

1335

1369

1369

1416

1414

TE D

RI PT dA

dT

dT,dC,bk(OPO)

d

831

ν (OPO)

889

d

920

d

d

dT

1241

1240

1240

1239

dT

—

1256

1256

1258

dA, dC

1305

1305

1302

1313

dA

1333

1337

1342

1332

dA, dG

1370

1374

1378

1375

dA, dG, dT

1414

1421

1413

1414

dA, δ(C2’H2) dA, dG

EP

AC C

dG

SC

784

dG

M AN U

729

ss-DNA +Al13

1480

1483

1483

1487

1488

1485

1508

1506

—

1508

1514

—

dA

1524

1521

1520

1531

1531

1531

dC

1573

1576

1575

1576

1576

1576

dA, dG

1607

1604

—

—

—

—

1666

1667

1668

1668

1662

1666

dC dT,ν(C4=O/C5=C6)

ACCEPTED MANUSCRIPT Table 4 Comparative studies on the effects of Al3+ and Al13 with ct-DNA and ss-DNA Samples

Binding Modes

Conformation (solution)

Binding Sites (solid state) Al3+…PO2

Al3+

electrostatic binding

Al3+…N7/guanine

Phosphate group

Al3+…C4=O/thymine

thymus DNA

Al13

electrostatic binding complexing

Double-helix distortion

PO2…Al13…N7-C8/guanine

RI PT

Calf

Al13…C4=O/thymine Al3+…PO2

Salmon

Al3+

electrostatic binding

Al3+…N3-O2/cytosine

Phosphate group

Al3+…N7-C8/guanine Al3+…C4=O/thymine

SC

sperm DNA

Double-helix distortion

complexing

Slight B-DNA to A-DNA

PO2…Al13…N7-C8/guanine

PO2…Al13…N1/ adenine Al13…C4=O/thymine

AC C

EP

TE D

M AN U

Al13

electrostatic binding

ACCEPTED MANUSCRIPT

0

a1

5 0.4 0.42

0.2

5

0.6

Absorbance

Absorbance

0.6

0.35

a2

0 0.4

0.2

0.0 220

235

240

240

245

260

280

300

0.0 220

320

240

Wavelength / nm

280

320

300

320

b2

SC

Absorbance

0.2

260

300

0

0.4

0.2

M AN U

Absorbance

5

240

280

5

0.6

b1

0.4

0.0 220

260

Wavelength / nm

0

0.6

RI PT

0.28

0.0 220

240

260

280

300

320

Wavelength / nm

Wavelength / nm

Fig. 1. The UV-vis spectra changes of Al3+ and Al13 interact with ct-DNA and ss- DNA, respectively. The inset of Fig. 1 a1 was amplification of the isosbestic point.

TE D

a1: Al3++ ct-DNA; a2: Al 13+ ct-DNA; b1: Al3++ ss-DNA; b2: Al 13 + ss-DNA. (0-5: cAl= 0, 1.3,

AC C

EP

2.6, 3.9, 5.2, 6.5×10-5 mol/L).

ACCEPTED MANUSCRIPT 6

CD (mdeg)

4 2

A

a b c d

0

-4 220

240

260 280 300 Wavelength (nm)

B

SC

0 -4

220

a b c d

M AN U

CD (mdeg)

4

-8

320

RI PT

-2

240

260 280 300 Wavelength (nm)

320

Fig. 2 CD spectra of DNA in the presence and absence of Al species. (A): ct-DNA; (B): ss-DNA.

AC C

EP

TE D

a: DNA; b: Al 3+ and c: nano-Al13; d: The CD spectra of buffer.

ACCEPTED MANUSCRIPT a b

1.0

0.8

(A-A0) / Af-A0

0.6 0.4 0.2

a c

0.8 0.6 0.4 0.2

a1

0.0 20

30

40

50

60

70

80

90

a2

0.0

100

20

30

o

0.6 0.4

a c

0.8 0.6 0.4 0.2

0.2

b1 50

60

70

80

o

Temperature / C

90

0.0

M AN U

0.0 40

70

80

SC

(A-A0) / Af-A0

(A-A0) / Af-A0

1.0

0.8

30

60

90

100

Temperature / C

a b

20

50

o

Temperature / C

1.0

40

RI PT

(A-A0) / Af-A0

1.0

100

20

30

40

50

60

70

b2 80

90

100

o

Temperature / C

Fig. 3 Melting curves of Al3+ and Al13 interact with ct-DNA and ss-DNA, respectively.

a1: Al3++

AC C

EP

TE D

ct-DNA; a2: Al 13+ ct-DNA; b1: Al3++ ss-DNA; b2: Al 13 + ss-DNA. (cAl= 3.9×10-5 mol/L).

AC C EP TE D B

1800

1600

1400

1200 1012

964

1057 1011 964

1057 1013 964

SC

1088

1200

1057

1085

a b c

1218

1400

1088

1218

-1

1000

Wavenumber / cm

1000

a: DNA; b: Al 3+ and c: nano-Al13. 800

964

1697

T

1697

964

1220

1369

1489

1083 1059

1369

1487

1646 1647 1609 1609 1578 1578

10611083

RI PT

1061 964

1083

1223 1220

1371

1487

1606 1576

1700

a b c

1222

1600

1419

1800

1419

1651

A

M AN U

1697 1696 1696 1650 1650 1650 1608 1604 1608 1578 1578 1533 1529 1532

T

ACCEPTED MANUSCRIPT

Wavenumber / cm

-1

800

Fig. 4 FTIR spectra of mixtures of Al species and DNA. (A): ct-DNA; (B): ss-DNA.

AC C EP TE D 800 1009

a b c

1200 1576

1666

1662

1414 1485

a: DNA; b: Al 3+ and c: Al13.

SC 1668

1487 1576

1240 1305 1337 1374 1421

1184

1092

1200

1576

1488

1239 1313 1332 1375

1094 1183

1022

800

1183 1240 1304 1342 1378

1094

782 802

B

888 965 1010 1053

668 680 748

1006 1060

872 874

1090

784

673 729

729

1005 1055 1092

872

679

-1

1600

Wavenumber / cm

RI PT

1668

1660

1176 1177 1174 1243 1242 1241 1306 1305 1306 1335 1333 1338 1369 1369 1416 1370 1416 1483 1480 1483 1508 1506 1576 1573 1575

1005 1060 1091

872

675 729

Raman Intensity

782

A

M AN U

731 782

676 734 782

Raman Intensity

ACCEPTED MANUSCRIPT

a b c

Wavenumber / cm

-1

1600

Fig. 5 Raman spectra of mixtures of Al species and DNA. (A): ct-DNA; (B): ss-DNA.

ACCEPTED MANUSCRIPT




Al3+ led to Hypochromic effect and nano-Al13 caused Hyperchromic effect.




DNA combined with Al3+ on phosphate group but with nano-Al13 on double-helix




FTIR and Raman spectra showed different binding sites of Al3+ and nano-Al13 to

AC C

EP

TE D

M AN U

SC

RI PT

DNA.