Vibrational dynamics of thin films of salt deoxyribonucleic acid

U ovoj doktorskoj disertaciji istraživan je utjecaj natrijevih i magnezijevih iona na strukturu i dinamiku deoksiribonukleinske kiseline (DNA) primjenom infracrvene spektroskopije s Fourierovom transformacijom na tankim filmovima DNA. Tanki filmovi DNA pripravljeni su iz niza otopina stalne koncentracije genomske DNA: Na-DNA (DNA s intrinsičnim kationima natrija) u čistoj vodi, 10 mM natrijevom kloridu i magnezijevom kloridu različitih ionskih jakosti, a Mg-DNA (DNA s intrinsičnim kationima magnezija) u čistoj vodi i magnezijevom kloridu različitih ionskih jakosti. Ovakav izbor uzoraka omogućio je istraživanje utjecaja, s jedne strane koncentracije dodane soli, a s druge strane kompeticije intrinsičnih i dodanih protuiona, na strukturu i konformaciju DNA. Pripravom tankih filmova iz otopina izbjegnut je utjecaj vibracija vode na spektar DNA u području baza. Spektri tankih filmova dobivenih iz Na-DNA u otopini natrijevog klorida te Na-DNA u čistoj vodi pokazali su da je konformacija dvostruke uzvojnice (ds) ostala sačuvana nakon provođenja protokola sušenja pa je za referentni spektar uzet spektar tankog filma dobiven iz čiste vodene otopine Na-DNA. U cilju razumijevanja utjecaja kationa magnezija na strukturu DNA istraživana su vibracijska svojstva tankih filmova Na-DNA u MgCl2 u širokom rasponu omjera koncentracija kationa i fosfata, [Mg]/[P], od 0,0067 do 30. Dobiveni rezultati pokazali su da kationi magnezija već pri niskim koncentracijama ostvaruju interakciju s fosfatnim skupinama, a s daljnjim povećanjem koncentracije i s bazama DNA preko dugodosežnih elektrostatskih sila. Pri najvećim omjerima [Mg]/[P] diskutiran je prijelaz DNA iz konformacije dvostruke uzvojnice u kompaktnu formu na osnovu eksperimentalnih rezultata, ali i teorijski predviđene neutralizacije fosfatnih skupina dobivene Manningovim dvoparametarskim modelom. Istraživana su i vibracijska svojstva tankih filmova Mg-DNA u čistoj vodi i Mg-DNA u MgCl2 u intervalu omjera [Mg]/[P] od 0,0067 do 10 te su rezultati uspoređeni s rezultatima dobivenim za Na-DNA u istom koncentracijskom rasponu. Spektri tankih filmova Mg-DNA u čistoj vodi pokazali su nešto drugačija vibracijska svojstva u odnosu na Na-DNA u čistoj vodi te je uočen znatno veći udio B forme DNA. Pri niskim koncentracijama magnezijevih iona ([Mg]/[P] < 1) spektri tankih filmova Na-DNA u MgCl2 i Mg-DNA u MgCl2 su različiti što se može pripisati utjecaju intrinsičnih natrijevih iona i intrinsičnih magnezijevih iona na DNA. S druge strane, pri višim koncentracijama magnezijevih iona, Na-DNA u MgCl2 i Mg-DNA u MgCl2 pokazuju slična vibracijska svojstva.In this doctoral thesis, the impact of sodium and magnesium ions on the structure and dynamics of deoxyribonucleic acid (DNA) was investigated utilizing Fourier transform infrared spectroscopy on DNA thin films. DNA thin films were obtained from a series of genomic DNA solutions of constant DNA concentration: Na-DNA (DNA with intrinsic sodium counterions) in pure water, 10 mM sodium chloride and magnesium chloride of various ionic strengths, and Mg-DNA (DNA with intrinsic magnesium counterions) in pure water and magnesium chloride of various ionic strengths. Such a choice of samples allowed the research of the effect of the concentration of the added salt as well as of the competition of intrinsic and added counterions on DNA structure and conformation. By preparation of thin films from solutions the influence of vibrational bands of bulk water that would impede the proper detection of DNA vibrational bands in the base region was avoided. Spectra of thin films obtained from Na-DNA in solution of sodium chloride and Na-DNA in pure water have shown that DNA retains its double helical structure (ds) under drying protocol so the spectrum of Na-DNA in pure water was chosen as a reference. In order to address how the behaviour of DNA evolves upon progressive addition of Mg2+ cations, an extensive investigation of infrared vibrational properties of Na-DNA in a broad range of added magnesium chloride salt concentration with cation to phosphate ratio, [Mg]/[P], between 0.0067 and 30 was performed. The obtained results revealed that at low magnesium concentrations the magnesium cations interact with the phosphate groups of the DNA, while with the further increase of magnesium concentration they interact with the base sites as well, via long-range electrostatic interactions. At highest [Mg]/[P] ratios the possibility of structural transition into a more compact form is discussed based on both the experimental results and calculations of DNA charge neutralization by Manning’s two variable condensation theory. The vibrational properties of Mg-DNA in pure water and Mg-DNA in a broad range of added magnesium chloride salt concentrations with cation to phosphate ratio, r=[Mg]/[P], between 0.0067 and 10 were investigated and compared with the results of Na-DNA in the same concentration range. The spectra of thin films Mg-DNA in pure water showed slightly different vibrational properties then Na-DNA in pure water and a significantly higher portion of B form DNA was observed. At low concentrations of magnesium ions (r < 1) spectra of Na-DNA in MgCl2 and Mg-DNA in MgCl2 show substantial differences which were attributed to the influence of the intrinsic sodium and magnesium ions on DNA. On the other hand, Na-DNA in MgCl2 and Mg-DNA in MgCl2 at higher concentrations of magnesium ions show similar vibrational properties

Structural and Oxidation Properties of Plasma Lipoproteins from Different Phenotypes: FT-IR and ESR Study

Subclasses of lipoproteins VLDL, LDL and HDL were isolated from plasma of two different phenotypes, A and B. Phenotype B is present in people with diabetes, atherosclerosis and higher risk for cardiovascular disease. Lipoproteins were studied by FT-IR spectroscopy to investigate conformational and structural differences reflected in spectra. Differences were observed in spectra of LDL and VLDL. In protein domain differences were observed in amide I band profile and discussed in terms of conformation¬al change of apo B-100 in small LDL and large VLDL particles. The changes in lipid bands from pheno¬type B are associated with differences in core composition and lipids\u27 ordering in monolayer. The study of slow oxidation was done by measuring the oxygen consumption in lipoprotein solutions. Oxidation was faster in HDL and VLDL from phenotype B, while for LDL it was similar for A and B samples and signif¬icantly faster than in other two lipoproteins. (doi: 10.5562/cca2236

Micro and Nano Structure of Electrochemically Etched Silicon Epitaxial Wafers

Silicon epitaxial wafers, consisting of 280 μm thick n-type substrate layer and 4–5 μm thick epitaxial layer, were electrochemically etched in hydrofluoric acid ethanol solution, to produce porous silicon samples. The resistivity of epitaxial layer was 1 Ω cm, while the substrate was much better conductor with resistivity 0.015 Ω cm. By varying the etching time, the micro- and nano-pores of different sizes were obtained within the epitaxial layer, and on the substrate surface. Due to the lateral etching the epitaxial layer was partially detached from the substrate and could be peeled off. The influence of etching time duration on the optical and structural properties of porous samples was investigated by Raman, infrared and photoluminescence spectroscopy. The samples were analysed immediately after the etching and six months later, while being stored in ambient air. The Raman spectra showed the shift in positions of transversal optical (TO) phonon bands, between freshly etched samples and the one stored in ambient air. Infrared spectra indicated the presence of SiHx species in the freshly etched samples, and appearance of oxidation after prolonged storage. Photoluminescence spectra were very weak in freshly etched samples, but their intensity has increased substantially in six month period. (doi: 10.5562/cca1971

Multivariate Analysis as a Tool for Quantification of Conformational Transitions in DNA Thin Films

The double-stranded B-form and A-form have long been considered the two most important native forms of DNA, each with its own distinct biological roles and hence the focus of many areas of study, from cellular functions to cancer diagnostics and drug treatment. Due to the heterogeneity and sensitivity of the secondary structure of DNA, there is a need for tools capable of a rapid and reliable quantification of DNA conformation in diverse environments. In this work, the second paper in the series that addresses conformational transitions in DNA thin films utilizing FTIR spectroscopy, we exploit popular chemometric methods: the principal component analysis (PCA), support vector machine (SVM) learning algorithm, and principal component regression (PCR), in order to quantify and categorize DNA conformation in thin films of different hydrated states. By complementing FTIR technique with multivariate statistical methods, we demonstrate the ability of our sample preparation and automated spectral analysis protocol to rapidly and efficiently determine conformation in DNA thin films based on the vibrational signatures in the 1800–935 cm−1 range. Furthermore, we assess the impact of small hydration-related changes in FTIR spectra on automated DNA conformation detection and how to avoid discrepancies by careful sampling

Conformational Transitions of Double-Stranded DNA in Thin Films

Conformational transitions of double-stranded DNA in different environments have long been studied as vital parts of both in vitro and in vivo processes. In this study, utilizing Fourier transform infrared spectroscopy (FTIR), we provide detailed analysis of dynamics of A- to B-form transitions in DNA thin films of different hydrated states based on a statistical analysis of a substantial number of spectra and band shape analysis (peak fitting) in both the phosphate (1150–1000 cm−1) and sugar–phosphate (900–750 cm−1) region. Hydration of DNA thin films is systematically controlled by the time spent in the desiccator chamber (from 3 min to 40 min) allowing conformation and hydration signatures, in addition to variations due to ambient conditions, to be resolved in the spectra. Conformation transition from A-form to more ordered B-form is observed if sufficient time in the desiccator chamber is allowed and is confirmed by changes on the bands at ≈890, 860, 837, and 805 cm−1. Phosphate vibrations at ≈1230 cm−1 and 1089 cm−1, and backbone vibrations at ≈1030 cm−1 and 765 cm−1 were found to be sensitive to changes in hydration rather than conformation. Additionally, we found that spectral variations caused by ambient conditions can be significantly reduced without inducing conformational changes, which serves as a good basis for quality assurance

Structural and Oxidation Properties of Plasma Lipoproteins from Different Phenotypes: FT-IR and ESR Study

Subclasses of lipoproteins VLDL, LDL and HDL were isolated from plasma of two different phenotypes, A and B. Phenotype B is present in people with diabetes, atherosclerosis and higher risk for cardiovascular disease. Lipoproteins were studied by FT-IR spectroscopy to investigate conformational and structural differences reflected in spectra. Differences were observed in spectra of LDL and VLDL. In protein domain differences were observed in amide I band profile and discussed in terms of conformation¬al change of apo B-100 in small LDL and large VLDL particles. The changes in lipid bands from pheno¬type B are associated with differences in core composition and lipids\u27 ordering in monolayer. The study of slow oxidation was done by measuring the oxygen consumption in lipoprotein solutions. Oxidation was faster in HDL and VLDL from phenotype B, while for LDL it was similar for A and B samples and signif¬icantly faster than in other two lipoproteins. (doi: 10.5562/cca2236

Possibility of Human Gender Recognition Using Raman Spectra of Teeth

Gender determination of the human remains can be very challenging, especially in the case of incomplete ones. Herein, we report a proof-of-concept experiment where the possibility of gender recognition using Raman spectroscopy of teeth is investigated. Raman spectra were recorded from male and female molars and premolars on two distinct sites, tooth apex and anatomical neck. Recorded spectra were sorted into suitable datasets and initially analyzed with principal component analysis, which showed a distinction between spectra of male and female teeth. Then, reduced datasets with scores of the first 20 principal components were formed and two classification algorithms, support vector machine and artificial neural networks, were applied to form classification models for gender recognition. The obtained results showed that gender recognition with Raman spectra of teeth is possible but strongly depends both on the tooth type and spectrum recording site. The difference in classification accuracy between different tooth types and recording sites are discussed in terms of the molecular structure difference caused by the influence of masticatory loading or gender-dependent life events