Josef Štěpánek

Metalation of 5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)porphyrin in silver colloids studied via time dependence of surface‐enhanced resonance Raman spectra

In SERS-active systems, when a free base porphyrin is adsorbed at a roughened metal surface, the metal ion may be incorporated into the porphyrin core. We have investigated the metalation kinetics of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP), reflected in the time evolution of its SERRS spectra. To establish proper metalation Raman markers, resonance Raman spectra of the free base and chemically Ag metalated TMPyP forms were measured under different conditions. The increase in intensity of the 395 cm-1 line, the decrease in intensity of the 329 cm-1 line, the reduction of the 1337+1360 cm-1 doublet to one strong line at 1340 cm-1 and the vanishing of the 1140 cm-1 medium line (the last exceptionally for 514.5 nm excitation) were found to be suitable markers for quantitative analysis. All other spectral changes related to metalation may coincide with effects caused by other variations of the porphyrin state. Time evolution of the porphyrin metalation was studied in various Ag colloid systems, with or without phosphate anions, citrate and/or Triton X-100. Time-dependent SERRS spectra of TMPyP were recorded within periods from minutes to several hours. Factor analysis of the SERRS spectra proved that the spectral changes were induced by only one phenomenon (metalation). The SERRS spectra were decomposed into the spectrum of the free base TMPyP and that of its Ag metalated form, and the metalation kinetics were determined by means of the time dependence of the metalated form portion in the SERRS spectrum. The results show a remarkable reliance on the state of the metal surface.

Laser ablation: Preparation of “chemically pure” Ag colloids for surface-enhanced Raman scattering spectroscopy

“Chemically pure” Ag colloids as active surfaces for surface-enhanced Raman scattering (SERS) spectroscopy were prepared by laser ablation of an Ag foil in pure water, in the presence of Cl− anions, and in the presence of an adsorbate. SERS-activity of the ablated colloids was proved using 2,2′-bipyridine [bpy] and phtalazine [pht] as adsorbates. Moreover, a SERS-active Ag colloid/adsorbate system was prepared by laser ablation of Ag foil immersed in an aqueous solution of pht. SERS spectra of pht obtained from this system are identical with those of pht added to Ag colloid ablated in pure water. The possibility of preparing a SERS-active system by laser ablation of Ag in the presence of an adsorbate was thus demonstrated. Furthermore, Ag colloid-bpy films were prepared from the laser ablated colloids and their SERS activity proved by obtaining a good quality SERS spectra of bpy.

Raman spectroscopic study of triplex‐like complexes of polyuridylic acid with the isopolar, non‐isosteric phosphonate analogues of diadenosine monophosphate

The formation of poly(rU) complexes with diadenosine monophosphate (3′–5′) and its four isopolar, non-isosteric phosphonate analogues was studied by means of Raman spectroscopic titration. A set of mixed samples containing the diadenosine monophosphate or monophosphonate and the poly(rU) at various concentration ratios was prepared. The room temperature spectra of mixed solutions were treated by factor analysis and decomposition of the Raman marker bands. For all the species studied, the results revealed an exclusive presence of the triplex-like complexes, i.e. complexes with a 1:2 stoichiometric ratio of the adenosine to uridine units, the Raman spectra of which exhibit all the features typical of the poly(rU)·poly(rA)·poly(rU) triple-helical structure. The individual molar fractions of the free ApA dimer, the free poly(rU) and the triplex-like complex do not fit classical equilibrium equation, because the binding ability of the adenosine dimers to poly(rU) falls significantly for small ApA molar fractions. This tendency is strongest for the natural diadenosine phosphate (3′–5′) and weakest for two analogues in which the —CH2— group in the modified internucleotide linkage is situated closer to the 5′-position. Copyright

Metallation kinetics of a free base porphyrin in surface-enhanced resonance Raman scattering active Ag colloid system as a probe of porphyrin–nucleic acids interaction

Abstract Complexes of the cationic free base 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin with DNA, double-stranded poly(dA-dT) and poly(dG-dC) polynucleotides have been studied by using surface-enhanced resonance Raman scattering spectroscopy (SERRS). SERRS spectra of these complexes have been recorded during time-scale varying from minutes to days, and the differences in the course of the porphyrin metallation, indicating different accessibility of the metal surface for the porphyrin core in the case of particular complexes, have been monitored. The results obtained for the three studied systems are in very good agreement with the expected dominant types of the porphyrin interaction with the nucleic acids, i.e., intercalation in the case of poly(dG-dC) and external binding in the case of poly(dA-dT).

METALATION OF POSITIVELY CHARGED WATER SOLUBLE MESOPORPHYRINS STUDIED VIA TIME-RESOLVED SERRS SPECTROSCOPY

Abstract Time-resolved SERRS spectra of 5,10,15,20-tetrakis[4-(trimethylammonio)phenyl]21 H ,23 H -porphine (TMAP) were recorded (using a multichannel Raman spectrometer) in various SERS-active Ag colloid/porphyrin systems. Data treatment based on a factor analysis was used to decompose all the SERRS spectra into two main components: SERRS spectrum of the free base TMAP and that of its Ag metalated form. The metalation kinetics obtained in this way was found to be highly dependent on the presence of phosphate anions, citrate and/or Triton X-100 in the colloidal system. The results are analogous to those previously obtained for 5,10,15,20-tetrakis(1-methyl-4-pyridyl)21 H ,23 H -porphine, a porphyrin with a substantially stronger tendency towards metalation.

Low temperature vibrational and vibronic spectra of adenine single crystals

Abstract Both polarized UV absorption and Raman spectra of adeninium sulphate single crystals were measured at 10 K on the same modular UV-VIS spectrometer. Sharp vibronic structure of the lowest-energy absorption band of N1,N7 diprotonated adenine is interpreted in terms of Herzberg-Teller theory. Slight change of adenine molecule geometry localized round the amino group, which is caused by the electronic excitation, and strong vibronic coupling of some vibrational modes with other electronic transitions have been found.

Surface plasmon resonance study on HIV-1 integrase strand transfer activity

AbstractUnderstanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats–IN complexes with the host DNA. HIV-1 integrase strand transfer activity was monitored in real time using a multichannel surface plasmon resonance biosensor.

5′-O-Methylphosphonate nucleic acids—new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes

Several oligothymidylates containing various ratios of phosphodiester and isopolar 5′-hydroxyphosphonate, 5′-O-methylphosphonate and 3′-O-methylphosphonate internucleotide linkages were examined with respect to their hybridization properties with oligoriboadenylates and their ability to induce RNA cleavage by ribonuclease H (RNase H). The results demonstrated that the increasing number of 5′-hydroxyphosphonate or 5′-O-methylphosphonate units in antisense oligonucleotides (AOs) significantly stabilizes the heteroduplexes, whereas 3′-O-methylphosphonate AOs cause strong destabilization of the heteroduplexes. Only the heteroduplexes with 5′-O-methylphosphonate units in the antisense strand exhibited a significant increase in Escherichia coli RNase H cleavage activity by up to 3-fold (depending on the ratio of phosphodiester and phosphonate linkages) in comparison with the natural heteroduplex. A similar increase in RNase H cleavage activity was also observed for heteroduplexes composed of miRNA191 and complementary AOs containing 5′-O-methylphosphonate units. We propose for this type of AOs, working via the RNase H mechanism, the abbreviation MEPNA (MEthylPhosphonate Nucleic Acid).

Bentonite/Iron Oxide Composites: Preparation and Characterization by Hyperfine Methods

Bentonite/iron oxide system is prepared by isothermal calcination of powder composed of bentonite clay and precursor containing ferric acetate. This preparation technique enables one to get the composite material directly, that is, iron oxide particles embedded in a bentonite matrix. Calcination temperature is varied from 320°C to 700°C. The resulting series of samples is characterized by local methods based on hyperfine interactions: 57Fe nuclear magnetic resonance (NMR) and the Mössbauer spectroscopy. The results show that the phase composition changes significantly in dependence on calcination temperature. The amount of maghemite phase rapidly increases up to °C and decreases abruptly for higher than 460°C.

A dual surface plasmon resonance assay for the determination of ribonuclease H activity

There is a demand for efficient tools for the monitoring of RNase H activity. We report on a new assay which allows for simultaneous (1) real-time monitoring of RNase H activity and (2) detection of cleavage reaction products. The dual assay is implemented using a multichannel surface plasmon resonance (SPR) biosensor with two independently functionalized sensing areas in a single fluidic path. In the first sensing area the RNA cleavage by RNase H is monitored, while the products of the cleavage reaction are captured in the second sensing area with specific DNA probes. The assay was optimized with respect to AON concentration and temperature. A significant improvement was obtained with special chimeric probes, which contain RNA substrate for RNase H and a longer deoxyribonucleotide tail, which enhances the SPR signal. It has been shown that RNase H stabilizes the RNA:DNA hybrid duplex before the cleavage. The potential of the assay is demonstrated in the study in which the ability of natural and modified oligonucleotides to activate RNase H is examined.