Josef Stepanek

A universal computer-controlled UV-VIS spectrometer with high resolution monochromator

Abstract A modular UV-VIS spectrometer based on a high-resolution monochromator was computerized using an 8-bit CBM 8296 microcomputer. The hardware of the microcomputer was extended with serial and 64-bit parallel interfaces and with graphics. To control the spectrometer a set of machine code routines was created; they are simply callable from the users program written in BASIC.

Advanced Microfluorescence Methods in Monitoring Intracellular Uptake of “Antisense” Oligonucleotides

Antisense strategy represents a promising molecular tool for efficient and selective chemotherapeutic action. It belongs among oligonucleotide strategies that employ specific single-stranded sequences of deoxyriboand ribonucleotides or their synthetic analogs to block or suppress expression of a pathogen in its early stage. This approach is also promising for studies of the biological function of the gene. However, the routine use of modified oligonucleotides in practice is complicated by non-ideal properties of currently available oligonucleotide analogs. A successful medical treatment requires not only proper binding of the modified oligonucleotide to its cellular target but also its efficient cellular uptake, stability and appropriate distribution in the intracellular environment. The latter processes can be effectively studied by various microfluorescence techniques. The paper reviews the current situation in the application of advanced microfluorescence methods in this field and gives a brief description of the oligonucleotide strategy and possibilities to support the cellular uptake, theoretical and technical basics of current fluorescence microimaging and fluorescence microspectroscopy including time-resolved measurements. Second part of the paper describes experiment preparation, surveys the most interesting studies published so far and outlines the perspectives.

Molecular dynamics simulations of the oligonucleotide with the modified phosphate/phosphonate internucleotide linkage

Impact of the internucleotide linkage modification by inserting a methylene group to the P-O bond (-O-PO2−-O- chain changed for -O-PO2−-CH2-O-), on the modified oligonucleotide binding ability to the natural DNA strand was studied by molecular dynamics simulation. Complex of (dT)11 with a deoxyadenosine undecamer containing alternating modified and natural internucleotide linkage was studied as a model system. The Amber force field was completed by a set of new parameters needed to model the modified part of the nucleotide. The simulations confirmed existence of a double-helical complex the melting point of which is considerably higher than 300 K. While the thymidine (unmodified) strand possesses a B-type secondary structure, the conformation of the adenosine (modified) strand is not stable at 300 K. The -ggt conformation of the modified linkages is highly preferred, temporary jumps to the -g-gt and ggt conformations were, however, observed.

Interaction of porphyrin/oligonucleotide complex with liposomes studied by drop coating deposition Raman spectroscopy

Drop coating deposition Raman (DCDR) microspectroscopy was used to investigate interaction of the complexed cationic copper 5,10,15,20-tetrakis(1-methyl-4-pyridyl) porphyrin (CuP) and phosphorothioate analog of dT15 oligonucleotide with liposomes, the lipid composition of which imitated the natural plasmatic membrane. Great advantage of dried drops on DCDR plates over a solution sample is that the specific drying process on the special hydrophobic surface efficiently separates liposomes from small species in the solvent. In our case, liposomes with bound CuP/oligonucleotide complexes formed a ring at the edge part of the dried drop while dried solution of this complex remained inside this ring. High quality spectra measured from the ring by using Raman confocal microspectrometer revealed unperturbed arrangement of lipid chains by the drying process, partial binding of the CuP/oligonucleotide complexes to liposomes, and a certain reorientation of lipid chains as a consequence of this interaction.

Monitoring of labeled antisense oligonucleotides within living cells by using a multifrequency phase/modulation approach for fluorescence lifetime measurements

A multifrequency phase/modulation method has been developed for our UV confocal laser microspectrofluorimeter (modulation frequency 1 ‐ 200 MHz) for fluorescence lifetime measurements. This technique enables excited state lifetimes of mixed fluorescent components to be resolved and the fluorescence spectral contribution of each species to be determined without using any model spectra. This approach is very efficient for analyzing intracellular multicomponent fluorescence signals. Our effort is focused on the elucidation of the intracellular behavior of synthetic modified oligonucleotides - potential drugs for antisense and/or antigene strategies of curing viral and malignant diseases. A novel type single stranded dT15 oligomer analogue containing isopolar, non-isosteric, phosphonate-based internucleotide linkages (3 0 -O‐P‐CH 2‐ O-5 0 ), labeled with tetramethylrhodamine dye at the 3 0 -end, has been utilized. This method, along with fluorescence micro-imaging, was used to monitor uptake, distribution and stability of our modified oligonucleotide inside living cells. Binding to Escorte vector leads to an homogeneous intracellular distribution of fluorescent labeled oligonucleotide, including nucleus staining, while point distribution only is achieved for its free form. q 2003 Elsevier Science B.V. All rights reserved.

Raman study of magnesium induced conversion of polyU·polyA duplexes to polyU·polyA·polyU triplexes

Raman titration experiment with magnesium salt added gradually to aqueous solution of duplexes formed by RNA homopolynucleotides polyU and polyA was performed to reveal its effect on homopolynucleotide complexes. Statistical analysis of obtained spectral set has confirmed the effect already found by less structurally sensitive methods [Nucleic Acids Res. 31(17) (2003), 5101–5107] that at sufficiently high concentrations magnesium causes transformation of polyU·polyA duplexes to polyU·polyA·polyU triplexes and single polyA strands. It was found that at relatively high polynucleotide concentrations used in Raman experiment, the threshold magnesium concentration for this effect is above the concentration of duplex basepairs in solution. Due to the strong spectral changes attributed to the varied percentages of duplexes, triplexes and single strands, it was not possible to register weaker direct Raman signs of the magnesium binding to polynucleotide strand.

Stability and Structure of Porphyrin Complexes Studied VIA Serrs Spectroscopy

Glycosides of sugars and 5,10,15,20-tetrakis-(4-hydroxyphenyl)-21H,23H-porphyrin, namely glucoside 5,10,15,20-tetrakis-(4-glucosephenyl)-21H,23H-wrphyrin (H2TPP-(0-Glc)4) and galactoside 5,10,15,20-tetrakis-(4-galactosephenyl)-21H,23H-poiphyrin (H2TPP-(0-Gal)4) (Fig.l) are in focus of our interest due to their potential application in photodynamic therapy (PDT) of cancer [1]. Despite the analogous molecular structures, the photo-chemical activities of H2TPP-(0-Glc)4 and H2TPP-(0-Gal)4 were found to differ slightly [1]. The aim of the present work was to find, by means of resonance Raman scattering (RRS) and surface enhanced resonance Raman scattering (SERRS) spectroscopies, a correlation between activity and structural properties of porphyrin self-aggregates formed in aqueous solutions.

Application of high-rate crystal growth technique to single crystals of nucleic acid bases

On the basis of the metastable zone investigations the optimal conditions for adenine sulphate solution stability were found. These made it possible to apply the novel high rate growth technique developed for KDP-family crystals group to the nucleic acid base crystals. The optical quality of adenine sulphate crystals grown at the rate about five times higher than the traditional one thus reducing the growth period six or seven times is sufficient not only for Raman spectroscopy but even for the normal reflection one.

Statistical signal processing in Raman spectroscopy of biological samples

The noise of Raman and elastic scattering has been studied by means of intensity value distribution, Fourier spectrum, autocorrelation function and correlation coefficient between both signals. Measurennts have been carried out for inhomogeneous liquid samples which are typical in biological applications of Raman spectroscopy . It. is shown how the statistical properties of noise ei serve for a check on an experimental set-up and a sample quality and for improvement in measuring process. A special procedure is proposed when the Raman and the elastic scatterings are measured simultaneously and the latter signal is used for decreasing of the noise in the Ranian spectriun.