Wyniki wyszukiwania - BazTech

1

Chromatograficzna analiza związków budujących kwasy nukleinowe

Studzińska S., Rola R., Łobodziński F., Krzemińska K.

Wiadomości Chemiczne

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2016

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[Z] 70, 9-10

633--656

EN

Understanding the characteristics, role and structure of nucleic acids allowed to answer questions about the disease processes. Today, nucleic acids and their constituents are tools, which are used by molecular biology in medicine and biotechnology. Antisense and gene therapy are intensively developing methods for possible treating or preventing disease. They use short fragments of DNA or RNA - oligonucleotides to silence the genes expression. They are not the only ones that allow analytical chemists to obtain information about the state of our body. Determination of modified nucleoside allows detection of cancer, while analysis of nucleotides allows the estimation of strengthening the immune system. There is a great need of sensitive, selective and precise methods of separation of nucleosides, nucleotides and oligonucleotides and their qualitative and quantitative analysis. Consequently liquid chromatography (LC) is the most commonly used for analysis of nucleic acid constituents. The most widely used modes of LC include Ion Exchange Chromatography (IEC) and Reversed Phase High Performance Liquid Chromatography (RP HPLC). Both techniques have their advantages and disadvantages in the analysis of nucleosides, nucleotides and oligonucleotides. In the case of IEC it is necessary to use high concentrations of the salt in the mobile phase or concentration gradients, which considerably limits the possibility of using MS detection. RP HPLC can be coupled with MS detection but only when volatile salts are mobile phase components. On the other hand there is a significant problem is the lack of sufficient selectivity for the most polar nucleosides and nucleotides. RP HPLC MS is still most often used in the determination of nucleosides and nucleotides, due to its high sensitivity and a comprehensive qualitative analysis. Another system used for the HPLC analysis of oligonucleotides is Ion Pair Reversed Phase High Performance Liquid Chromatography (IP RP HPLC). These compounds can not be analyzed by RP HPLC due to their high polarity. The advantage of IP RP HPLC is selectivity, achieved by a suitable choice of mobile phase composition and the possibility of using MS. A disadvantage of IP RP HPLC in the analysis of oligonucleotides is however lower sensitivity compared to RP HPLC. During the last few years Hydrophilic Interaction Liquid Chromatography (HILIC) was applied for the separation of mixtures of nucleosides, nucleotides, oligonucleotides extracted from a biological or food samples. The presented results demonstrate the usefulness of this method, however, the resolving power is limited due to the asymmetric peak shape. On the other hand proper selection of the mobile and stationary phase can lead to a high selectivity in the analysis of the most polar nucleosides, nucleotides and oligonucleotides, which can not be separated by RP HPLC.

2

Techniki sekwencjonowania jako nowej generacji analityka w omice

Szymańska S., Studzińska S., Pareek Ch. S., Buszewski B.

Analityka : nauka i praktyka

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2012

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nr 3

27--36

PL

Sekwencjonowanie pozwala na ustalenie kolejności nukleotydów i wymaga dalszych analiz dających możliwości wyciągania inspirujących wniosków pozwalających na odkrycia mogące mieć kluczowe znaczenie podczas walki z chorobami cywilizacyjnymi ludzkości.

3

Wykorzystanie spektrometrii mas do analizy modyfikacji nukleotydów i adduktów DNA

Hanus J., Jelonek K., Pietrowska M.

Wiadomości Chemiczne

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2011

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[Z] 65, 3-4

191-205

EN

Chemically modified nucleotides, which are not normally present in genetic material, are called DN A adducts. This type of DN A modifications (damage) is directly related to processes of mutagenesis and carcinogenesis. Elevated levels of DN A adducts present in genetic material reflect exposure of humans to carcinogenic factors and are markers of increased risk of cancer [1]. For this reason different methods useful for quantitative and qualitative analyses of DN A adducts are used in the field of cancer prevention and research (Tab. 1). Enzymatically-catalyzed methylation of cytosine, observed mostly in so called CpG islands, is a frequent endogenous modification of genetic material. Such a DN A methylation is a key factor involved in regulation of gene expression, and methylation status of oncogenes and tumor supressor genes is an important biomarker of carcinogenesis. As such, analytical methods for assessment of DN A methylation are of great importance for molecular diagnostics of cancer. During the last decade significant progress has been made in methods available for quantitative, qualitative and structural analyses of biological molecules. Among intensively developed tools for bioanalyses are methods of mass spectrometry. Spectrometers that are based on two methods of ionization, namely electrospray ionization (ESI ) [30] and matrix-assisted laser desorption-ionization (MALDI ) [48], are particularly suitable for analyses of biological macromolecules: proteins and nucleic acids. Currently available mass spectrometers, together with microscale methods for sample preparation and separation, significantly increased sensitivity and accessible mass range of analyses. New generation of “user-friendly” instruments is developed to bring the techniques directly into the workplaces of biological and clinical investigators. This review demonstrates representative examples of mass spectrometry techniques used for qualitative analyses of nucleotide modifications and adducts present in genetic material of humans. In this field several methods base on spectrometers with electrospray ionization. Generated ions are separated according to their mass-to-charge ratio in an analyzer by electric fields; among different ion analyzers frequently used in this methods are single or triple quadrupole and ion traps (Fig. 1). Among other methods available for assessment of DN A adducts is so called Accelerator Mass Spectrometry (Fig. 2) [41]. The most frequently applied method for the assessment of DN A methylation is based on methylation-specific PCR reaction. Products of such PCR reactions are analyzed using MALDI mass spectrometry [54] (Fig. 3). In summary, new powerful methods of mass spectrometry that made available qualitative analyses of damage and modifications of human genetic material found their important place in modern biological and medical laboratories.

4

Wizualizacja algorytmów optymalnego dopasowania sekwencji nukleotydów i aminokwasów

Skowron A., Mrozek D.

Studia Informatica

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2011

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Vol. 32, nr 2A

245-258

PL

Celem prac przedstawionych w niniejszym artykule była konstrukcja narzędzia do wizualizacji wybranych algorytmów optymalnego dopasowania sekwencji nukleotydów i aminokwasów. Zasadę działania zbudowanego narzędzia można sprowadzić do trzech kroków. W kroku pierwszym określane są parametry wejściowe. W kroku drugim następuje wizualizacja dopasowania sekwencji biopolimerowych. Na koniec wyznaczane jest optymalne dopasowanie, zobrazowane ścieżką przejścia oraz wartością liczbową.

EN

The aim of the work reported in this paper was to develop a tool for visualization of optimal alignment algorithms of nucleotide and protein sequences. Functioning of the developed tool is based on three steps. In the first step, input parameters are determined. In the second step, the tool visualizes alignment of biopolymers according to the chosen algorithm. At the end, an optimal alignment is illustrated as an alignment path with appropriate similarity measure.

5

Efektywność grup donorowych nukleotydów w reakcjach z jonami metali w układach podwójnych i potrójnych z udziałem tetramin

Gąsowska A.

Wiadomości Chemiczne

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2007

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[Z] 61, 5-6

339-359

EN

Nucleotides, being multifunctional ligands with donor nitrogen and oxygen atoms, take part in the majority of selective and specific processes occurring in nature [1-15]. It has been established that nucleotides react with the polyamines (biogenic amines) present in the living organisms and take part in genetic information transfer [16-24]. Nucleotides are composed of a purine or pyrimidine base, sugar residua and phosphate groups (Fig. 1) [25-27]. Each of the three components have potential centres of interaction with metal ions [28-29]. Because of the wide diversity of coordination possibilities there are often controversies as to the mode of coordination even in simple complexes with metal ions. Some authors claim that only nitrogen atoms of the nucleotide are effectively engaged in the metallation [30-43], while others maintain that it requires a combined engagement of nitrogen atoms and phosphate group [44-71]. There are also researchers who point to the involvement of only phosphate group of the nucleotide in the metallation [72-77]. The reaction of nucleotides with tetramines results in the formation of molecular complexes (Fig. 3) [78-88]. In the literature to date, there is no agreement as to the character of interactions and effectiveness of nucleotide donor groups in the formation of adducts with polyamines [80-82, 85-87, 89, 90]. According to some authors, the interaction between a nucleotide and polyamines in the metal-free systems has a noncovalent ion-ion or ion-dipole nature and the stability of molecular complexes is determined by the number of active centres in the reagents and the structural factor [80-84, 87]. According to other authors, it is a typical electrostatic interaction and the adduct stability is determined by the charge of the reagents [85, 89]. In the adducts formed by nucleotides with polyamines, the main interaction centres of a nucleotide are endocyclic nitrogen atoms and a phosphate group (the latter undergoes deprotonation already at a low pH), while in the case of tetramine the interaction centres are the NHx+ groups [77, 80-87, 89-91]. In the ternary systems of metal/nucleotide/tetramine, the following heteroligand molecular complexes are formed: MLźźźźźźHxL' (x = 4, HxL'-fully protonated polyamine) (Fig. 4) [80-82, 91, 94, 96], mixed protonated complexes MLHxL' (x = 1, 2, 3) (Fig. 5) [81, 82, 92, 96] and MLL' type complexes (Fig. 6) [81, 82, 91]. A significant influence of polyamines on the character of interactions of nucleotides with metal ions has been noted [80-82, 90-96]. In molecular complexes, the fully protonated polyamine is located in the outer coordination sphere. In the MLHxL' type complexes, the deprotonated nitrogen atoms of tetramine are involved in the coordination, while its protonated centres -NHx+ take part in noncovalent interactions that additionally stabilise the complex [81, 82, 92, 96, 97]. In the MLL' type complexes, oxygen atoms of nucleotide phosphate group and deprotonated nitrogen atoms of tetramine are in the inner coordination sphere, while nucleotide donor nitrogen atoms do not take part in the metallation [81, 82, 91].

6

Chemiczna synteza oligorybonukleotydów

Milecki J.

Wiadomości Chemiczne

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2002

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[Z] 56, 3-4

255-277

EN

Basic issues and problems of chemical synthesis of oligoribonucleotides are presented. The paper describes three methods for construction of oligonucleotide chains: (1) a triester method which involves activation of nucleoside phosphodiesters with different azole sulfonates, and currently widely used methods employing PIII synthetic intermediates: (2) a phosphoramidite method which makes use of activation of nucleoside phosphoramidites by weakly acidic azoles or azole salts, (3) an H-phosphonate method, which uses nucleoside H-phosphonates activated by acid chlorides, both PIII intermediates are applied mainly in solid-support synthetic methodology. Problems of choosing appropriate protecting group for the synthesis are discussed. The article presents properties of basic types of protecting groups for lactam, exo-amino (base-labile protection), and hydroxyl groups (acid-labile for 5' protection, acid- or specific reagent-labile for 2' protection). The problem of 2'OH protection is described in detail. In this respect acid-labile groups and alkylsilyl groups are compared and their advantages and disadvantages are discussed. More detailed discussion is devoted to the phenomenon of the silyl group migration during the synthesis of monomeric units for oligonucleotide chain assembly. Basing on the NMR study of the isomerisation reaction it was possible to determine limits of safety of the reaction conditions.

7

Zastosowanie porfirynowych faz stacjonarnych w HPLC

Biesaga M.

Analityka : nauka i praktyka

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2002

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nr 4

13--19

PL

Jednym z podstawowych problemów chemii analitycznej jest selektywność metody oznaczania danego związku, zwłaszcza gdy występuje on w niskim stężeniu w próbce o skomplikowanej matrycy.

8

Thin layer chromatography of nucleotides on aminopropyl bonded silica plates

Szumiło H., Flieger J.

Chemia Analityczna

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2000

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Vol. 45, No. 3

363-370

EN

The conditions of mono-di and triphosphate nucleotide separation on thin layers of silica gel with chemically bonded aminopropyl groups (HPTLC-NH2) were elaborated on. The separation was achieved by analysing the effect of pH, and concentration of various inorganic salts in water and water-methanol mobile phases.The results were analysed using the stoichiometric displacement model developed for ion-exchange chromatography. It was shown that the model may be useful for optimising elution conditions of polianions on amine plates.

PL

Opracowano warunki rozdzielania nukleotydów mono- di- i trifosforanowych na cienkich warstwach żelu krzemionkowego z chemicznie związanymi fazami aminopropylowymi (HPTLC-NHa). Analizując wpływ pH, stężenia i rodzaju soli nieorganicznych w wodnych i wodno-metanolowych fazach ruchomych uzyskano rozdział badanych związków. Do analizy uzyskanych wyników zastosowano model steąhiometrycznej wymiany opracowany dla chromatografii jonowymiennej. Wykazano, że model ten może być przydatny do optymalizowania warunków elucji polianionów na płytkach ze związaną fazą aminową.

9

Chromatographic separation of nucleotides using stationary phases modified with metal ion complexes

Flieger J., Szumiło H., Jóźwiak K.

Chemia Analityczna

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1998

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Vol. 43, No. 4

669-675

EN

Complexes of metal ions Ag(I), Cu(II) and Mg(III) with di(2-ediylhexyl) orthophosphoric acid (DEPH) were immobilized on the RP l8 W plates via hydrophobic interactions. The resultod stationary phases were testod with TLC for selected nucleotides. Effects of mobile phase, its pH end the kind of the metal complex on the retention behaviour of nucleotides were studied. A possible mechanismes of interactions of solute molecules with stationary and mobile phases were discussed.

PL

Kompleksy jonów metali (Ag(I), Cu(II) i Mg(II) z kwasem di(2-etyloheksylo) ortofosforowym (DEHP) immobilizowano na płytkach ze związaną fazą oktadecylową wykorzystując niespecyficzne oddziaływania hydrofobowe. Uzyskane fazy stacjonarne testowano, analizując wybrane nukleotydy metodą TLC. Badano wpływ rodzaju eluentu, jego pH oraz rodzaju jonów metali na retencję nukleotydów. Zaproponowano prawdopodobny mechanizm interakcji nukleotydów z fazą stacjonarną i ruchomą.