2. PŘEDNÁŠKA 7.10. 2013 ....ještě doplněk scientometrie Index h navrhl Jorge Hirsch, University of California, San Diego (Nature 436 (2005) 900) je to číslo, ktere udává počet n prací (určitého autora, instituce, apod.) které byly citovány nejméně n-krát h-index 22 21 22. 18. 21. 22. Index h The journal impact factor is a measure of the frequency with which the "average article" in a journal has been cited in a particular year. The impact factor will help you evaluate a journal's relative importance, especially when you compare it to others in the same field. NOTE: Title changes and coverage changes may result in no impact factor for one or more years. Impact factor 2011 Cites in 2011 to articles published in: 2010 = 100 2009 = 132 Sum: 232 Number of articles published in: 2010 = 31 2009 = 22 Sum: 53 Calculation: Cites to recent articles: 232 IF = 4.377 Number of recent articles: 53 Impact Factor (IF) charakterizuje průměrnou citovanost prací publikovaných v daném časopise Vybráno z Nature: The Swiss journal Folia Phoniatrica et Logopaedica has a good reputation among voice researchers but, with an impact factor of 0.655 in 2007, publication in it was unlikely to bring honour or grant money to the authors' institutions. Now two investigators, one Dutch and one Czech, have taken on the system and fought back. They published a paper called: 'Reaction of Folia Phoniatrica et Logopaedica on the current trend of impact factor measures' (H. K. Schutte and J. G. Švec Folia Phoniatr. Logo. 59, 281-285; 2007). This cited all the papers published in the journal in the previous two years. As 'impact factor' is defined as the number of citations to articles in a journal in the past two years, divided by the total number of papers published in that journal over the same period, their strategy dramatically increased Folia's impact factor this year to 1.439. Hrátky s IF Problémy časopisů s nízkým IF 1. Nedostatek vysoce kvalifikovaných recenzentů a vyšší pravděpodobnost publikace nekvalitních prací (záleží na šíři problematiky/scope of the journal) 2. Slabé ocenění publikovaných prací při evaluacích a financování výzkumu 3. Menší zájem čtenářů 4. Nestabilita IF atd. ALE San Francisco Declaration on Research Assessment Putting science into the assessment of research There is a pressing need to improve the ways in which the output of scientific research is evaluated by funding agencies, academic institutions, and other parties. To address this issue, the group of editors and publishers of scholarly journals listed below met during the Annual Meeting of The American Society for Cell Biology (ASCB) in San Francisco, CA, on December 16, 2012. The group developed a set of recommendations, referred to as the San Francisco Declaration on Research Assessment. We invite interested parties across all scientific disciplines to indicate their support by adding their names to this declaration. The Journal Impact Factor is frequently used as the primary parameter with which to compare the scientific output of individuals and institutions. The Journal Impact Factor, as calculated by Thomson Reuters, was originally created as a tool to help librarians identify journals to purchase, not as a measure of the scientific quality of research in an article. With that in mind, it is critical to understand that the Journal Impact Factor has a number of well-documented deficiencies as a tool for research assessment. These limitations include: A) citation distributions within journals are highly skewed [1-3]; B) the properties of the Journal Impact Factor are field-specific: it is a composite of multiple, highly diverse article types, including primary research papers and reviews [1, 4]; C) Impact Factors can be manipulated (or “gamed”) by editorial policy [5]; and D) data used to calculate the Journal Impact Factors are neither transparent nor openly available to the public [4, 6, 7]. IF je pouze předběžný údaj o významu určité práce DŮLEŽITĚJŠÍ je CITOVANOST DANÉ PRÁCE v určitém OBORU IF, počty citací a další scientometrické údaje by neměly být využívany ke srovnávání úspěšnosti vědců z různých oborů Citace jinými autory x autocitace Nobel Extra třída bio ČEŠI a. Fyzici b.Chemici c.Bio Vesmír, září 2006 Když si budete vybírat téma své diplomky/dizertace snažte se zjistit jak je váš budoucí školitel ve vědě úspěšný a jak aktuální a zajímavé jsou problémy, které řeší Vědecké týmy, vědecká spolupráce Společné a vlastní a publikace ÚLOHA JEDNOTLIVCE Již během svého studia můžete dělat vědecké objevy! Již během svého studia můžete dělat vědecké objevy! ‹#› 13 This year we commemorate the 90th Anniversary of the invention of polarography by J. Heyrovsky. In 1941 he invented oscillographic polarography with controlled a.c. (cyclic a.c. chronopotentiometry). By the end of the 1950‘s oscillographic polarography was the method of choice for the DNA electrochemical analysis: 1958: Nucleic acid bases, DNA and RNA are electroactive 1960: Relations between the DNA structure and electrochemical responses 90 years of polarography and ~55 years of nucleic acid electrochemistry E. Palecek, Nature 188 (1960) 656-657 E. Palecek, Naturwiss. 45(1958)186-187 Electrochemistry of nucleic acids is now a booming field WHY? 112 (2012) 3427-3481 1960-66 Relation between the DNA structure and electrochemical responses 1974 DNA unwinding at negatively charged surfaces 1981-83 Electroactive markers covalently bound to DNA 1986-88 DNA-modified electrodes progress in GENOMICS increasing importance of parallel nucleotide sequencing electrochemistry can complement optical detection in arrays and particularly in chips for decentralized analysis IF ~40 1958: Nucleic acid bases, DNA and RNA are electroactive 50 years of nucleic acid electrochemistry ...part of the guanine ring important for the anodic signal is near to the surface whereas the the analogous part of cytosine is hidden inside the DNA double helix participating in the hydrogen bonding.... (showing a cathodic signal in ssDNA but not in dsDNA) E. Palecek, Nature 188 (1960) 656-657 E. Palecek, Fifty years of nucleic acid electrochemistry, Electroanalysis 2009 H. BERG Bchem Z. 1957: DNA and RNA are INACTIVE Recently we have found that peak H is produced by proteins adsorbed at mercury and solid amalgam electrodes modified by different kinds of thiol self-assembled monolayers (SAMs). For practical reasons we were primarily interested in DTT SAMs. Electrochemical analysis of proteins and peptides at Hg electrodes in the presence of large excess of thiols was difficult or impossible. Temperature, at which the electrode process is taking place, greatly influences the electrochemical behavior of the surface-immobilized proteins. protein-modified electrode V. Ostatná, H. Cernocká, E. Palecek (2010) J.Am. Chem. Soc., 132, 9408-9413 Thiol SAM V posledních letech se zabýváme interakcemi bílkovin a jejich elektrochemii Tumor suppressor protein p53 declared „The Molecule of the Year“ by Science magazine in 1993 perhaps the most important protein in the development of cancer. This protein p53 plays a critical role in the cellular response to DNA damage by regulating the expression of genes involved in controlling cell proliferation, DNA repair, and apoptosis. P53 protein is inactivated by mutation in about 50 % of human malignancies. Most mutations are located in the DNA-binding core domain of the protein. p53 protein is biologically active in its reduced state and is usually stored with mM concentrations of reducing agent - dithiothreitol (DTT). EU 6th FP: Mutant p53 as target for improved cancer therapy ‹#› 17 15.9 17.9 20.3 °C 8.6 11.1 13.9 °C p53 core domain Mutation in R175H induces structural perturbation at the zinc-binding site, destabilizes the core domain by 3 kcal/mol and eliminates p53 sequence specific DNA binding. The same effect can be observed in the wt core domain upon removal of the zinc ion. We tested other mutants such as V145A, F270L, R273H and Y220C and we always observed CPS responses different from the wt protein H1 H2 E. Palecek et al. JACS 2011, 133, 7190–7196 The Protein Group Hana Cernocka Mojmir Trefulka Petra Mittnerova Emil Palecek Lida Rimankova Martin Bartosik Veronika Ostatna Veronika Vargová 288 10304 35.78 57 Společné a vlastní publikace ÚLOHA JEDNOTLIVCE Když si budete vybírat téma své dizertace snažte se zjistit jak je váš budoucí školitel ve vědě úspěšný ‹#› 20 This year we commemorate the 90th Anniversary of the invention of polarography by J. Heyrovsky. In 1941 he invented oscillographic polarography with controlled a.c. (cyclic a.c. chronopotentiometry). By the end of the 1950‘s oscillographic polarography was the method of choice for the DNA electrochemical analysis: 1958: Nucleic acid bases, DNA and RNA are electroactive 1960: Relations between the DNA structure and electrochemical responses 90 years of polarography and ~55 years of nucleic acid electrochemistry E. Palecek, Nature 188 (1960) 656-657 E. Palecek, Naturwiss. 45, 186-187 Electrochemistry of nucleic acids is now a booming field WHY? 112 (2012) 3427-3481 1960-66 Relation between the DNA structure and electrochemical responses 1974 DNA unwinding at negatively charged surfaces 1981-83 Electroactive markers covalently bound to DNA 1986-88 DNA-modified electrodes progress in GENOMICS increasing importance of parallel nucleotide sequencing electrochemistry can complement optical detection in arrays and particularly in chips for decentralized analysis S přáním co nejlepší scientometrické váhy všem přítomným Acad Sci, Brno Masaryk Univ, Brno 2010 + elektrochemie poly- a oligosacharidů, glykoproteiny 273 8.836 32,37 54 Společné a vlastní a publikace ÚLOHA JEDNOTLIVCE Elektrochemie biomakromolekul bílkoviny, glykoproteiny DNA, RNA 1958: Nucleic acid bases, DNA and RNA are electroactive 50 years of nucleic acid electrochemistry ...part of the guanine ring important for the anodic signal is near to the surface whereas the the analogous part of cytosine is hidden inside the DNA double helix participating in the hydrogen bonding.... (showing a cathodic signal in ssDNA but not in dsDNA) E. Palecek, Nature 188 (1960) 656-657 E. Palecek, Fifty years of nucleic acid electrochemistry, Electroanalysis 2009 H. BERG Bchem Z. 1957: DNA and RNA are INACTIVE 50 Paleček Emil 7 524 248 Elektrochemie nukleových kyselin a bílkovin; Nádorové supresory, zejména protein p53; Agregace bílkovin v neurodegenerativních chorobách (zejména agregace α-synucleinu v Parkinsonově chorobě) Chemická reaktivita a struktura nukleových kyselin. Lokální struktury DNA stabilizované superhelikálním vinutím; Interakce DNA a bílkovin s povrchy; Interakce DNA-protein; ‹#› 26 2. přednáška 7.10. ‹#› 27 LEFT-HANDED Z-DNA alternating pu-py CRUCIFORM inverted repeat CURVATURE 4-6 A’s in phase with the helix turns SINGLE-STRANDED region AT-rich Text TRIPLEX structure homopu.homopy HAIRPIN SUPERCOIL Negative SUPERCOILING stabilizes local DNA structures Physical methods such as NMR and X-ray analysis indispensable in the research of linear DNA structures are of limited use in studies of local structures stabilized by supercoiling Problems of life origin What was first - DNA, RNA or protein? Well-known Oxford zoologist Professor Richard Dawkins (who declares himself to be passionate fighter for the truth) writes in his book River out of Eden: “At the beginning of Life Explosion there was no mind, no creativity, no intent, there was only chemistry” Let us try to summarize what chemistry it was ‹#› 30 PROBLEMS OF LIFE ORIGINS The Miller-Urey experiment attempted to recreate the chemical conditions of the primitive Earth in the laboratory, and synthesized some of the building blocks of life but geologists showed that prebiotic atmosphere was not strongly reducing and not oxygen-free, differring from that expected by Miller and Urey S. Miller and H. Urey subjected mixture of methane, ammonia and hydrogen to an electric discharge and led the product into water ... Cytosine synthesis would not be possible even strongly in reducing prebiotic atmosphere. Similar problems arise with the abiotic synthesis of nucleotides Abiotic synthesis of a complicated molecule such as RNA is highly improbable ‹#› 33 ‹#› 34 NOBEL laureate Christian de Duve has called for “a rejection of improbablities so incomensurably high that they only can be called miracles, phenomena that fall outside the scope of scientific inquiry”. DNA, RNA and PROTEINS must then be set aside as participants in the origin of life. ‹#› 35 ‹#› 36 ‹#› 37 DNA drugs come of age - new DNA vaccines Viral gene(s) are brought to the cells via an appropriate plasmid. Once plasmids are inside, the cells manufacture the protein encoded by the gene. In the case of an antiviral DNA vaccine, the resulting viral proteins elicit an immune response that prevents future infection by that virus. MAKING THE VACCINE PROTEINS: A DNA vaccine delivered into the skin enters (tranfects) local skin cells and some immune cells. The transfected cells make the plasmid-encoded viral protein; immune cells engulf the antigen proteins as they are exiting cells. A GOOD IDEA The immune system does not perceive the plasmids as foreign material. The protein encoded by the plasmid gene elicits proper immune reaction against this protein. However, the early DNA vaccines evoke a weak immune response only. The reasons were (i) vaccine plasmids were not getting into enough cells, (ii) the cells were not producing enough of the encoded proteins. Simply, the immune system was not being sufficiently stimulated. 2007 FAILURE (STEP TRIAL) Merck tried to use as vector an adenovirus called AdHu5 to deliver HIV viral genes. Unfortunately, people who got the vaccine were no better protected than those who received the placebo, and eventually they appeared to be more vulnerable to being infected by HIV. The discouraging result was 49 out of 914 men in the vaccine group became HIV positive, whereas 33 out of 922 men in the placebo group did not. THE REBIRTH OF DNA The search for improvement was directed into several directions how to boost all aspects of the plasmids´ activity namely new methods of getting them into cells or new ways of increasing protein production. New vaccine delivery methods get considerably more cells – including immune cells themselves – to take up plasmids. For instance, needle-free systems, such as GeneGun and Bioject that use pressurized air to inject vaccine, deliver plasmids into the skin where immune entries called antigen-presenting cells are highly concentrated. To achieve a similar result the injection can be followed by electroporation. That is application of a series of electrical pulses that cause cell membranes to temporarily open pores that allow plasmids to enter more easily. Electroporation can increase uptake of plasmids by as much as 1,000 fold. The plasmid-gene constructs themselves have also been improved through refinements to the DNA sequences of the genes they carry. Codon optimization results in the gene´s instructions in a way the cell will execute most readily. Certain amino acids are designated by more than one codon, but cells typically favor one of these synonymous codons and translate it more efficiently than the others. Thus optimal codons increase production of the desired protein. Also the stability and accuracy the messenger RNA gene transcripts plays a role. A leader sequence near the start of each gene is the first to be translated by the cell into the beginnings of a protein molecule, and optimizing a gene´s leader sequence can improve the stability of the final protein molecules. It is even possible to mark a protein as one that the cell should secrete. The search for improvement was directed into several directions how to boost all aspects of the plasmids´ activity namely new methods of getting them into cells or new ways of increasing protein production. New vaccine delivery methods get considerably more cells – including immune cells themselves – to take up plasmids. For instance, needle-free systems, such as GeneGun and Bioject that use pressurized air to inject vaccine, deliver plasmids into the skin where immune entries called antigen-presenting cells are highly concentrated. To achieve a similar result the injection can be followed by electroporation. That is application of a series of electrical pulses that cause cell membranes to temporarily open pores that allow plasmids to enter more easily. Electroporation can increase uptake of plasmids by as much as 1,000 fold. A final important improvement involves additions of adjuvants, which are typically added to traditional vaccines to boost immune system responses. An adjuvant can even steer the immune system toward one response over another. For instance, greater production of T cells can kill pathogen-infected cells. On the other hand antibody proteins block pathogens from entering cells. Vaxfectin has been shown to increase antibody responses to a DNA vaccine against influenza 200-fold. Resiquimod provokes strong reaction including T cells and antibodies. It is also possible to incorporate the gene for an adjuvant molecule directly into a vaccine plasmid. A MULTIPURPOSE TECHNOLOGY Unlike classical drugs that often take the form of small chemical molecule, DNA therapies deliver a gene to treat an ailment. The plasmid does not integrate permanently into the recipient´s cellular genome. Successes in plasmid-based therapies have been in animals. In pigs the application of growth hormone-releasing hormone supported the gestating fetuses´ survival. A single injection was sufficient. Large clinical trials for human DNA therapies are now under way. One of them is growth factors that mobilize stem cells to treat congestive heart failure. • critical limb ischemia • new blood vessels • hepatitis C virus • veterinary applications are more advanced than human – e.g. melanoma in dogs (achieved six-fold increase of the medial survival time • flu vaccine: in animals protects against common flu strains inclusive the highly lethal H5N1 avian flu • main reason: plasmids contain so-called consensus sequences of flu virus genes • experimental DNA version of an H1N1 vaccine is now in early human trials with encouraging results • HIV: Pennvax-B: 3 viral genes + adjuvant molecules, applied by electroporation • HIV in NIH: DNA-based HIV vaccine with one of two adenovirus-based HIV vaccines as boosts With optimized plasmids and improved delivery methods we can see a comeback by the start of the STEP trial. The DNA approach has begun to show promise for uses beyond classical vaccination, including plasmid delivery of some medications an of immune therapies targeted at cancers. Cells typically favor one of the synonymous codons and translate it more efficiently than the others WHY? DNA vaccine projects continues but original ideas may not be so simple ....... KEY CONCEPTS • Scientists long assumed that any DNA mutation that does not change the final protein encoded by a gene is effectively “silent”. • Mysterious exceptions to the rule, in which silent changes seemed to be exerting a powerful effect on proteins, have revealed that such mutations can affect health through a variety of mechanisms. • Understanding the subtler dynamics of how genes work and evolve may reveal further insights into causes and cures for disease. MUFFLED MESSAGE A synonymous mutation was found to affect pain sensitivity by changing the amount of an important enzyme that cells produced. The difference results from alteration in the shape of mRNA that can influence how easily ribosomes are able to unpackage and read the strand. The folded shape is caused by base-pairing of the mRNA´s nucleotides; therefore, a synonymous mutation can alter the way nucleotides match up. ‹#› 47 Or did life come from another world? The hypothesis of F. Crick is discussed in November issue of Scientific American 2005. It is concluded that microorganism could have survived a journey from Mars to Earth DNA DENATURATION and RENATURATION/HYBRIDIZATION J. Marmur and P. Doty, around 1960 Harvard Univ, Cambridge, Mass Microbiologist, biochemist and molecular biologist Julius Marmur – dicovered renaturation of DNA *22 March, 1926 Bialystok (Poland) – +20 May, 1996 New York, NY Oswald Avery 1944 - DNA is a genetic material (Rockefeller Institute, New York, NY) Rollin D. Hotchkiss Julius Marmur 1993 Nature 248(1974) 766 Francis Crick 21 years after invention of the DNA double helix structure about the discovery of DNA renaturation IFFY stories On this day 50 years ago, Watson and Crick published their double-helix theory. But, what if... By Steve Mirsky (2003) “I am now astonished that I began work on the triple helix structure, rather than on the double helix,” wrote Linus Pauling in the April 26, 1974 issue of Nature. In February 1953, Pauling proposed a triple helix structure for DNA in the Proceedings of the National Academy of Sciences (PNAS). He had been working with only a few blurry X-ray crystallographic images from the 1930s and one from 1947. If history´s helix had turned slightly differently, however, perhaps the following timeline might be more than mere musing... August 15, 1952: Linus Pauling (finally allowed to travel to England by a US State Department that thinks the words “chemist” and “communist” are too close for comfort) visits King´s College London and sees Rosalind Franklin´s X-ray crystallographs. He immediately rules out a triple helical structure for DNA and concentrates on determining the nature of what is undoubtedly a double helix. February 1953: Pauling and Corey describes the DNA double helix structure in PNAS ..... Triple helix with bases on the outside and sugar-phosphate backbone in the interior of the molecule My IFFY story: If L. PAULING had in his lab an oscillopolarograph in 1952 he would never propose this structure. Polarography clearly showed that bases must be hidden in the interior of native DNA molecule and become accessible when DNA is denatured/single-stranded REALITY ‹#› 54 LEFT-HANDED Z-DNA alternating pu-py CRUCIFORM inverted repeat CURVATURE 4-6 A’s in phase with the helix turns SINGLE-STRANDED region AT-rich Text TRIPLEX structure homopu.homopy HAIRPIN SUPERCOIL Negative SUPERCOILING stabilizes local DNA structures Physical methods such as NMR and X-ray analysis indispensable in the research of linear DNA structures are of limited use in studies of local structures stabilized by supercoiling DNA and RNA are Electroactive Species producing faradaic and other signals on interaction with electrodes Cytosine (C) Adenine (A) A, C, G are reduced at MERCURY electrodes Guanine (G) reduction product of guanine is oxidized back to G All bases (A, C, G, T, U) yield sparingly soluble compounds with mercury and can be determined at concentration down to 10-11M. Solid amalgam electrodes can be used instead of the mercury drop electrodes. ____________ A and G as well as C and T are oxidized at CARBON electrodes _________________________________________________________ PEPTIDE NUCLEIC ACID (PNA) BEHAVES SIMILARLY TO DNA AND RNA _________________________________________________________________________________________________ Microliter volumes of the analyte are sufficient for analysis _____________________________________________________________ Electroactive Labels can be Introduced in DNA Fojta, M., et al.. (2007): „Multicolor“ electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes. Anal. Chem. 79, 1022-1029 Trefulka, M., et al. (2007): Covalent labeling nucleosides, RNA and DNA with VIII- and VI-valent osmium complexes. Electroanal. 19, 1281-1287 Electrochemistry of Nucleic Acids is a Booming Field Oscillographic polarography at controlled a.c (cyclic a.c. chronopotentiometry) complete analyses on a single mercury drop 1941 Nobel Prize 1959 Jaroslav Heyrovský 1890-1967 invented POLAROGRAPHY in 1922 Present electrochemical analysis stems from Heyrovský’s polarography J Heyrovsky S Ochoa A Kornberg Electrodes Heyrovsky’s polarography was based on mercury electrodes. At present a number of different electrodes is used in electrochemical analysis, incl. bimacromolecule studies, such as liquid mercury and solid mercury-containing electrodes (such as film and solid amalgam, incl. dental amalgam electrodes), carbon, gold, indium-tin oxide, silver, etc. Only with mercury-containing and carbon electrodes well-behaved NA electroactivity has been observed. Mercury electrodes and most of the solid electrodes greatly differ in their potential windows Hg electrodes thus suits better for reductions while solid electrodes (e.g. carbon, Au,,,) are better for oxidation processes. Material of the electrode is also very important. Hydrophobicity/hydrophilicity as well reactive functional groups may greatly affect adsorption of DNA and proteins -2 V Hg 0 V Carbon, Au, Ag, Pt…. -1 V +1 V DNA and RNA are polarographically inactive. H. Berg, Biochem. Z. (1957) E. Palecek,Naturwiss. 45, 186-187 OSCILLOGRAPHIC POLAROGRAPHY At controlled alternating current (constant current chronopotentiometry) dE/dt E ssDNA dsDNA CA G anodic cathodic sparingly soluble compounds with Hg LITERATURE in 1958: Adenine is polarographically reducible at strongly acid pH while other NA bases as well as DNA are inactive J.N.Davidson and E.Chargraff: The Nucleic Acids, Vol. 1, Academic Press, New York 1955 Palecek E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958), 186 Palecek E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960), 656 D.c. polarography vs. oscillopolarography (OP) Why d.c. polarography was rather poor in DNA analysis? (a) no DNA accumulation at the electrode (b) DNA adsorption at negatively charged DME (~-1.4V) compared to open current potential in OP We developed methods of chemical probing of the DNA structure based on osmium tetroxide complexes (Os,L). Some of the Os,L complexes react with single-stranded DNA but not with the double-stranded B-DNA. These methods yielded information about the distorted and single-stranded regions in the DNA double helix at single-nucleotide resolution. DNA probed both in vitro and directly in cells. In the beginning of the 1980‘s Os,L complexes were the first electroactive labels covalently bound to DNA. These complexes produced catalytic signals at Hg electrodes allowing determination of DNA at subnanomolar concentrations Electroactive labels can be introduced in nucleic acids Os(VIII)L complexes are sensitive to the DNA structure (CHEMICAL PROBES OF THE DNA STRUCTURE) they react with single-stranded and distorted but NOT with intact double-stranded DNA in vitro and in cells In 1986 we proposed Adsorptive Transfer Stripping Voltammetry (AdTSV) based on easy preparation of DNA-modified electrodes AdTSV has many advatages over conventional voltammetry of NAs: 1) Volumes of the analyte can be reduced to few microliters 2) NAs can be immobilized at the electrode surface from media not suitable for the voltammetric analysis 3) Low m.w. compounds (interfering with conventional electrochemical analysis of NAs) can be washed away 4) Interactions of NAs immobilized at the surface with proteins and other substances in solution and influence of the surface charge on NA properties and interactions can be studied, etc. ADSORPTIVE STRIPPING NA is in the electrolytic cell and accumulates at the electrode surface during waiting ADSORPTIVE TRANSFER STRIPPING NA is attached to the electrode from a small drop of solution (3-10 μl) NA is at the electrode but the electrolytic cell contains only blank electrolyte OSCILLOGRAPHIC POLAROGRAPHY At controlled alternating current (constant current chronopotentiometry) dE/dt E ssDNA dsDNA CA G anodic cathodic sparingly soluble compounds with Hg LITERATURE in 1958: Adenine is polarographically reducible at strongly acid pH while other NA bases as well as DNA are inactive J.N.Davidson and E.Chargraff: The Nucleic Acids, Vol. 1, Academic Press, New York 1955 Palecek E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958), 186 Palecek E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960), 656 J Heyrovsky S Ochoa A Kornberg J. Heyrovsky invented POLAROGRAPHY in 1922. After 37 years he was awarded a Nobel Prize Nobel Prizes 1959 In difference to most of the electrochemists I met in the 1960‘s and 1970‘s, J Heyrovsky was interested in nucleic acids and he greatly stimulated my polarographic studies of DNA J M at the 40th Anniversary of the Discovery of the DNA Double Helix In 1960 when I published my NATURE paper on electrochemistry of DNA I obtained invitations from 3 emminent US scientists: J. Marmur - Harvard Univ. L. Grossman - Brandeis Univ. J. Fresco - Princeton Univ. To work in their laboratories as a postdoc In 1960 new techniques were sought to study DNA Denaturation and Renaturation. To those working with DNA Oscillographic Polarography (OP) appeared as a very attractive tool. Invented by J. Heyrovsky, it was fast and simple, showing large differences between the signals of native and denatured DNA. The instrument for OP was produced only in Czechoslovakia. I accepted the invitation by Julius Marmur but for more than two years I was not allowed to leave Czechoslovakia. In the meantime JM moved from Harvard to Brandeis Univ. By the end of November 1962 I finally got my exit visa and with Heyrovsky Letter of Reccommendation in my pocket I went to the plane just 24 hours before expiration of my US visa. Before my departure I sent my OP instrument by air to Boston. It arrived after 9 months completely broken. I nstead of OP I had to use ultracentrifuges and microbiological methods. oscilak Julius Marmur discovered DNA Renaturation/Hybridization and proposed (in JMB) a new method of DNA isolation which was widely applied. His paper was quoted > 9000x. At the end of my stay at Brandeis I did some OP experiments which I finished in Brno and published in J. Mol. Biol. in 1965 and 1966. CI-2 E RENATURATION OF RNA AS DETECTED BY DPP Time dependence Early evidence of DNA Premelting and Polymorphy of the DNA Double Helix B. sublilis and B. brevis DNAs have the same G+C content and different nucleotide sequence B. subtilis B. brevis J. Mol. Biol. 20 (1966) 263-281 Before my departure to the US I observed Changes in the polarographic behavior of DNA far below the denaturation temperature. These changes were later called DNA Premelting POLAROGRAPHIC BEHAVIOR OF dsDNA At roomand premeltig temperaturse depended on DNA nucleotide SEQUENCE 1976 What the people said: Before 1980 No doubt that this electrochemistry must produce artifacts because we know well that the DNA double helix has a unique structure INDEPENDENT of the nucleotide SEQUENCE After 1980 Is not it strange that such an obscure technique can recognize POLYMORPHY OF THE DNA DOUBLE HELIX? What the people said Meeting F. Crick in Copenhagen and Arhus, 1977 (B. Clark) We developed methods of chemical probing of the DNA structure based on osmium tetroxide complexes (Os,L). Some of the Os,L complexes react with single-stranded DNA but not with the double-stranded B-DNA. These methods yielded information about the distorted and single-stranded regions in the DNA double helix at single-nucleotide resolution. DNA probed both in vitro and directly in cells. Probing of DNA structure with osmium tetroxide complexes In the beginning of the 1980‘s Os,L complexes were the first electroactive labels covalently bound to DNA. These complexes produced catalytic signals at Hg electrodes allowing determination of DNA at subnanomolar concentrations Firsts in Electrochemistry of Nucleic Acids during the initial three decades 1958 DNA and RNA and all free bases are electrotractive 1960-61 assignment of DNA electrochemical signals to bases, relation between the DNA structure and electrochemical responses 1961 adsorption (ac impedance) studies of DNA (IR Miller, Rehovot) 1962-66 DNA premelting, denaturation, renaturation/hybridization detected electrochemically, traces of single stranded DNA determined in native dsDNA. Nucleotide sequence affects dsDNA responses 1965 Association of bases at the electrode surface (V. Vetterl) 1966 application of pulse polarography to DNA studies 1967 detection of DNA damage 1967-68 Weak interactions of low m.w. compounds with DNA (P.J. Hilsson, M.J. Simons, Harrow, UK and H. Berg, Jena) 1974 DNA is unwound at the electrode surface under certain conditions (EP and H.W. Nürnberg, Jülich, independently) 1976 Evidence for polymorphy of the DNA double-helical structure For two decades only mercury electrodes were used in NA electrochemistry 1978 Solid (carbon) electrodes introduced in nucleic acid research (V. Brabec and G. Dryhurst, Norman) 1980 Determination of bases at nanomolar concentrations by cathodic stripping 1981-83 Electroactive markers covalently bound to DNA 1986-88 DNA-modified electrodes Results obtained at: IBP, Brno or elsewhere (author’s name is given); the results which have been utilized in the DNA sensor development are in blue ‹#› 73 NPP dcP DPP native denatured 1974 DNA unwinding at negatively charged surfaces ‹#› 74 In native DNA its NPP responses depended on the initial potential, Ei DME HMDE TIME TIME Ered Ei E A1 1s 60 s III II a b I c 3 1 d 3 3 2 1 e f E B A2 ssDNA dsDNA ‹#› 76 Effect of pH on DNA unwinding ‹#› 77 Effect of nucleotide sequence on DNA unwinding In 1986 we proposed Adsorptive Transfer Stripping Voltammetry (AdTSV) based on easy preparation of DNA-modified electrodes AdTSV has many advatages over conventional voltammetry of NAs: 1) Volumes of the analyte can be reduced to few microliters 2) NAs can be immobilized at the electrode surface from media not suitable for the voltammetric analysis 3) Low m.w. compounds (interfering with conventional electrochemical analysis of NAs) can be washed away 4) Interactions of NAs immobilized at the surface with proteins and other substances in solution and influence of the surface charge on NA properties and interactions can be studied, etc. ADSORPTIVE STRIPPING NA is in the electrolytic cell and accumulates at the electrode surface during waiting ADSORPTIVE TRANSFER STRIPPING NA is attached to the electrode from a small drop of solution (3-10 μl) NA is at the electrode but the electrolytic cell contains only blank electrolyte ‹#› 79 ‹#› 80 ‹#› 81 denatured DNA native DNA peak G Scheme 1 x Potential region U (around -1.2 V) (first seconds) (tens of seconds) B C Potential region T A Figure 19 DNA unwinding at negatively charged Au surfaces was recently observed by R. Georgiadis et al. and applied in DNA sensors Heaton RJ, Peterson AW, Georgiadis RM, PNAS 98 (2001) 3701 Electrochemistry of Nucleic Acids At present electrochemical detection of any nucleotide sequence, including detection of point mutations is possible in PCR-amplified DNAs. Detections of DNA methylation and microRNA’s are gradually getting ground. Challenges: 1) Detection of a specific nucleotide sequences in biological materials without PCR amplification. Exploitation of natural amplification of DNA and RNA sequences for electrochemical analysis of DNA and RNA. High sensitivity (signal amplification) and specificity (elimination of non-specific interactions) of the analysis is required. 2) Development of electrochemical sensors for DNA-protein interactions for genomics, proteomics and biomedicine Electrochemical sensors for DNA hybridization are coming of age Ternary layers with dithiols Binary layer … new ternary interface involving hexanedithiol (HDT) co-immobilized with the thiolated capture probe (SHCP) on gold surfaces, followed by the incorporation of 6-mercapto-1-hexanol (MCH) as diluent. The new SHCP/HDT+MCH monolayer led to a 80-fold improvement in the signal-to-noise ratio (S/N) for 1 nM target DNA in undiluted human serum over the common SHCP+MCH binary alkanethiol interface, and allowed the direct quantification of the target DNA down to 7 pM (28 amol) and 17pM (68 amol) in undiluted/untreated serum and urine, respectively. ‹#› 87 SUMMARY Electroactivity of nucleic acids was discovered about 50 years ago Reduction of bases at Hg electrodes is particularly sensitive to changes in DNA structure. The course of DNA and RNA denaturation and renaturation can be easily traced by electrochemical methods At present electrochemistry of nucleic acids is a booming field, particularly because it is expected that sensors for DNA hybridization and for DNA damage will become important tools in biomedicine and other regions of practical life in the 21st century DNA-modified electrodes can be easily prepared; microL volumes of DNA are sufficient of its analysis but miniaturization of electrodes decreases these volumes to nL. Sensitivity of the analysis has greatly increased in recent years. ‹#› 88 DNA surface denaturation ‹#› 89 NPP dcP DPP ‹#› 90 ‹#› 91 Recently we have found that peak H is produced by proteins adsorbed at mercury and solid amalgam electrodes modified by different kinds of thiol self-assembled monolayers (SAMs). For practical reasons we were primarily interested in DTT SAMs. Electrochemical analysis of proteins and peptides at Hg electrodes in the presence of large excess of thiols was difficult or impossible. Temperature, at which the electrode process is taking place, greatly influences the electrochemical behavior of the surface-immobilized proteins. protein-modified electrode V. Ostatná, H. Cernocká, E. Palecek (2010) J.Am. Chem. Soc., 132, 9408-9413 Thiol SAM Tumor suppressor protein p53 declared „The Molecule of the Year“ by Science magazine in 1993 perhaps the most important protein in the development of cancer. This protein p53 plays a critical role in the cellular response to DNA damage by regulating the expression of genes involved in controlling cell proliferation, DNA repair, and apoptosis. P53 protein is inactivated by mutation in about 50 % of human malignancies. Most mutations are located in the DNA-binding core domain of the protein. p53 protein is biologically active in its reduced state and is usually stored with mM concentrations of reducing agent - dithiothreitol (DTT). EU 6th FP: Mutant p53 as target for improved cancer therapy ‹#› 94 15.9 17.9 20.3 °C 8.6 11.1 13.9 °C p53 core domain Mutation in R175H induces structural perturbation at the zinc-binding site, destabilizes the core domain by 3 kcal/mol and eliminates p53 sequence specific DNA binding. The same effect can be observed in the wt core domain upon removal of the zinc ion. We tested other mutants such as V145A, F270L, R273H and Y220C and we always observed CPS responses different from the wt protein H1 H2 E. Palecek et al. JACS 2011, 133, 7190–7196 The Protein Group Hana Cernocka Mojmir Trefulka Petra Mittnerova Emil Palecek Lida Rimankova Martin Bartosik Veronika Ostatna Veronika Vargová ‹#› 96 Chemie, struktura a interakce nukleových kyselin Fyzikální vlastnosti a izolace DNA Denaturace, renaturace a hybridizace DNA Biosyntetické polynukleotidy Souhrn přednášky z minulého týdne V posledních letech jsou k dispozici komerčně dostupné kolonky využívající imobilizaci DNA na pevném podkladu. K separaci DNA jsou rovněž používány magnetické kuličky (magnetic beads) konec 7.10.09 Characterize your DNA sample: ds x ss, circular x linear circular: nicked, oc; covalently closed, cc, cd linear: cohesive or blunt ends number of base pairs, purity: protein, RNA .... content analytical methods Denaturation x degradation aggregation renaturation/hybridization DNA DENATURATION and RENATURATION/HYBRIDIZATION J. Marmur and P. Doty DNA renaturation/reassociation depends on the concentration of the DNA molecules and the time allowed for reassociation. Often imperfect matches may be formed which must again dissociate to allow the strands to align correctly. C0t value of DNA is defined as the initial concentration C0 in moles nucleotides per Litre multiplied by time t in seconds. C0t reflects complexity of DNA. Methods: S1, hydroxyapatite - dsDNA binds more strongly Microbiologist, biochemist and molecular biologist Julius Marmur – dicovered renaturation of DNA 22 March, 1926 Bialystok (Poland) – 20 May, 1996 New York, NY Oswald Avery 1944 - DNA is a genetic material (Rockefeller Institute, New York, NY) Rollin D. Hotchkiss Julius Marmur 1993 Nature 248(1974) 766 Francis Crick 21 years after invention of the DNA double helix structure about the discovery of DNA renaturation Syntetické oligonukleotidy Dr. L. Havran, 1. předn. Důležité modely vlivu sekvence nukleotidů na vlastností DNA nukleosid-difosfáty nevyžaduje primer ani matrici nukleosid-trifosfáty poly(A) poly(rC) poly(dG) poly(U) poly(rT)