POKRAČOVANÍ 22.10.08/předn.3 Progress in genomics affects electroanalysis Many areas of science are influenced by the fast development of the genomics and by the success of the Human Genome Project. Classical sequencing of individual human genomes with 3xl09 base pairs is too difficult. Sequencing by DNA hybridization is gaining importance Relatively expensive DNA hybridization ARRAYS with optical detection are currently applied in research-krbs It is believed that electrochemistry can complement the optical detection providing new LESS EXPENSIVE hybridization detection for decentralized DNA analysis in many areas of practical life LOW DENSITY CHIPS DNA isolation PCR electrochemical detection LAB-ON-A-CHIP Double-surface technique Few years ago we proposed a new technique in which (in difference to previous techniques) DNA hybridization is separated from electrochemical detection. Optimum properties of the hybridization surface (H) and the detection electrode (DE) are not identical. We used magnetic beads optimized for hybridization as surface H and chose optimum DE for the given electrode process. With spherical magnetic beads non-specific binding of NAs is minimized. 20 microL of the bead suspension gives 3 to 7 cm2 area. Beads can be incorporated into microf luidic systems and chips Electrochemical sensors/detectors for DNA hybridization Single-Surface Technologies: PROBE CCTTATCCTCCAAT TARGET : CC.AaTACCACCTTA (COM PLE M E ISTÁ R Y) CCTTATCCTCCAAT CCTTATCCTCCAAT DUPLEX FORMATION PROBE : TARGET ■*■ NO DUPLEX {HYBRID NÍXT FORMED} ACAATACCATTCC { NON-COM PLE M E NT A R Y > DNA IIVBRIDIZATION AS DELECTRDľllĽMItľAL DETECTTOrt ATTHE SAME SIRTACĽ ľ i; ■ K ttfget DNA REDCX INDICATCR COVALENTLY BOUND LABEL ENZYME, etc. LUIIIKIM HkCJl't.kJ'lt.SUl l>YV Ifl I'l.lAhv SlSiil.K M'kAMl Au J K Barton =1 Surface-attached molecular beacons liluim- 0|HkiJ H- AiJ______j .i í \ -.£* ™% i \ J V \ JjF A Heeger In the last decade nucleic acid electrochemistry was oriented predominantly to DNA sensors for (a) DNA hybridization and (b) DNA damage. This trend has been accompanied not only by interesting discoveries but also by a number of poor papers lacking the necessary control experiments,claiming sequence detection without PCR amplification but using synthetic oligos as target DNA, etc. Electrochemical sensors for DNA hybridization At present both single- and double-surface techniques can be used for DNA sequencing of longer oligonucleotides and PCR products. Electrochemical detection of point mutations is also possible. Optimization of the procedures are now necessary to develop commercially successful devices. Challenges: 1) Sequencing eukaryotic DNA without amplification (by PCR). Great sensitivity and specificity of the analysis is required 2) Development of electrochemical sensors for DNA-protein protein-protein interactions for proteomics and biomedicine The results of the DNA electrochemistry studies and development of the electrochemical DNA hybridization sensors in the last decade suggest that these sensors can complement DNA sensors with optical detection How and when the DNA electrochemistry begun? Science in Czechoslovakia after the Und World War After February 1948 life in Czechoslovakia was increasingly affected by the Stalinist ideology and heavily controlled by the Party and Government. Many scientists and scholars were fired from Universities but some of them got employment in the Institutes of the Czechoslovak Academy of Sciences established in 1952. This was possible particularly at the Institutes whose Directors were influential Party members but serious scientists. PRAHA/PRAGUE institute of Organic Chemistry and Biochemistry/ Director: F. Sorm Chemistry and Biochemistry of Proteins and Nucleic Acids B. Keil, B. Meloun, O. Mikes, J. Doskočil, D. Grunberger, A. Holy, I. Rychlík, J. Riman, J. Sponar, V. Paces, Z. Sormová, S. Zadrazil For many years Czech scientists were efficiently isolated from the West In this respect the situation in Brno was much worse than in Prague Institute of Biophysics, Brno Director: F. Hercik Founded in 1955 for radiobiological research it gradually turned into an institute devoted mainly to DNA For a long time we received 50 -100 US $ for materials/chemicals per year and Department. The orders of materials from the West had to be planned 1-2 years ahead Taking part in meetings in western countries was difficult not only because of currency problems OSCILLOGRAPHIC POLAROGRAPHY At controlled alternating current (constant current chronopotentiometry) dE/dt dsDNA ssDNA cathodic CA paringly soluble :ompounds with Hg anodic H-E 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 Paleček E.: Oszillographiche Polarographie der Nucleinsauren und ihrer Bestandteile; Naturwiss. 45 (1958), 186 Paleček E.: Oscillographic polarography of highly polymerized deoxyribonucleic acid; Nature 188 (1960), 656 600 i 50 years of nucleic acid electrochemistry 5twiiHir>HirK At;* ~ 1958: Nucleic acid bases, DNA and RNA are electroactive 500- Mr« ...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 nnrtirinntinn in thť». hvrlrnnp.n hnnrlinri (showing a Cathodic Signal in SSĎNA m c o s 400- i um "" ' ■ ■ !l ■ . I iliifm I-4JJ Ihre "ľ ......I" !'■■ - II MUM. J17.*, *J* 11-M II ,„,l X gvn-huNh*! i r,,[,(,.(, i, | r, Mtl. ■Í *fff*rllÍFFUPTm| fir.....I ■klliulji.....trhmrJll i I,. ""■«r II Wh I.,i...... - wUmJKlin, i-.l ....„..., -, Kbm ScfUan ..n.lk-.lnh Wb I...!, J*U m.ni ^||t r|1|,r |Umi]i JHTlllnjv.LtHL^ifJtlKd Jlirľl ľ™ ^""1. W*«J Bl^r, VVI IVI VU^ I I IV I I IV Ml IMIVSy4VSU*J LSMI I VS I WT I [participating in the hydrogen bonding. %but not in dsDNA) E. Paleček, Nature 188 (1960) 656-657 300- 0) E 200- 100- «■i™,,, ,]..„vi.y,i,],li,. ,„.„] ,.....llf] hi „. ,|lrl|(.,:u|i. is inactive. TliiH phonumrnon. nmv tjo perhaps interpreted in the following mannen one part nf the £»«""■'■'"■.....ring, which isofgr.......nportanoe fbi t Me l,.rinnt um nf the anodic indentation (apparently an .immo.Srci„p ,„ position 1 ie involved'), is found m the molecule neur thf> surf«», tt]„,reas the „naJoc-oua pari »f the pyrimidine rint; of cviosino í «»par-anthr the O.am,no-gro.,|.>) is hidden inside the iiinlcCTlL. where it participate» in the Formation of "»hyfogen bond, l„ aparmic „,id. where the i nuble.fiehcaJ etructurc is vomplelelv destroyed, (leoxycytulyJie acid ú not blocked itericallj in «re way so [hut its oacfflc-polsrnfcrnphi^ activity irm-v muoift-r .t,.,.!!. F».....leonyadenylic acid and deoxy. "'■..... ■■......."' '"'> ""I »how the ehimicterinli,. Mentation in ammonium formate as medium, and conseniif-nl ly no indentations due to these nucleotides »n be -,|,„-,„] if [lK.y ,in, ,,„,„„, ;„ |hr. |n„| , ol deoxynln)iiiii'lL-k- ncid. t hav,, aba -r,r.....ti deoxyribonucleic acid and Si!"""','........l -M Slldi,,m chloride aa medium roth suhetanoa. yMJd oscillogram* that differ only -li^hi K. In 1 .1/ iodiam hydroxide, tl.....«rill.....ami or deoxynbonuoleio ncid differ eonaiderably from Uu» ol apurmic acid. |" '■"''"li nti.i nirtiri wiluti,,,«', proteine aive an »dentation even at .1 vory low concentration'. Since tne deoxyribonucleic „cid indentation is produced this medium nt a different potent isj, 1 vra, abk to use (he oseillopoliiroin-nphic reaction For iMectin« 'itswyribrnnielei.: aeid and proteins in the presence ľf I ™n other. In this manner, protein« can be detected J»«n fa the presenoe of an abundance ofdeoxvribo nucleic acid (Fip, 4). Emu. Pasbokx .1— J in—r~if—tr—ip-^r^i^-in 1960 1970 1980 1990 2000 year E. Paleček, Fifty years of nucleic acid electrochemistry, Electroanalysis 2009, in press ^ oi- o__rLi/ ^ ^ H V* Bb ■1.5 -1.0 -0.5 DPP DUL SWY HMDL C.7SA 2ftnA 3 jiA G™ I ■ ň ! í 2 s/V -1.5 -1.0 1.4 -0.9 0.9 1-1J E (V) -1.5 -1.0 1.4 -0.9 E (V) 0.9 1.1 J. Heyrovsky invented POLAROGRAPHY in 1922. After 37 years he was awarded a Nobel Prize J Heyrovsky S Ochoa A Kornberg 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 Polarographie studies of DNA res 1959 - . i— • ■ -. -u----------------- "vr" ■ i . : ■ I - -■ 5 i ig. 1. de polarogiams of native and denatured calf thymus DNA" (a) native DNA at a concentratmn of 500 „g/ml in 0.5.1/ ammonium formate with 0.1,1/ sodium phosphate (pH 7.0)j 6) denatured DNA at a concentration of 500 «/ml in 0.5A/ ammonium formate with 0.0/ sodium phosphate CpH 7.0). DNA was denatured by heat at the Tn nlrl™ °Lf6 "g/ml ÍU ()M7M NaCI with °-7 mM citrftte. Both curves start at U.U V, 100 mV/scale umt, capillary I, saturated calomel electrode. 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. 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. J M at the 40th Anniversary of the Discovery of the DNA Double Helix Rrprinlrii Trom Caiv rtrmrfn Hjiuoit Sr**wcA un QrA^nTATTVi BioLoor Vokna XXVIII. IMS /Vim«* im VJIJt. Specificity of the Complementary RIVA Formed by Bacillus sub t His Infected with Bacteriophage SP8 At the end of my stay at Brandeis I did some OP experiments which I finished in Brno nd published in J. Mol. Biol, in 1965 and 1966. J. Mabklr", C. If. GASCxarAN, E. Paucckk, F. M, Kahaj*+h J. LfcYisB. ind M. Hammel* On+lwtt fbp>trtmr*t vf fíiurhpm.i*ryt Bmwt*Í* Ľiifrrrntf, IVnttiuim. Wt*~ŕŕÁu*ŕíl* INCREASING peak II CI-2, ÍNCREASINÔ\ peak II and III CI-2 . only peak III CI-2 peak II no CI-2 Native DNA A, B C melted DNA D H melti r^ quick cooling quick cooling slow cooling renaturation denatured DNA RENATURED DNA Temperature peak II no CI-2 only peak III CI-2 peak II no CI-2 DNA Premelting and Polymorphy of the DNA Double Helix Li Í976 Reprinted trom; FTOCtESS I« hUCUlC ACID HfiEAJKH ■*?-G «OLÍCULAI B.C!.:: jv v'J: 1| ■*ŕ IÍÍ4 *OÚÉMJC F««, |Mf Before my departure to the US I observed Changes in the Polarographie behavior of DNA far below the denaturation temperature. These changes were later called DNA Premelting J. Mol. Biol. 20 (1966) 263-281 ľ. ľli.Kl Y Y, B. siiblilis and B. brevis DNAs have the same G+C content and different nucleotide sequence B. subtilis Premefring Changes in DNA C. F íí-í j>íiLáhix.'opri, dropping mercury aloclroa poliriied with repute] cj-tL** at A.C. Th* n'.«uu»m«il4 «rcn r-nri^l i^iii i= rl:> Ln!^.it*rorj'ůf Prof. J. Slirrňiir, D*p*rtriLtr.i t-ľ läiucliMüijiry. rEHt.dŕii Vniwríily. TCilÜism, J]*«., V.S.A. POLAROGRAPHIC BEHAVIOR OF dsDNA At roomand premeltig temperaturse depended on DNA nucleotide SEQUENCE A 6. PoLVMDBi'iEY of DNA Seco.sdahy Structufe On the basis of the preceding discussion, a schematic picture of the structure of natural linear D\A in solution under physiological conditions (e.gr, ní 36 ZC. moderate ionic strength* and n H 7) can be down. We cm assume that the double-helical structure of the very long f A + T)-rich regions differs; from the structure- of thr mftjor part at the molecute and ťhat some of the (A -f-T)-rich segments are open (Fig. 20). An open ds-structure con be assumed in the region ní chain termini and/or in the vicinity of ss-breaks und other anomalies in the DNA primary structure. The eiract changes in the open ds-regions \vi\l depend on the nucleotide sequence as well as on ihe chemicaE nature of the atiomnlv. Most of the molecule will exhibit an "Y[j"°E Willfflirfifflli fi^J"? ""ft llfíl dťviatiúiu yivúu by UiĽ iiuckuLiuY- iuuucucfc. ETe^'atmg tiie- temperature in tlie premelting region í Fig. 20) is likelľ to lenid to the opening of other regions, and. eventualit, to expansion of the existing distorted ds-xegions and to further structural changes. Thus the course of the con^ formational changes as a function of temperature (premelting) will be determined by the distribution of the nucleotide sequences and anomalies in the primary structure, and mav have an almost continuous oh iractfir. Consequently, even if we do not consider ''breathing," not only the architecture of a DMA double-helical molecule, but also its^ mechanics or dynamics can be taken into account. To determine whether, e.g.., only the (A + T)'rich molecule ends will be open at a certain temperature Or also long A + T regions in the center of the molecule, further experimental research with better-defined samples of vinil and siynthetic nueleic aeids will be necessary. Further wort will undoubtedly provide new information on the details of the local arrangement of nucleotide residues in the double helix, as well us Qn DNA conformational motility. Thus a more accurate picture of DNA structure will emerge, whose characteristic feature will be poly-můrphy of the double helix, in contrast to the classical, highly regular DNA structure models. Meeting F. Crick in Copenhagen and Arhus, 1977 (B. Clark) December Jj 197a polydiA-'VMiA-T) What the people said Sefore 1980 No doubt that this electrochemistry must produce artifacts because we know well that the DNA double helix has i unique structure INDEPENDENT After 1980 Is not it strange that such an obscure can recognize POLYMORPHY OF THE DNA DOUBLE HELIX? technique if the nucleotide SEQUENCE Professor lU.il Paltet); InAtLtute Of biophytici CtfrchOtlOvak Acadeay of Sci*nc*f Brno It, Kralovopotska LIS iľzť-hns Lov a It la Dear Professor Patectk* I do apologise for tftkinq (0 Lonq to reply to your letter or Septtwbtr 29 and the very interesting tvViaw ycu lant with it- Mil for t und tel y I myself will not be aolt CO attvnd th* Symposiu* yon plan for September, 197 7 And my Cambridge COlLed?ua Aůťůri Klug tells; bi« that he too is unAbl* to be present. Had you COAtldtrtd th* possibility of asking or. Hank Soball.? H« has ju*t pub-:■...::■.-'. In iJ ::.■■-: an account of (.hu uí'híi CtaM - pi i rtd) kink and has ideas about presetting eonformationa. I have no Uu whether he vould b* able to cone but should you with to invitn hist his address Lti Depaetotnt of Ch*4niltryT Th* University of Rochtstttn ftivtŕ £tatiOnr Rochastar, Ua-j York I -; -.. J 7. Voura sincarely. f. Mr C. Cťick Pvrkauf Poundacion Visiting Professor z: g 40 g 20 LU d SRNA, SSC, 55 C d&RNA, SSC, 85 ^C dsRNA, 2.SxSSC, 85"C peak MIR peak HR - T 20 —i— 40 60 80 TIME (MINUTES) 100 RENATURATION OF RNA AS DETECTED BY DPP Time dependence 120 Fig. 10, Time-course of renaturation of phage \1 dsRNA. (A) Thermally denatured ssRNA was incubated (•- *) at 85°C in 2.5 x sodium saline citrate {SSC) or (o o) at 85JC in SSC, and (x x) at 55X\ Samples were withdrawn in time intervals given in the graph and quickly cooled. DPP measurements were performed at room temperature at a RNA concentration of 3.2ug/mL in (UM ammonium formate with 0.2M sodium acetate, pH 5.6; PAR 174. (B) (o—o) peak IIR, (•—*) peak IHR. ssRNA (108 ug/mL) in 0.01 x SSC was heated for 6 min at 100~C, Then it was placed into a thcrniostated Polarographie vessel with the same volume of 0,6M ammonium formate with 0.2 M sodium phosphate, pH 7, preheated to 58ÜC. The pulse polarograms were measured at 5^C in times given in the graph. Southern-Harwell A 3100, amplifier sensitivity 1/8. Adapted from Paleček and Doskočil (1974). Copyright 1974, with permission from Academic Press, 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 ĎNA (IR Miller, Rehovot) 1962-66 DNA premelting, denaturation, renaturation/hybridization detected electrochemically, traces of single stranded DNA determined in native dsĎNA. Nucleotide sequence affects dsĎNA 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. Ďryhurst, Norman) 1980 Determination of bases at nanomolar concentrations by cathodic stripping 1981-83 Electroactive markers covalently bound to DNA 1986-88 ĎNA-modif ied 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 Elektrochemie nukleových kyselin r\er\\ omezena jen na sensory. Může se zabývat napr. - strukturními přechody DNA (a) v roztoku (b) na elektrodě - adsorpcí DNA na elektricky nabitých površích - interakcemi DNA (a) s nízkomolekulárními látkami včetně mutagenních látek (b) s bílkovinami (včetně enzymů (c) s jinými makromolekulami - stanovením DNA v roztocích - elektrickými vlastnostmi DNA (napr. vodivost) atd. S jakými DNA v současnosti zpravidla pracujeme: DNA molecules A. GENOMIC (chromosomal) molecularly Dglydisperse. nucleotide sequence unknown B. PLASMID OR VIRAL monodisperse, nucleotide stejmeinige_Jmojj|tfi rel sc oc 'in ds ss motif* rs I usually 3-4 Kb mw ca2xi0 6 « i PCR "PRo^ccTs C. BIOSYNTHETIC POLYNUCLEOTIDES polydisperse, simple repeated sequence motifs or homopolymers ATATATATATATATATATATAT AAAAAAAAAAAAAAAAAAAAA aS TATATATATATATATATATATA III I I I I l I HTTTTTi I I 1 n T TT ss CCCCCCCCCCCCCCCCCCCCCCCC average mw 10 5 10 6 D. SYNTHETIC OLIGONUCLEOTIDES monodisperse, programmed nucleotide sequence chemically modified bases and backbone possible GCGCATTTCCGG CGCGATATCGCG CGCGTATAGCGC us lengths 10-20 nucleotides and ds lUbCTlMX lEIlMK AL MI m H) D S RISftXiNI»! KMALLCIIANCiliSIX DXASIW I II *. AND DimWMIML TRACKS OK IMPĽUmiLH IK DNA SAMPULS *^r MERCľl'RY ELirTRODENAREPARTTfl'LARLY9HXMT1YE » /M\ ULTLHM1NA110N (>ľ TVACKK (< H41 or i llLJXA *\^IXA ťHIJLIJhb ťHIJLIJhS IX liMAKiJLMSOk- ill U*A IKTÜMALATDILH / ťOVAľ i N T MOMHIW CiRIX)VILniNDI!RS AĎSORPTIVE STRIPPING ADSORPTIVE TRANSFER STRIPPING NA is in the electrolytic cell and accumulates NA is attached to the electrode NA is at the electrode but the at the electrode surface during waiting from a small drop of solution electrolytic cell contains only blank (3-10 fl) electrolyte 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. Probing of DNA structure with osmium tetroxide complexes fco. ^SSSUHB**** £>£T£tTi0M tifillTS i frfftppwc /"V fag/*»/ OsC> H g míÍh »f f U* DM rtMBhb 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 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. Critical ftrviěvs in Eiochtmiitry andMvlecular Biology, 2ů(í):]5l—2!S (1091) Local Supercoll-Stabilized DNA Structures E Pafŕčeŕí MH-Phndi HM für Kafaiyakslsef» Chemie, GBKínflon, BHD vnt Instate o! Hopfiysia, Czechoslovak Aiadarny ol Sosnm, 6i ?65 aino, C5FH [17] Probing of DNA Structure in Cells with Osmium Tetroxide—2,2'-Bipyridine By Emil Paleček 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. Hľ-Tiinn^rN i;n7,vmolovy, vín 31: CcvyiVú D IW2 bf Acadenfc i*«*, lne All rif hi^ vi reiKVkliiťih*» in mnf ŕínn nc^nťJ END-LABELING of DNA and RNA Electroactive labels such as ferrocene, daunomycin, viologen, thionine, etc. were covalently bound to DNA to obtain electrochemical signals closer to zero charge and/or to increase the sensitivity of the analysis. These labels are expensive and can hardly be used for labeling of longer NAs, such as plasmid or chromosomal DNAs. Already in 1981 we proposed osmium tetroxide complexes with nitrogeneous ligands (Os^11,!^) as DNA electroactive labels. They can be introduced in any DNA in an average biochemical or biological laboratory without any special equipment. DNA-Os^11^ adducts produce redox signals at mercury, amalgam, carbon and gold electrodes; in addition, electrocatalytic signals can be obtained at mercury and amalgam electrodes. Multiple labels can be easily introduced. specific sequence Os OsoPp?S Trefulka, M., et al. (2007): Covalent labeling of nucleosides, RNA and ONA with VIII- and VI-valent osmium complexes. Electroanalysis 19 (No. 12) 1281-1287. u «H, ^ / OH OH Different reaction» of Otwl,blpy' With six-valent Os(VI)L ribose residue m************ can be modified 98 End-labeling of DNA with OsVIII,L mercury amalgam Catalytic hydrogen evolution -1200 r -800 ■Ung-mľ1—►, -400 - 0 ng.mľ' Em -900 -1200 Er mV -1500 OjO -02 -04 -06 -OS -U) E[V] AdTS CVof CT ss DNA (20 (g/ml) modified by 2 mM Os04 bipy, electrolyte 0.3 M ammoniumformate and 0.05 M sodium phosphate ,pH 6.90 AdTS DPV CT ss DNA modified by 2 mM Os04bipy, electrolyte: 0.1M acetate buffer pH 4.8 We generated mononoclonal antibodies against DNAbase-Os(VIII)bipy and recently also against RNAsu9ar-Os(VI)bipy AdTS SWVof (GAA)7T50 (460 nM) modified by 2 mM Os04 bipy 0.2 M acetate buffer pH 5.0 ■*03i|C T?8,L Large number of papers since 1981 reviewed in E. Paleček, Meth. Enzymol 212 (1992) 139 Paleček E., Scheller F., Wang J., Eds. Electrochemistry of nucleic acids and proteins.. Towards electrochemical sensors for genomics and proteomics.; Elsevier: Amsterdam, 2005 B. Yosypchuk, M. Fojta, L. Havran, M. Heyrovsky, E. Paleček, Electroanalysis 18:186 (2006). Fojta M., Havran L., Kizek R., Billová S., Paleček E. Biosensors & Bioelectromcs 20 (5): 985-994 2004 L. Havran, M. Fojta, E. Paleček, Bioelectrochemistry 63:239 (2004). Paleček, E., etal.. (2002). Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor. Anal. Chim. Acta 469,73-83 Reactions of different Os(VIII)L complexes with DNA yield peaks at different potentials X CT N CH, phen Os, blpy HN' dT-Os,blpy C" -\ /? Ik o SO.H H3C 303H H.C bpds neoc íl i "V, H3C^CH3 tern Carbon electrodes neoc neoc -0.8 -0.6 -0.4 -0.2 0.0 Fojta, M., et al. (2007): „Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes. Anal. Chem. 79, 1022-1029 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 (PNA5). 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..... Ä-l CIIEiíĽirStľ: FAľijyC- A AW LV&&Ľ F'EC*. N. A a rmixyShv ítuí-Ci ex k Km m k ?ti-ct^.sCACit)S U* lílM'i F.'.Ľi.C^G 4NL> Hi^ikici 11, 0>ifi:v <'..'. t err, a<-i Ü-fcm.r.j^ I.A.nriR.AT\iRniH mi CcirutrsTnv,'1 Cjtuniitľix Ljecrmrju í y TĽĽnSdľ.dliV CroiöiDni=L[cd DcKmbcríi;, líl.K liä UI&UISTRk': PA.SUKG ASW í-OREi' ľjuOC. N. A. S. VřlíLdi itc iijvůlvtd ill efltŕŕ IĹnĽi#ts. This distiMtuíiD of the úJiosphat* RTiütip ftoiti ti» fsffutir tíŕtraledral coDĚKuratiaD ia Jtot supported br direct estperiitisütal evidence; imfojtiuiatelr no precise structure djctmniimtijons have beai jiiuíů of anr phosphate di-estcra. Toe diEtoiTion, wbJŕti cor-reapojids to a larger amount of double bond cbarac-tcT for the inner oxygen atoniEttüm for the onrgic-n atoms involved in the c&tcr iuitages, is a reason- HIhii nr lliŕ .»iiitHľ-l» sŕťl Miiijŕihir, *1iß»liig sí*wrnl niiŕ-fcľiiMŕ--Tť\-iť1i»í*>j. Triple helix with bases on the outside and sugar-phosphate backbone in the interior of the molecule My IFFY story: If L. PAULINO had in his lab an oscillopolarograph in 1952 he would never proposed this structure. Polarography clearly showed that bases must be hidden in the interior of native DNA molecule and become accessible when DNA is denatured 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 easily traced by electrochemical methods. Nucleic acids can be labeled; osmium complexes were the first electroactive labels covalently bound to DNA. At present Os labels are perhaps the most sensitive DNA end-labels. DNA-modif ied 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. 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 28 Chemie, struktura a interakce nukleových kyselin 2008-09 3.EP/6. PŘEDNÁŠKA 22.10.08 Fyzikální vlastnosti a izolace DNA Ďenaturace, renaturace a hybridizace DNA Biosyntetické polynukleotidy Fyzikální vlastnosti DNA Studium tyz. vlastnosti DNA in vitro vyžaduje její Izolaci z buněk či virů do zřerf. vodných roztoků, v nichž nejsou přítomny ostatní celulární komponenty. Takto ztrácíme sice Informace o jejich uspořádání in vivo (interakce s RNA, bílkovinami» atd.) - získáváme však možnost zodpovědět jiné otázky jako m. v., sekundární struktura ap. Izolace DNA - pokrok v poznání vlastností DNA postupoval souběžně s pokrokem Izolačních technik. Napr, zjištění lámavostl dlouhých molekul DNA dfky působení střižných sil (shear degradation) - čím větší molekula, tím snadnější degradace (vyfouknuti 1 ml roztoku pipetou o průměru 0,25mm za 2 s zlomí DNA T2 na poloviny. Při vysoké konce. (500 ng/mt) DNA je možnost zlomení menší. Začátkem 60 let byl vypracovány metody umožňující Izolaci nedegradované DNA T2a TjOSO.IO6). Tyto DNA se pak staly standardem pro kalibraci metod stanoveni mol. hmotnosti DNA. Důležitým krokem při izolací DNA je odstranění bílkovin: vysoká konc. solí, detergent, CHCI3- isoamyl, emulsifíkace, proteasy a fenolová extrakce. CHCI3-opakované třepáni, degradace; lepší je fenol - DNA o m.v. blízké celému chromosomu E.coř/(~109) - nebezpečí znečištění fenolu peroxidy (destilace). laolace DNA z bakteriofága a) purifikace tága diferenční centrifugací a/nebo v grád CsCi b) deproteinace (většinou fenolem) Dnes ne]častě|i je používaná plasmidová DNA. Stupeň čistoty a volba metody izolace jsou velmi závislé na účelu, ke kterému má být DNA použita. V posledních letech jsou k dispozici komerčně dostupné kolonky využívající imobilizaci ĎNA na pevném podkladu. K separaci ĎNA jsou rovněž používány magnetické kuličky (magnetic beads) pokrač. 29.10.08/předn. 4 demo: textbooks+monographic series Tissue Cold dilure TCA er PCA Organic solvente IZOLACE OEfiRADQVÁNÝCH NA Acid-soluble + lipid fraction Nucleic ocid + prořein residue Alkaline digesNon AadificaNon Soluble exrracr containing RNA \ Residue containing ONA Hoľ TCA Of PCA Soluble exfraď Residue containing (protein) RNA + ONA Extraction and fractionation of nucleic acids from tissues. *TCA - trichloroacetic acid, ŤPCA - perchloric acid. IZOLACE INTAKTNl DNA J. Marmur a. z virů a bakteriofágů b. z bakterii c. z eukaryotních buněk Plasmldová DNA RNA Chromosomal DNA Plasmid DNA Separation of closed-circular DNA of plasmid pBR322 from £. coli chromosomal DNA by isopycnic ultracentrifugation in a CsCl density ßraojejjt in the presence of C^icjjtjp htQrrydff The band marked 'chromosomal DNA" may also contain nicked plasmid DNA molecules. J. MARMUR, Harvard UnlvVBrandels Univ., Boston, Mass. Izolace DNA z bakterií: 1. Ivsa bunék a) mechanicky b) enzymaticky (lysozym) c) detergenty (SDS) 2. deprotelnace a) CHCI3 b) fenol c) enzymaticky d) uttracentrifugace v grád CsCI 3. odstraněni RNA a) enzymaticky ( RNasa) b) diferenční sráženi c) ulracentrifugace v grád CsCI Jednotlivé kroky při Izolaci DNA jsou často kombinovány se srážením etan o lem 4. dlalysa Dnes Jsou k dispozici komerčně dostupné přípravky (většinou různé druhy kolonek) pro izolaci DNA z prokaryotních \ eukaryotnfch buněk, které jsou vhodné zejména pro rutinní, sériové izolace DNA A Procedure tor the Isolation of Deoxyribonucleic Acid from Micro-organisms f J. MabmurJ Department of Ohemistry, Harvard University, Cambridge. Massachusetts, U.S.A. (Received 6 December 1960) T A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparation relatively free from protein, and RJSTA. Alternative methods of cell disruption and D1STA isolation have been described and compared, DNA capable of transforming homologous strains has been tise d to test various steps in the procedure and preparations have been obtained possessing high specific activities. Representative samples have been characterized for their thermal stability and sedimentation behaviour, 1, Introduction ■ To facilitate the study of the biological, chemical and physical properties of DNA it is necessary to obtain the material in a native, highly polymerized state. Several procedures have described the isolation of DNA from selected groups of micro-organisms (Hotehkiss, 1957: Zamenhof. Reiner, DeGIovanni & Rich, 1956; Chargaff, 1955). However, no detailed account is available for the isolation of DNA from a diverse group of micro-organisms. The reason for this is that micro-organisms vary greatly - — Origin 3000-2000- a. j. 1000- c E £ CD E BOG'S c n c 3 100 \ Characterize your DNA sample: ds x ss, circular x linear circular: nicked, oc; covalently closed, cc, cd \ \ (b) 10 20 Distance of migration (mm) 30 linear: cohesive or blunt ends number of base pairs, purity: protein, RNA .... content analytical methods Agarose gel electrophoresis of DNA. (a) Separation of: I, different forms of DNA of plasmid pBR322; 2. fragments of DNA (lengths indicated in kbp) derived from plasmid pBR322 by double-digestion with restriction endonuclease Bam HI and flgM; (b) Plot of length of DNA fragment (log scale) against distance of migration (line a r scale) of data from (a) 2, illustrating linear relationship. W Construct gradient eintritt Appl v [oyer of RNA to top of gradient Bottom of gradient Fro et \on number Top of gradient Rate zonal centrifugation of RNA through a sucrose density gradient. A sucrose density gradient is constructed in a centrifuge tube (a) and the RNA solution applied as a layer on top (b). During ultracentrifugation the main components of the RNA separate into zones, primarily on the basis of molecular weight (c). Th ese zo nes may be recovered by pu n c mri ng t he bottom of the tube and col ľect i ng diŕfe re nt fractions in separate tubes (d). The separated RNAsmay be visualized andquantitated by measurement of the absorbance at 260 nnt (e). Steps (d) and (e) may be conveniently combined by pumpingthe gradient through the flow-cellof a recording spectrophotometer. Síly ovlivňující konformaci DNA a) Elektrostatické síly podmíněné ionizací. V rozmezí pH 5-9, kdy nedochází ve větším stupni k ionizaci baží je, DNA a n iontovým poly elektrolytem - poly a n iontem, díky záporným nábojům, které nesou fosfátové skupiny). V roztocích solí jsou záporné náboje vystíněny kladnými náboji kationtů (např. Na+), které vytvářejí kolem každého zápornéno náboje iontovou atmosféru. Jestliže je koncentrace kationtů nízká, nabývá na významu odpuzováni fosfátových skupin. U dvousroubovicové DNA se toto odpuzování stává faktorem ovlivňujícím významně vlastnosti molekul teprve při iontových silách nižších než 0,1. Při velmi nízkých iontových silách (kolem 1CT4-10"5) jsou odpudivé síly již tak velké, že mohou zapříčinit zhroucení dvousroubovicové struktury (denaturaci). Jednoretézcová DNA (a podobně i RNA) je velmi citlivá ke změnám v koncentraci iontů již při Iontových silách nižších jak 1,0; snižování iontové síly vede ke zvětšování prostoru zaujímaného polynukleotidovým řetězcem. b) Síly plynoucí z vertikálního uspořádání baží (vrstvení baží, stacking). Síly působící mezi bázemi pravidelně uspořádanými ve dvojité ěroubovicí jsou zejména interakce typu dipól - dipól, dipól - indukovaný dipól a Londonovy síly, Existují teoreticky odvozené důkazy, že tyto síly jsou postačující pro stabilizaci šroubovice; jejich volná energie odpovídá asi -7 kcal na mól párů baží. Naproti tomu volná energie vodíkových můstků činí asi -3 kcal pro (G.C) a -2 kcal pro (A.T) pár (na mó! párů baží). C) Vodíkové Vazby (můstky)-představují Jediný známý způsob zajišťující specif I citu párování bázi Jsou tedy součástí mechanismu jímž DMA realizuje svoji biologickou funkcí. Zpočátku se o nich soudilo, že jsou nejdůležitějším činitelem pro stabilitu dvojité šroubovice; experimentálně i teoreticky bylo však dokázáno, že tomu tak není d) Hydrofobní S íly tento termín se týká stability dvousroubovicové DNA plynoucí z její archItekrury: polární skupiny jsou na povrchu, zatímco hydrofobní baze jsou uvnitř molekuly a mají větší tendenci interagovat mezi sebou nežli s molekulami vody. Toto uspořádání stabilizuje tedy dvoušroubovicovou molekulu DNA ve vodném prostředí. Je známo, že molekula DNA je ve vodném roztoku obklopena hydratační vrstvou, která hraje významnou úlohu ve stabilizaci dvojité šroubovice, Podrobné znalosti o této hydratační vrstvé jsou nyní získávány zejména díky výsledkům rtg. strukturní analýzy krystalů DNA. benaturation x degradation aggregation renaturation/hybridization DNA ĎENATURATION and RENATURATION/HYBRIĎIZATION Native DNA AR melti A260 D r f premelting / -A-------------B-------- ^ quick cooling melted DNA D melti % slow cooling renaturation quick cooling denatured DNA RENATURED DNA Temperature J. Marmur and P. Ďoty i and renaturation : L 70 21 Pneumococcal s (36%) £. coli (52%) S. mcrescens (5Ba/c) M phlei (66°/o) —I----------------------1---------- 80 90 Temperalure (°C) —I 100 l^turarion by heat of DNAs isolated from different sources. The figures in brackets indicate the : the DNA in G + C (%) (from Molecular Generics by G. S. Stent. W. H. Freeman and Co. I-after [116]). STRAND SEPARATION AND SPECIFIC RECOMBINATION IN DEOXYRIBONUCLEIC ACIDS: BIOLOGICAL STUDIES By J. Marmur and D. Lane CONANT LABORATORY, DEPARTMENT OF CHEMISTRY, HARVARD ľMVĽRSITY Communicated by Paul Doty, February 86, 1960 It is clear that the correlation between the structure of deoxyribonucleic acid (DNA) and its function as a genetic determinant could be greatly increased if a means could be found of separating and reforming the two complementary strands. In this and the succeeding paper1 some success along these lines is reported. This paper will deal with the evidence provided by employing the transforming activity of DNA from Diplococcus -pneumoniae while the succeeding paper' will summarize physical chemical evidence for strand separation and reunion. 6 80 o U ■f (D 60 40 20 f. coli Serratia ^^ ^ \jrM- phi ei Calf my m us Salmon ^_ * _ sperm Tí* Pneumococcus _L 1,69 1.70 7.71 1.72 1.73 1.74 Densíľy Fig. 2.21 Relationship of density to content of guanine plus cytosine in DNAs from various sources [64]. Percentage Source of DNA (G + C) Plasmodium falciparum (malarial parasite) 19 Dictyostelium (slime mould) 22 M. pyogenes 34 Vaccinia virus 36 Bacillus cereus 37 B. megaterium 38 Haemophilus influenzae 39 Saccharomyces cerevisiae 39 Calf thymus 40 Rat liver 40 Bull sperm 41 Diplococcus pneumoniae 42 Wheatgerm 43 Chicken liver 43 Mouse spleen 44 Salmon sperm 44 B. subtilis 44 Tlphage 46 £. coli 51 Tl phage 51 T3 phage 53 Neurospora crassa 54 Pseudomonas aeruginosa 68 Sarcina lutea 72 Micrococcus luteus 72 Herpes simplex virus 72 Mycobacterium phlei 73 Nucleotide pairs ,1 ,n ,n2 m3 Im4 m4 m6 I m7 me in91 1T 10 10 10 MO 10 f 10 T 10 10 10 T 10 i______i______i______i______i______-í____—i—--------1-----------1-----------1-----------1 100 1000 10000 CQr (mole x s/L) Fig. 2.20 The rate oť reassociation of double-stranded polynucleotides from various sources showing how the rate decreases with the complexity of the organism and its genome (from [60]). 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. Cot value of DNA is defined as the initial concentration Co in moles nucleotides per Litre multiplied by time t in seconds. Cot reflects complexity of DNA. Methods: SI, hydroxyapatite - dsĎNA binds more strongly Biosyntetické polynukleotídy Syntetické olígonukleotídy Dr. L. Havran, I3«ffififffflftfiftllflljrô p(8>Hffiai«MQfiiftllátf ■ modely pro výzkum fyzikálních a chemických vlastností a struktury nukleových kyselin Důležité modely vlivu sekvence nukleotidů na vlastností ĎNA POLYR1BONUKLEOTIDY byly syntetizovány většinou pomocí polvnukteotid fosforvtázy. která polymerizuje nukIeot1d-5'-dllosfáty {pň čemž se uvolňuje anorganický fosfát) Po počáteční syntetické fázi, dochází k rovnováze mezi syntézou a degradací (tosforolyzou) a vytvářeli se polymery s pomerné malým rozptylem délek Polynukleotid losforyláza polymerizuje mnohá analoga nukleojid dlfoafátú Jako 2'-0-metyl, 2*-chIoro-, 2'-fluoro- a dokonce 1 arabinonukleosid-5 -dltoefáty a nukleotid difosfáty s různě modifikovanými bázemi. Nukleozldy mající konformaci syn- (např. 8-bromoguanosin) polymerizovány nejsou. Enzym vyžaduje konformaci cukru 3'-endo. Tento enzym nevyžaduje pro svoji funkci matrici (někdy očko/primer)-Vhodný zejména pro syntézu homopolvnufcleotídů. Heteropolymery mají náhodnou sekvenci nukleotidů. I Příprava polynukleotldú s definovanou sekvencí nukleotidů vyžaduje RNA-DOlymerá2U fzávistou na DNA) nebo DNA-DOlymerazu furo syntézu polydeoxyribonukleotidů) nukleosid-difosfáty nevyžaduje primer ani matrici nukleosid-trifosfáty ^ňmopotvnufckKmdv PolyiUl a oolyfcmoH pokojové teplote map aiáto výraanou sekundární strukturu, prt vyisf teplot« tuto strukturu ztrácej* PolvfCi v kyselém prostředí tvoří dvojretězovou protoníiovanou strukturu s paralelními řetězci. V neutrálním prostředí tvoří jednořetězovou strukturu stabilizovanou vetttkálmm vrstvením baží (stacking) Polvf*) tvoří v kyselém prostředí dvojretězovou strukturu s paralelními řetězci (podobně jako poly (C). Párování baží Je ve struktuře paly(A) zajištěno jinak než v poly(C). V neutrálním prostředí má poly(A) strukturu jed no řetězovou. Ppi y < Gl a PolvJu tvoří čtyřvláknové struktury poly(A) poly(rC) poly(dG) poly(U) poly(rT) PrtynuhtootktOY» Komptexy Smícháním poly nuk kotidú (za vhodných iontových podmínek) vznikají dvou- a víceřetězové komplexy Pol¥(A)poly(U) tato dvojitá šroubovice vzniká při fyziologické iontové sile za nepřítomnost ale2* Při vyšších iontových silách může vzniknout trojřetéxová struktura poíy(A)poly(U) poly (U) [poly(A) 2 poly(U)] (Hoogsteen) PoíylGV polv. PoW (n>(cÍC) > (dlHdC) > (dí)*{rC) poly(dl-dC) a poly (dG-dC) jsou stabilnější nežli odpovídající komplexy homopoly nukleotidů Směsné křivky: