Chemické vlastnosti, struktura a interakce nukleových kyselin Bi7015 •Struktura DNA • •Báze, nukleosidy, nukleotidy •Párování bazí •Dvoušroubovice DNA •Víceřetězcové struktury • •Chemická reaktivita NK. • •Hydrolýza NK, redukce, oxidace, nukleofily, elektrofily, alkylační činidla. •Mutageny, karcinogeny, protinádorově účinné látky. •Interakce DNA s UV a ionizujícím zářením. •Maxamovo a Gilbertovo sekvencování, chemické strukturní sondy. •Poškození DNA a jeho detekce. •Základní mechanismy opravných procesů •Nekovalentní interakce DNA s malými molekulami. • •Iontové interakce, vazba do žlábku, interkalace. •Bisinterkalátory, „provlékající se“ (threading) interkalátory, metalointerkalátory. •Přenos náboje/elektronů zprostředkovaný dvoušroubovicí DNA. •Analytické využití interkalátorů a látek vázajících se do žlábku; chemické nukleázy. •Interakce DNA s proteiny. • •Principy, funkční skupiny DNA a proteinů zprostředkující vzájemné interakce. •Nespecifické a sekvenčně specifické interakce. •Příklady sekvenčně specifických interakcí (např. helix-otáčka-helix, zinkové domény). HMG proteiny. •Příklady: Mechanismus interakce restrikčních endonukleáz s DNA. Interakce proteinu p53 s DNA. •Základní metody studia interakcí bílkovin s DNA; EMSA, imuno-techniky, footprinting DNA. •Enzymy účastnící se metabolismu NK. • •DNázy, RNázy, polynukleotidfosforyláza. •Endonukleázy rozpoznávající jednořetězcové NK •Enzymy účastnící se opravných procesů. •Topoizomerázy, helikázy. •Ligázy, polynukleotid kináza. •Restrikční endonukleázy, metyltransferázy; metylované baze, metylace DNA u prokaryot a eukaryot. •Využití enzymů při studiu struktury a interakcí DNA. •Struktura a funkce ribonukleových kyselin a regulace genové exprese. • •Ribozomální, transferové a informační RNA. •Sestřih prekurzorových RNA. •Katalytické funkce RNA, samosestřih. •RNA interference. Regulace genové exprese, úloha RNA; vztah k metylaci DNA. Metodické přístupy. •Elektrochemie nukleových kyselin a DNA biosenzory. • •Vztah mezi strukturou DNA a jejím chováním na rtuťových a uhlíkových elektrodách. •Elektrochemické biosenzory pro hybridizaci DNA. Značení DNA elektroaktivními skupinami. •Elektrochemické biosenzory pro poškození DNA. You and chemistry? J Some basic terms •hydrogen bond •ionic interactions •hydrophobic interactions, stacking •ester bond •N-glycosidic bonds •condensation, hydrolysis •oxidation, reduction •electrophile, nucleophile •tautomers, enol-keto, amino-imino • • Hydrogen bond d- d+ d- electronegative atom (O, N) electronegative atom with lone electron pair (O, N) Hydrogen bond •crucial importance for biology •properties of water •nucleobase pairing •protein structures •protein-DNA interactions •many others https://upload.wikimedia.org/wikipedia/commons/thumb/e/e2/Beta-D-Ribofuranose.svg/2000px-Beta-D-Rib ofuranose.svg.png D- und L-Ribose in Fischer-Projektion b-D-ribofuranose N-glycosidic bond anomers: a (hemiacetal hydroxyl „down“) b („up“ as here) hemiacetal (more exactly: in b anomer the hemiacetal hydroxyl is on the same side of the furanose ring as the -CH2OH group) https://upload.wikimedia.org/wikipedia/commons/thumb/e/e2/Beta-D-Ribofuranose.svg/2000px-Beta-D-Rib ofuranose.svg.png D- und L-Ribose in Fischer-Projektion b-D-ribofuranose - H2O N-glycosidic bond HO-R O-glycosides N-glycosides substitution of hemiacetal OH (condensation reaction) https://upload.wikimedia.org/wikipedia/commons/thumb/e/e2/Beta-D-Ribofuranose.svg/2000px-Beta-D-Rib ofuranose.svg.png D- und L-Ribose in Fischer-Projektion b-D-ribofuranose - H2O N-glycosidic bond nucleoside nucleoside formation Ester bonds •esters: products of condensation of acids with alcohols •substitution of –OH of the acid with –OR • • H H phosphoric acid (dihydrogen phosphate) - H2O - H2O phosphodiester https://upload.wikimedia.org/wikipedia/commons/f/f6/Ester-general.png carboxy ester -OH H- Tautomers •isomers enol-keto, amino-imino •double bond switch plus hydrogen/proton migration •in nucleobases: critical effect on pairing properties •6-substituents in purines + 4-substituents in pyrimidines: oxygenous=keto, nitrogenous=amino •hydrogen yes or not on the neighboring ring nitrogen • •relation to chemical mutagenesis 6 4 http://www.atdbio.com/img/articles/UV-melting-curve-large.png http://www.perkinelmer.com/CMSResources/Images/44-148452nucleic_acid_principle.jpg http://spin.niddk.nih.gov/clore/Structures/MEF2A-DNA/2.gif enthalpy (reaction heat) disordered Gibbs energy absolute temperature ordered less ordered ordered network of „weak“ bonds– released heat (H-bonds, electrostatic...) hydrogen bonds stacking (heat released) bonds broken (consumes heat) entropy (disorder) <0 Energetics of interactions (including structure) DNA structure 1953: James Watson, Francis Crick, Rosalind Franklin, Maurice Wilkins: the DNA double helix 1962: Nobel Prize (JW, FC, MW) basic principle of the preservation, transfer and expression of genetic information explained main_watson_crick Click for larger picture! Click for larger picture! DNA model (A Barrington Brown/Science Photo Library) Mendel 1864: „elements of heredity“, Mendel laws Miescher 1871: discovered „nuclein“, a substance occuring in cell nuclei DNA is the genetic material (1944 Avery, 1952 Hershey) •amino pairs with keto •purine pairs with pyrimidine •consequently, A pairs with T and G with C •nitrogen in the ring: donor or acceptor of H bond Tautomers •isomers enol-keto, amino-imino •double bond switch plus hydrogen/proton migration •in nucleobases: critical effect on pairing properties •6-substituents in purines + 4-substituents in pyrimidines: oxygenous=keto, nitrogenous=amino •hydrogen yes or not on the neighboring ring nitrogen • 6 4 http://www.dnacoil.com/wp-content/uploads/2013/08/wrong_dna_pauling.png https://paulingblog.files.wordpress.com/2011/01/triple-helix.jpg http://undsci.berkeley.edu/images/dna/pauling_model.jpg Linus Pauling – suggested triple helix structure with bases outside - INCORRECT Other concepts: ladder (not interwound) structure (to overcome topological problems with unwinding the double helix) Sugar numbering in nucleosides: 1‘, 2‘....5‘ Building blocks of nucleic acids: bases and pentoses RNA DNA always b anomer ATP methyl donor for methylation reactions C:\Users\Uživatel\Documents\dokumenty\prezentace\knihy-Biochemie\přednášky VUT\620px-Archaeosine_Archaeosin.svg.png archaeosine pseudouridine https://upload.wikimedia.org/wikipedia/commons/thumb/b/b7/Dihydrouridine.svg/170px-Dihydrouridine.s vg.png dihydrodouridine 5 Unusual bases and nucleosides in tRNA adenine deamination guanine deamination catabolism of purines nucleoside=inosine (I) tRNAPhe in archeae http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/structure/tRNA/trna_diagram_small.gif C:\Users\Uživatel\Documents\dokumenty\prezentace\knihy-Biochemie\přednášky VUT\281px-Wobble.svg.png Watson-Crick base pairs („canonical“) wobble pairs (examples) (in fact canonical) Stacking -interakce mezi paralelně orientovanými páry bazí -překryv p-orbitalů aromatických kruhů -interakce s jinými molekulami (interkalátory, zbytky aromatických aminokyselin při interakcích DNA-protein) Multistranded DNA structures • • • •triplexes •tetraplexes (quadruplexes) Hoogsteen base pairs (triads) cytosine protonation Triplex DNA (homopurine·homopyrimidine stretch of suitable sequence) e.g. TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (intermolecular triplex) Guanine tetrad Guanine tetraplex tetramolecular parallel bimolecular antiparallel intramolecular antiparallel intramolecular parallel cytosine tetraplex (i-motif) hemiprotonated C+•C pair metal ion-mediated pairing (non-physiological) Chemical reactivity of DNA Chemical reactivity of DNA •DNA chemistry is derived from chemistry of its costituents •phosphodiester bonds •N-glycosidic bonds •deoxyribose •nitrogenous bases • 2-deoxyribose phosphate •Chemical modification of DNA: • •damage to the genetic material • •analytical use •both phosphodiester and N-glycosidic bonds susceptible to acid hydrolysis •N-glycosidic bond more stable toward hydrolysis in pyrimidine than in purine nucleosides (and more in ribo- than in deoxynucleosides) •stable in alkali (unlike RNA) •alkali-labile sites: upon DNA damage •enzymatic hydrolysis (N-glycosylases, nucleases, phosphodiesterases) • DNA hydrolysis •two main sites susceptible to oxidation attacks: • • •C8 of purines (ROS) • •C5-C6 of pyrimidines Oxidation + reactions with nucleophiles •C4 and C6 are centres of electron deficit in pyrimidine moieties (electrophile centres) • • • • • •reaction with hydrazine: pyrazole derivative and urea residue bound to the sugar •with T the reaction is disfavored in high salt: Maxam-Gilbert sequencing technique reactions with nucleophiles •hydroxylamine: cytosine modification •the products‘ preferred tautomer pairs with adenine →mutagenic • • • • • •bisulphite: cytosine modification inducing its deamination to uracil →mutagenic •5-methyl cytosine does not give this reaction: genomic sequencing • of 5mC • reactions with electrophiles •attacking N and/or O atoms •nitrous acid (HNO2) causes base deamination (C→U, A→I) – affecting base pairing, mutagenic • • • •aldehydes: reactions with primary amino groups •formaldehyde: two step reaction DNA alkylation •hard or soft alkylating agents •hard ones attack both N and O atoms, soft only N •dimethyl sulfate: typical soft alkylating agent • • • • •N-alkyl-N-nitroso urea: typical hard alkylating agent •modifies all N + O in bases as well as phosphate groups (forming phosphotriesters) • •analytical use (sequencing, foorprinting) Biological consequences of base alkylation •N-alkylation: the primary site = N7 of guanine (accessible in both ss and dsDNA) •does not change base pairing; easily repairable •N3 of adenine or guanine: located in minor groove •cytotoxic modification (DNA/RNA polymerization blocked) •N1 of guanine: interferes with base pairing • •O-alkylation (G-O6, T-O6) the bases „locked“ in enol forms → improper base pairing → mutagenic Tautomerization, base pairing and chemical mutagenesis similarly uracil is deamination product of cytosine •chloro- (bromo-) acetaldehyde: two reactive centres (aldehyde and alkylhalogenide) •reaction with C or A •chemical probes (react only with unpaired bases) • • • • •diethyl pyrocarbonate: acylation of purines (primarily A) or C •modification leads to opening of the imidazole ring •chemical DNA probing Metabolically activated carcinogens •some substances became toxic after their metabolic conversion •detoxifying machinery of the organism acts here as a bad fellow •microsomal hydroxylase complex, cytochrome P450 •the role of this system is to introduce suitable reactive groups into xenobiotics enabling their conjugation with other molecules followed by removal from the organism •but…. Metabolically activated carcinogens •aromatic nitrogenous compounds (amines, nitro- or azo- compounds): • • • •aromatic amines are converted into either (safe) phenols, or (dangerous) hydroxylamine derivatives •azo- compounds: „cleaved“ into amines •nitro- compounds: reduced into hydroxylamines • Metabolically activated carcinogens •polycyclic aromatic hydrocarbons like benzo[a]pyrene: three-step activation •P450 introduces epoxy group •epoxide hydrolase opens the epoxide circle •P450 introduces second epoxy group •DNA adduct formation (primarily -NH2 of guanine, then G-N7, G-O6 and A-N6) • •similar pathway of • aflatoxin activation • bay region anticancer drugs •some types of antineoplastic agents act via formation of DNA adducts •metallodrugs: mainly platinum complexes (ineffective) cisplatin: reaction with DNA in certain sequence motifs some adduct types preferred (and/or more stable than others) 1,2-GG and 1,2-AG IACs = the main cytotoxic lesions other platinum complexes tested as cytostatics Photochemical DNA modifications •mainly pyrimidines •excitation at 240-280 nm: reactive singlet state •water addition at C5-C6 • • •excitation at 260-280 nm: photodimerization of pyrimidines • • • • • • •photoproducts of C can deaminate to U (mutagenic effects) effects of ionizing radiation •mostly indirect – through water radiolysis •each 1,000 eV produces ~27 •OH radicals that attack DNA •sugar damage:abstraction of hydrogen atoms from C-H bonds •a series of steps resulting • in strand breakage effects of ionizing radiation •base damage: hydroxylation and/or (under aerobic conditions) peroxylation pyrimidine products purine products (mainly C8 hydroxylation or opening of the imidazole ring) chemical nucleases species containing redox active metal ions mediating production of hydroxyl radicals (or othe reactive oxygen species) via Fenton and/or Haber-Weiss processes bleomycine Fe or Cu Men + H2O2 → Men+1 + •OH + OH- iron/EDTA complex Cu(phen)2 complex Chemical approaches in DNA studies (several examples) Maxam and Gilbert method of DNA sequencing HCOOH (acid depurination) DMS (preferential methylation of G at N7; spontaneous depurination) G A T C hydrazine (modification &breakdown of the pyrimidine ring) hydrazine+NaCl (selective modification of cytosine) at sites of base modification (removal) the sugar-phosphate backobone is labile towards alkali treatment with hot piperidine → cleavage at such sites •DNA fragment is end-labeled (radionuclide, fluorophore) • •the sample is divided into four reactions (HCOOH, DMS, hydrazine, hydrazine + NaCl) • •the conditions are chosen to reach only one modification event per DNA molecule HCOOH G A T C G A T C A T C G A T C G T C G A T C G A T C A T C G A T C G T C or or or only the „subfragment“ bearing the label is „visible“ in the following detection step G A T C A T C piperidine G A T C A T C G A T C G A T C A T C G A T C T C G A T C A T C T C G A T C G A T C DNA „footprinting“: determination of binding sites of other molecules (e.g. proteins) at the DNA sequence level complex free DNA DMS (guanine) piperidine DNaseI UO2 UV Cu(phen)2 OH radicals single strand-selective chemical probes Open local structures in negatively supercoiled DNA relaxed circular DNA negatively supercoiled DNA (linking deficit) stress related to the negative superhelicity (the linking deficit) can be absorbed in local open structures DNA segments of specific sequence can adopt „alternative“ local structures cruciform DNA (inverted repeat) Open local structures in negatively supercoiled DNA unpaired bases Triplex DNA (homopurine·homopyrimidine stretch with mirror symmetry) e.g. TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (intermolecular triplex) Open local structures in negatively supercoiled DNA Otevřené lokální struktury v negativně nadšroubovicové (sc) DNA Intramolecular triplex (homoPu•homoPy segment within negatively supercoiled DNA) T T T T T T A A A A A A A Chemicals selectively reacting with unpaired bases: osmium tetroxide complexes (Os,L) (T, more slowly C) chloroacetaldehyde (CAA) (A, C) diethyl pyrocarbonate (DEPC) (A, G) footprinting of CGC+ triplex by DMS steric clash steric clash carbodiimide carbodiimide Using the Maxam-Gilbert technique, it is possible to determine with a high preciseness which nucleotides are forming the local structure Ø modification of supercoiled DNA Ø restriction cleavage, radiactive labeling Ø hot piperidine Ø sequencing PAGE the structure can be deduced from the modification pattern TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA