Electroactivity of DNA and effects of DNA structure Miroslav Fojta Institute of Biophysics Department of Biophysical Chemistry and Molecular Oncology Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis Elektrochemické metody … polarografie -I -E Xm-n Xm n e- identifikace elektrochemicky aktivní látky (co to je) kvantifikace (jaká je koncentrace) kapající rtuťová elektroda Elektrochemické metody … -I -E Xm Xm-n n e- •visicí rtuťová kapková elektroda •pevné elektrody Xm Xm-n n e- Elektrochemické metody … voltametrie 3’ 5’ purine bases thymine (T) pyrimidine bases cytosine (C) adenine (A) guanine (G) DNA double helix base pairs sugar-phosphate backbone major groove minor groove C•G T•A A B C D 2-deoxyribose phosphate minor groove major groove tn_Emil late 1950s, Emil Paleček: DNA polarography nature1 nature2 DSCF0004 nucleic acids are electroactive •at mercury electrodes, bases A,C and G undergo redox processes •at carbon electrodes, purine bases can be oxidized •sugar residues in nucleic acids can be oxidized at copper electrode Singhal, P.; Kuhr, W. G.: Anal. Chem. 1997, 69, 3552-3557; Anal. Chem. 1997, 69, 4828-4832. Adenine and Cytosine are Reduced at the Mercury Electrode CA Guanine is reduced at the mercury electrode at highly negative potentials... …and its reduction product yields anodic peak in cyclic voltammetry G Guanine and adenine residues yield specific oxidation peaks at carbon electrodes PSA Gox Aox Reduction DNA signals at the mercury electrodes are strongly influenced by DNA structure •this is due to location of the A and C electroactive sites within the Watson-Crick hydrogen bonding system Reduction DNA signals at the mercury electrodes are strongly influenced by DNA structure square-wave voltammetry DNA oxidation at carbon electrodes is less influenced by DNA structure •oxidation sites of guanine and adenine in dsDNA are located closer to the double helix surface and are accessible via the double helix grooves DNA oxidation at carbon electrodes is less influenced by DNA structure At mercury electrodes in weakly alkaline media, adsorption-desorption (tensammetric) signals of nucleic acids can be detected (e.g., using AC polarography, voltammetry, AC Z) •depending on the conditions and on DNA structure, individual components of the polynucleotide chains may be involved in adsorption/desorption processes background electrolyte -at moderate ionic strenght, double-stranded DNA yields peak 1 due to desorption/reorientation of DNA segments adsorbed via the sugar-phosphate backbone 1 double-helix -distorted or regions of double-stranded DNA yield peak 2 double-helix 2 background electrolyte 2 background electrolyte single-stranded (denatured) DNA yields peak 1 (due to the sugar-phosphate backbone) and peak 3 due to desorption/reorientation of DNA segments adsorbed via freely accessible bases 3 5 adsorption/desorption behavior of DNA at electrodes is strongly related to negative charge of its sugar-phosphate backbone (together with a strong adsorption of nucleobases via hydrophobic forces) peptide nucleic acid: DNA analogue with neutral backbone DNA PNA (decamers, identical base sequence) AC Z at HMDE differential pulse polarography •used in nucleic acid studies in the 60-70´s •discrimination between ss and dsDNA differential pulse polarography •peak II: high sensitivity to subtle changes of dsDNA structure (&dynamics) • •DNA premelting PREMELT DPP peak II A260 differential pulse polarography •polymorphy of DNA double helix: its structure depends on the nucleotide sequence Ø B. sublilis and B. brevis DNAs have the same G+C content and different nucleotide sequence B. subtilis B. brevis differential pulse polarography •strand breaks Ø ioniz differential pulse polarography Ødouble helix distortions due to nucleobase photoadducts UV differential pulse polarography Øchemical modification of DNA: platinum adducts Ø distinction of the kind of structural change caused by modification with different Pt complexes peak II: conformation distortion, base pairing preserved peak III: base unpairing (Brabec et al.) Changes of DNA structure at electrically charged surface HMDE 1 3 2 -1.5 -0.5 E (V) DME (SMDE) 1 2 -1.5 -0.5 E (V) 3 U intensities of ssDNA-specific signals prolonged exposure to (accumulation at) potential given on x-axis pH close to neutral (bases not ionized): region T: – negligible structural changes due to adsorption region U: surface denaturation dsDNA dsDNA ssDNA positive or neutral base sugar phosphate • close to the duplex ends (or single-strand breaks), some bases can be unpaired and make contact with the mercury surface • phosphates repelled from negatively charged surface • randomly adsorbed bases represent relatively firm anchor sites • constraints in the double helix cause its (slow) unwinding • more (unpaired) bases are coming into contact with the electrode (in real situation the strand must rotate around one another; the process requires repeated adsorption/desorption events) effects of initial potential and scan direction alternative models •not double helix unwinding but conformation transition of dsDNA •potential induced „p-state“ of dsDNA involving B-A transition at the surface (Berg) •„ladder DNA“ structure (Nurnberg) • •these models do not accord with numerous experimental data that support the unwinding model: covalently closed circular DNAs: limited unwinding duplex with covalently cross-linked strands: limited unwinding DNA with or without ends detection of DNA strand breaks using supercoiled DNA and mercury electrodes supercoiled open (nicked) circular linear supercoiled DNA structural transitions induced by DNA supercoiling sc-EM sc oc Vinograd, 1960‘s: two forms of circular viral DNA (sedimentation velocity or sedimentation equilibrium studies) ribbon model: upon introducing torsional and/or bending stress, DNA behaves like an elastic ribbon relaxed covalently closed circular DNA negatively supercoiled (sc) DNA open circular (oc) DNA 18 turns 20 turns 20 turns (constraint due to the twist deficit causes formation of the superhelix) negative superhelicty is absorbed by local opening of the double helix cruciform triplex no constraint – free rotation at the strand break DNA topoisomers differ in the superhelicity level agarose gel elfo peak 1 peak 3* overall shape of the molecules DC around pzc peak 1 peak CA peak G peak 3* is due to local helix opening (base unpairing) transition 1 transition 2 peak CA (similar dependence on –s as peak 3*) is known to be sensitive to DNA denaturation peak G (no transition 2) is less sensitive to DNA structure 1 1 3 2 2 3* 3* -1.5 -0.5 -1.5 -0.5 E (V) E (V) 200 nA 200 nA 3 Studies with DME: peak 3* is due to helix opening in solution (in difference to peak 3 produced by oc or linear dsDNA that is observed only at the HMDE DME HMDE circular and linear DNAs pUC19 DNA four different plasmids (3-6 kbp) peak 3 peak 1 nicked circular (sb accumulation) linearization upon adsorption fragmentation upon adsorption agarose gel elfo shows ocDNA as the only product of DNA cleavage during the experiment sc oc lin •monitoring of DNA cleavage in solution using electrochemistry • •DNase I introduces single strand breaks •DNA treated with the enzyme, then adsorbed at HMDE different cleavage mechanisms in solution and at the surface? correlation between peak 3 and peak 1 peak heights E/V -0.8 -1.6 3 1 cleavage time „solution“: as previous slide „surface“: DNA adsorbed at HMDE, then treated with the enzyme sc, 3 kbp lin, 3 kbp lin, ≤500 bp HMDE GC/MFE •at the GC/MFE: more pronounced effects of the DNA molecule length •poor responses of long dsDNAs •steeper dependence of peak heights on DNA cleavage extent (from the second phase) GC/MFE: not a smooth surface like HMDE long rigid ds DNA: poor contact with the electrode surface shorter DNA fragments can fit better the surface shape peak 1 peak 3 DNA structural changes due to intercalation CQ conc.: a-0, b-10 mM, c-50 mM Due to intercalation (e.g., of chloroquine) in solution/during DNA immobilization intDNA is more resistant to surface denaturation within the region U DNA containing numerous strand breaks, adsorbed in the presence of an intercalator, does not yield responses characteristi for intDNA strand breaks abundance peak 3 peak 2 no intercalator int DNA -1.2 V -0.4 V -1.2 V -0.4 V time of exposure to the potential DNA in the absence of intercalator: B-form, bases hidden in the double-helix interior, relatively far from the electrode surface DNA saturated with the intecalator (intDNA): untwisted and lengthened double helix, less deep grooves – bases closer to the surface, contacts between the surface and the base pair edges after removal of the intercalator, the intDNA conformation is preserved the adsorbed untwisted regions of intDNA yield the AV voltammetric peak 2 X break X break intDNA with intercalator intDNA after intecalator removal X break dsDNA without intercalator with intercalator intDNA after intecalator removal dsDNA without intercalator X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X single-stranded oligonucleotides: effects of nucleobase composition and/or nucleotide sequence •Dračka, Trnková •current measured in voltammetry is a sum (linear combination) of partial current components (diffusion, capacitive, kinetic…) •these componets depend diversely on scan rate •possibility of numerical elimination of any component based on measurements at several different scan rates •studies of electrode processes •separation of overlapped signals •different EVLS responses for ODNs with identical base content but differing in their sequence 2D condensation of homopyrimidine oligos at mercury-based electrodes Prezentace_2a Ø capacitance pits indicate formation of condesed films ØNA bases, nucleosides, nucleotides for decades known to form such 2D-condensed layers (V. Vetterl) Øup to recently, no observations with DNA, polynucleotides or ODNs ØS. Hason: homopyrimidine ODNs (30 to 90-mers studied) can form such condensed films at negatively charged mercury or amalgam surfaces transfer (ex-situ) experiment: the condensed film is formed of reoriented ODN molecules already adsorbed (not due thickening the adsorbed layer by extra molecules form the bulk) mixed A+G or C+T ODNs silver amalgam electrode HMDE around pzc: random orientation of the ODN chain negatively charged surface: sugar-phosphate backbone repelled pyrimidine bases udergo self-assembly these phenomena may affect behavior of ODNs anchored via terminal thiol group Electrochemistry of G quadruplexes Guanine is reduced at the mercury electrode at highly negative potentials... …and its reduction product yields anodic peak upon its oxidation (e.g., in cyclic voltammetry) GHMDE Effects of DNA structure: duplex versus single-stranded double-helix •accessibility of nucleobases for electrode processes •mainly at mercury- based electrodes carbon electrode mercury electrode CA CA Watson-Crick and Hoogsteen base pairing Watson-Crick base pair (G•C) C•G•C+ triad (Watson-Crick C•G + Hoogsteen G•C+ pairing) guanine tetrad M+ 7 8 3 4 X. H. Cai, G. Rivas, H. Shirashi, P. Farias, J. Wang, M. Tomschik, F. Jelen and E. Palecek, Anal Chim Acta 1997, 344, 65-76 Guanine quadruplexes M+ parallel tetramolecular antiparallel bimolecular parallel monomolecular (telomeric DNA) •biological importance (binding sites of proteins, targets for small molecules = drugs) •aptamers (including electrochemical aptasensors) •DNA nanotechnology Guanine quadruplexes Ana Brett et al. (e.g. Anal Chem 2014) Gox at carbon (GCE) parallel tetramolecular antiparallel bimolecular parallel monomolecular (telomeric DNA) M+ Studies of homoligonucleotides and oligonucleotides with „segmental“ sequences Series of papers by L. Trnkova et al. on reduction of C+A (EVLS studies) A set of 15-mers with internal dGn segments of different lengths Oxidation on carbon (PGE) A set of 15-mers with internal dGn segments of different lengths Cyclic voltammetry on HMDE Sufficiently long dGn segments form parallel G-guadruplexes G0 G1 G2 G3 G5 G7 G9 G15 G0 G1 G2 G3 G5 G7 G9 G15 L M CD spectra native PAGE denaturing PAGE •starting from G5, quadruplex structures are observed under native conditions •starting from G7, the structures resist denaturing conditions Different behavior of rigid quadruplex segments on negatively or positively charged surfaces unstructured stable (rigid) negatively charged HMDE positively charged PGE anchored by strongly adsorbed ss tails repulsion attraction ineffictive guanine reduction facile guanine oxidation guanines only in quadruplex AC voltammetry (adsorption/desorption) Conclusions •segmental-sequence oligonucleotides: excellent tools for studies of DNA structure/dynamics on electrically charged surfaces • •extension towards quadruplexes: for the first time we performed a study of electrochemical reduction of the G-quadruplex forming DNA ( • • • • • •further systematic studies in progress