Modified DNA Duplexes Research Articles

Ultrafast Formation of a Delocalized Triplet-Excited State in an Epigenetically Modified DNA Duplex under Direct UV Excitation.

Epigenetic modifications impart important functionality to nucleic acids during gene expression but may increase the risk of photoinduced gene mutations. Thus, it is crucial to understand how these modifications affect the photostability of duplex DNA. In this work, the ultrafast formation (<20 ps) of a delocalized triplet charge transfer (CT) state spreading over two stacked neighboring nucleobases after direct UV excitation is demonstrated in a DNA duplex, d(G5fC)9•d(G5fC)9, made of alternating guanine (G) and 5-formylcytosine (5fC) nucleobases. The triplet yield is estimated to be 8 ± 3%, and the lifetime of the triplet CT state is 256 ± 22 ns, indicating that epigenetic modifications dramatically alter the excited state dynamics of duplex DNA and may enhance triplet state-induced photochemistry.

5-Formylcytosine weakens the G-C pair and imparts local conformational fluctuations to DNA duplexes.

DNA epigenetic modifications such as 5-methyl (5mC), 5-hydroxymethyl (5hmC), 5-formyl (5fC) and 5-carboxyl (5caC) cytosine have unique and specific biological roles. Crystallographic studies of 5mC containing duplexes were conducted in the A-, B- or the intermediate E-DNA polymorphic forms. 5fC-modified duplexes initially observed in the disputed F-DNA architecture were subsequently crystallized in the A-form, suggesting that epigenetic modifications enable DNA sequences to adopt diverse conformational states that plausibly contribute to their function. Solution-state studies of these modifications were found in the B-DNA form, with marked differences in the conformational flexibility of 5fC containing duplexes in comparison to C/5mC containing duplexes, compromising the DNA duplex's stability. Herein, we systematically evaluate sensitive and commonly inaccessible NMR parameters to map the subtle differences between C, 5mC, and their oxidized (5hmC/5fC) counterparts. We observe that 15N/1H chemical shifts effectively report on the weakening of 5fC-G Watson-Crick base-pair H-bonding, extending the instability beyond any achievable within the sequence-specific changes in DNA. Triple 5fC containing sequences propagate the destabilization farther from the site of modifications, explaining reduced duplex stability upon multiple modifications. Additionally, scalar and residual dipolar coupling measurements unravel local sugar pucker fluctuations. One-bond 13C-1H scalar coupling measurements point towards a significant deviation away from the anticipated C2'-endo pucker for the 5fC modified nucleotide. Structural models obtained employing 13C-1H residual dipolar couplings and inter-proton distances corroborate the sugar pucker's deviation for 5fC modified DNA duplexes. The changes in the sugar pucker equilibria remain local to the 5fC modified nucleotide sans additive/long-range effects arising from multiple contiguous modifications. These observations highlight the impact of a major groove modification that alters the physical properties of DNA duplex without disturbing the Watson-Crick face. The changes observed in our studies for the 5fC containing DNA contrast with the perturbations induced by damage/lesion highlight the varied conformational preferences that modified nucleobases impart to the DNA duplex. As sequence-specific DNA transactions are rooted in the base-pair stability and pucker deviations, the observed structural perturbations for 5fC-modified DNA potentially play critical functional roles, such as protein-DNA recognition and interactions.

MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Å from the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2′-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

Specificity of MLL1 and TET3 CXXC domains towards naturally occurring cytosine modifications

CXXC domains have traditionally been considered as CpG specific DNA binding domains that are repelled by cytosine modifications. This view has recently been challenged by the demonstration that CXXC domain of TET3 has relaxed sequence specificity and binds with the highest affinity to symmetric DNA duplex containing 5caCpG. Here, we present a comparative analysis of the MLL1-CXXC and TET3-CXXC sequence specificity and tolerance to cytosine modifications (5-methyl, 5-hydroxymethyl, 5-formyl, 5-carboxyl) in CpG and non-CpG context. For the first time, we take into consideration possible interference from cytosine bases elsewhere in the sequence. We show that despite similar overall structure, MLL1-CXXC has greater sequence and modification specificity than TET3-CXXC. MLL1-CXXC is specific only for CpG and does not tolerate any cytosine modifications. In contrast, TET3-CXXC does not require the CpG context of cytosine bases. Methyl-, formyl- and carboxyl-modifications are tolerated by TET3-CXXC, but only preceding G. Based on our and other data we propose a parsimonious model of MLL1-CXXC and TET3-CXXC DNA binding. This model explains why the binding of modified DNA duplexes by TET3-CXXC requires in some cases a register shift and is therefore context-dependent.

Excision of Carbohydrate-Modified dNMP Analogues from DNA 3' end by Human Apurinic/Apyrimidinic Endonuclease 1 (APE1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1)

We have studied the excision efficiency of human apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) on matched or mismatched bases located at the 3' end of DNA primers. We have used model DNA duplexes, which mimic DNA structures that occur during either replication (DNA with a 3' recessed end) or repair (DNA with a single-strand break). Both APE1 and TDP1 are more efficient in removing ribose-modified dNMP residues from mismatched pairs rather than canonical pairs. Thus, both of these enzymes may act as proofreading factors during the repair synthesis catalyzed by DNA polymerases including DNA polymerase β (Polβ). The design of new DNA polymerase inhibitors, which act as DNA or RNA chain terminators, is one of the main strategies in the development of antiviral agents. The excision efficacy of APE1 and TDP1 has also been studied for 3'-modified DNA duplexes that contain ddNMP or phosphorylated morpholino nucleosides (MorB) commonly used as terminators in the DNA synthesis. We have also investigated the insertion of ddNTP and morpholino nucleotides catalyzed by Polβ and human immunodeficiency virus reverse transcriptase. This experiment has pointed to MorCyt, cytosine-containing morpholino nucleoside, as a potential antiviral agent.

5-Formylcytosine does not change the global structure of DNA.

The mechanism by which the recently identified DNA modification 5-formylcytosine (fC) is recognized by epigenetic writer and reader proteins is not known. Recently, an unusual DNA structure, F-DNA, has been proposed as the basis for enzyme recognition of clusters of fC. We used NMR and X-ray crystallography to compare several modified DNA duplexes with unmodified analogs and found that in the crystal state the duplexes all belong to the A family, whereas in solution they are all members of the B family. We found that, contrary to previous findings, fC does not significantly affect the structure of DNA, although there are modest local differences at the modification sites. Hence, global conformation changes are unlikely to account for the recognition of this modified base, and our structural data favor a mechanism that operates at base-pair resolution for the recognition of fC by epigenome-modifying enzymes.

Structural stability of the photo-responsive DNA duplexes containing one azobenzene via a confined pore.

Herein, the structural stability of single azobenzene modified DNA duplexes, including the trans form and cis form, has been examined separately based on their distinguishable unzipping kinetics from the mixture by an α-hemolysin nanopore. Furthermore, the accurate isomerization efficiency between the trans and cis form was obtained with single molecule resolution.

DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm=-3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis.

DNA-Based Oligochromophores as Light-Harvesting Systems.

The chromophores ethynyl pyrene as blue, ethynyl perylene as green and ethynyl Nile red as red emitter were conjugated to the 5-position of 2'-deoxyuridine via an acetylene bridge. Using phosphoramidite chemistry on solid phase labelled DNA duplexes were prepared that bear single chromophore modifications, and binary and ternary combinations of these chromophore modifications. The steady-state and time-resolved fluorescence spectra of all three chromophores were studied in these modified DNA duplexes. An energy-transfer cascade occurs from ethynyl pyrene over ethynyl perylene to ethynyl Nile red and subsequently an electron-transfer cascade in the opposite direction (from ethynyl Nile red to ethynyl perylene or ethynyl pyrene, but not from ethynyl perylene to ethynyl pyrene). The electron-transfer processes finally provide charge separation. The efficiencies by these energy and electron-transfer processes can be tuned by the distances between the chromophores and the sequences. Most importantly, excitation at any wavelength between 350 and 700 nm finally leads to charge separated states which make these DNA samples promising candidates for light-harvesting systems.

139 Analysis of structure and thermodynamics of modified DNA duplexes using molecular dynamics simulation

Molecular dynamics (MD) simulation is a unique method for studying structure and dynamics of nucleic acid complexes. It is widely used for the investigation of conformational properties of native N...

Combined nucleobase and backbone modifications enhance DNA duplex stability and preserve biocompatibility

DNA strands containing a triazole linkage flanked on its 3′-side by an aminoethylphenoxazine nucleobase analogue (G-clamp) have been prepared by solid-phase synthesis followed by CuAAC-mediated click oligonucleotide ligation. The stability of the doubly modified DNA duplexes and DNA–RNA hybrids is greatly increased, whereas a single base pair mismatch located at or adjacent to the modifications is strongly destabilising, making triazole G-clamp a potent mismatch/point mutation sensor. A DNA strand containing this unnatural combination was successfully amplified by PCR to produce unmodified copies of the original template, with deoxyguanosine inserted opposite to the G-clamp-triazole nucleotide analogue. This study shows for the first time that a polymerase enzyme can read through a combined backbone/nucleobase modification surprisingly well. These favourable properties suggest new applications for oligonucleotides containing the G-clamp triazole modification in biotechnology, nanotechnology, diagnostics and therapeutics.

Saturation-Recovery EPR with Nitroxyl Radical–Dy(III) Spin Pairs: Distances and Orientations

We describe a method for measuring the distance between a radical and a Dy(III) ion using saturation-recovery electron paramagnetic resonance (EPR) and demonstrate its application using four chemically modified DNA duplexes. The four DNA duplexes contain a terminal nitroxide spin-label and a midsequence, EDTA-bound Dy(III) ion but differ in the nitroxyl radical (NO)-Dy(III) distance. Distances can be determined with high precision because of their sixth-root dependence on the experimentally determined dipolar rate constant. Furthermore, the orientation of the NO-Dy(III) interspin vector in the Dy(III) g-tensor reference frame can be determined for two of the DNA duplexes. The shortest mean NO-Dy(III) distance, 18.3 ± 0.3 Å, and the longest, 50.3 ± 2.4 Å, are near the lower and upper distance limits of what can be measured with the NO-EDTA(Dy(III)) pair at X-band. These methods are applicable to structural studies of nucleic acids, proteins, and their complexes.

DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

Discovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 and human cell membranes at micro- or submicromolar concentrations. Respective inhibitors adopted an aptamer pattern instead of a base-pairing interaction pattern. Structure-activity relationship studies of the respective DNA duplexes showed that the rigid and negatively charged DNA skeletons, in addition to the presence of hydrophobic groups, were crucial to the anti-HIV-1 activity of these compounds. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interacted with the primary pocket in the gp41 N-terminal heptad repeat (NHR) instead of interacting with the lipid bilayers.

Structural, Dynamical, and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

Among the distinct strategies proposed to expand the genetic alphabet, size-expanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. The most relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMO-LUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs

Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection.

Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent extract of HeLa cells. Synthesis and application of long model DNA

Long linear DNA analogs of nucleotide excision repair (NER) substrates have been synthesized. They are 137-mer duplexes containing in their internal positions nucleotides with bulky substitutes imitating lesions with fluorochloroazidopyridyl and fluorescein groups introduced using spacer fragments at the 4N and 5C positions of dCMP and dUMP (Fap-dC- and Flu-dU-DNA) and DNA containing a (+)-cis-stereoisomer of benzo[a]pyrene-N2-deoxyguanidine (BP-dG-DNA, 131 bp). The interaction of the modified DNA duplexes with the proteins of NER-competent HeLa extract was investigated. The substrate properties of the model DNA in the reaction of specific excision were shown to vary in the series Fap-dC-DNA << Flu-dU-DNA < BP-dG-DNA. During the experiments on affinity modification of the proteins of NER-competent extract, Fap-dC-DNA (137 bp) containing a (32)P-label in the photoactive nucleotide demonstrated properties of a highly efficient and selective probe. The set of the main targets of labeling included polypeptides of the extract with the same values of apparent molecular weights (35-90 kDa) as when using the shorter (48 bp) Fap-dC-DNA. Besides, some of the extract proteins were shown capable of specific and effective interaction with the long analog of NER substrate. Electrophoretic mobility of these proteins coincided with the mobilities of DNA-binding subunits of XPC-HR23B and PARP1 (~127 and ~115 kDa, respectively). The 115-kDa target protein was identified as PARP1 using NAD+-based functional testing. The results suggest that the linear Fap-dC-DNA is an unrepairable substrate analog that can compete with effective NER substrates in the binding of the proteins responsible for lesion recognition and excision.

DNA Oligomers Containing Site-Specific and Stereospecific Exocyclic Deoxyadenosine Adducts of 1,2,3,4-Diepoxybutane: Synthesis, Characterization, and Effects on DNA Structure

1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is considered the ultimate carcinogenic species of BD because of its potent genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. Mutagenesis studies suggest that DEB adducts formed at adenine bases may be critically important, as it induces large numbers of A → T transversions. We have recently identified three types of exocyclic DEB-dA lesions: N⁶,N⁶-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N⁶,N⁶-DHB-dA), 1,N⁶-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N⁶-γ-HMHP-dA), and 1,N⁶-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N⁶-α-HMHP-dA) [Seneviratne, U., et al. (2010) Chem. Res. Toxicol. 23, 118-133]. In the work presented here, a postsynthetic methodology for preparing DNA oligomers containing stereospecific and site-specific N⁶,N⁶-DHB-dA and 1,N⁶-γ-HMHP-dA adducts was developed. DNA oligomers containing site-specific 6-chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to generate oligomers containing N⁶-(2-hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine adducts, followed by their spontaneous cyclization to 1,N⁶-γ-HMHP-dA lesions. N⁶,N⁶-DHB-dA containing strands were prepared analogously by coupling 6-chloropurine containing DNA with (3S,4S)- or (3R,4R)-pyrrolidine-3,4-diols. Oligodeoxynucleotide structures were confirmed by ESI-MS, exonuclease ladder sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy studies of DNA duplexes containing N⁶,N⁶-DHB-dA and 1,N⁶-γ-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when an adenine residue was placed opposite 1,N⁶-γ-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.

2′-aldehyde oligonucleotides: Synthesis and use for affinity modification of DNA-recognizing proteins

Oligonucleotides with 1,2-diol grouping were prepared from 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxo-ethyl]uridine 3'-phosphoramidite. The thermal stability of modified DNA duplexes and their ability to form complexes with the p50 subunit of the NF-kappaB transcription factor and (cytosine-5)-DNA methyltransferase SsoII were studied. The periodate oxidation of the l,2-diol grouping of the oligonucleotides resulted in reactive 2'-aldehyde derivatives. The opportunity of their use for the affinity modification of DNA-recognizing proteins was studied.