DNA quality control by conformational readout on the undamaged strand of the double helix

Synthetic DNA probes were incubated in human cell extracts to dissect the early step of bulky lesion recognition in the nucleotide excision repair pathway. Excision was induced upon combination of the target adduct with either a two-sided bulge, involving both the damaged sequence and its undamaged partner strand, or a one-sided bulge, affecting exclusively the undamaged complementary sequence. Surprisingly, the same adduct became refractory to repair when only the modified strand was bulged out of the double helix. Adduct removal was further dependent on an intact opposing strand and, at carcinogen-DNA adducts, the assembly of excision complexes was triggered by a single flipped-out deoxyribonucleotide in the complementary sequence. These findings describe a mechanism of molecular readout in DNA repair that, unexpectedly, is entirely confined to the undamaged side of the double helix.     

A versatile toolbox for variable DNA functionalization at high density

To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.     

Gap site-specific rapid formation of fluorescent silver nanoclusters for label-free DNA nucleobase recognition

Silver nanoclusters (Ag NCs) templated with DNAs have attracted much attention as novel fluorophores because of their convenient emission tunability by the sequence and length of the template DNAs. However, the precise production of Ag NCs in a site-specific manner still remains a challenge to attain highly selective and label-free DNA recognition. Here we exploited the availability of a gap site in DNA duplexes as a new scaffold for the synthesis of Ag NCs. Compared to the commonly used DNA templates for the creation of Ag NCs, the gap site in DNA duplexes was found to facilitate the rapid formation of the fluorescent Ag NCs without sacrifice of their bright emission and excellent stability. We found that fluorescent Ag NCs were highly selectively formed when cytosine faced toward the gap site in DNA duplexes, and they were in situ utilized as readout by signal-on manner for the DNA mutation assays. This base-selective growth of the fluorescent Ag NCs at the gap site would find promising applications in practical detection of single nucleotide polymorphism (SNP) and construction of DNA-based functional sensors with label-free and cost-effective merits.     

Torsional deformation of double helix in interaction and aggregation of DNA

We incorporate sequence-dependent twisting between adjacent base pairs and torsional elasticity of double helix into the theory of DNA-DNA interaction. The results show that pairing and counterion-induced-aggregation of nonhomologous DNA are accompanied by considerable torsional deformation. The deformation tunes negatively charged phosphate strands and positively charged grooves on opposing molecules to stay "in register", substantially reducing nonideality of the helical structure of DNA. Its cost, however, makes interaction between nonhomologous DNA less energetically favorable. In particular, interaction between double helical DNA may result in sequence homology recognition and selective pairing of homologous fragments containing more than 100-200 base pairs. We also find a weak, but potentially measurable, increase in the expected counterion concentration required for aggregation of nonhomologous DNA and slightly higher solubility of such DNA above the critical concentration.     

Investigation of DNA-protein sequence-specific interactions with a ds-DNA array

The sequence specific recognitions between DNAs and proteins play important roles in many biological functions. The use of double-stranded DNA arrays (ds-DNA arrays) for studying sequence specific recognition between DNAs and proteins is a promising method. Here we report the use of a ds-DNA probe with multi operation sites of restriction proteins in the middle sequence to investigate DNA-protein sequence-specific interactions including methylation. We arranged EcoR I site and Rsa I site on the same duplex DNA probe to fabricate ds-DNA arrays. We used the ds-DNA arrays to study DNA-restriction enzyme reactions before and after duplex DNA methylation under different probe concentration and reaction time conditions. Our results indicated that the ds-DNA arrays can be further biochemically modified and made accessible for interactions between DNAs and proteins in complex multi-step gene-regulation processes.     

Electrochemical analysis of the self-complementary B-DNA decamer d(CCAGGCCTGG)

The self-complementary decamer d(CCAGGCCTGG), native and denatured calf thymus DNAs were studied by means of differential pulse polarography with the DME and by cyclic voltammetry with the HMDE. The decamer (which represents one turn of the DNA double helix in the B form) produced cathodic and anodic signals similar to those yielded by high-molar mass DNAs. By measuring the anodic peak (due to guanine residues), it was possible to detect the decamer at subnanomolar concentrations by adsorptive stripping cyclic voltammetry at relatively short waiting times. The high sensitivity of the electrochemical analysis for small changes in the DNA double helix observed earlier in high-molar mass DNAs, together with the low requirements for the amount of the analyzed decamer sample, suggest that electrochemical techniques may become useful also in oligonucleotide studies.     

On the stability of single- and double-stranded DNA helices: The application of the PPT-MCF method on large fragments of DNA

The pseudo-polarization tensor mutually consistent field (PPT -MCF ) method recently introduced [1] has been applied to study the stacking interactions between the nucleotide bases in large periodic B-DNA fragments. The effects on the global and local binding properties caused by replacing one base in the periodic sequence by another base are investigated. The increase in the stability for comparable fragments owing to this base substitution is further enforced in the case of periodic alternating helices. The most important results are that the stacking interaction between two bases is slowly converging with the interbase distance and that the average contribution per base to the binding energy is repulsive. Furthermore, the energetical properties of double helix models in B- and Z-DNA configurations, respectively, consisting of up to five base pairs have been compared. It turns out that the G C G C sequence in Z-DNA is significantly more stable than either in periodic or periodic alternating B-DNA. In these cases the average energy contribution of a single Watson–Crick-type base pair is predicted also to be positive. From the calculations it follows that the double helix is not stabilized owing to the hydrogen bonding between the bases belonging to both strands, in contradiction to most other investigations.     

Distinct conformations of DNA-stabilized fluorescent silver nanoclusters revealed by electrophoretic mobility and diffusivity measurements

Silver-DNA nanoclusters (Ag:DNAs) are novel fluorophores under active research and development as alternative biomolecular markers. Comprised of a few-atom Ag cluster that is stabilized in water by binding to a strand of DNA, they are also interesting for fundamental explorations into the properties of metal molecules. Here, we use in situ calibrated electrokinetic microfluidics and fluorescence correlation spectroscopy to determine the size, charge, and conformation of a select set of Ag:DNAs. Among them is a pair of spectrally distinct Ag:DNAs stabilized by the same DNA sequence, for which it is known that the silver cluster differs by two atoms. We find these two Ag:DNAs differ in size by ～30%, even though their molecular weights differ by less than 3%. Thus a single DNA sequence can adopt very different conformations when binding slightly different Ag clusters. By comparing spectrally identical Ag:DNAs that differ in sequence, we show that the more compact conformation is insensitive to the native DNA secondary structure. These results demonstrate electrokinetic microfluidics as a practical tool for characterizing Ag:DNA.     

Growth and Origami Folding of DNA on Nanoparticles for High‐Efficiency Molecular Transport in Cellular Imaging and Drug Delivery

A novel three‐dimensional (3D) superstructure based on the growth and origami folding of DNA on gold nanoparticles (AuNPs) was developed. The 3D superstructure contains a nanoparticle core and dozens of two‐dimensional DNA belts folded from long single‐stranded DNAs grown in situ on the nanoparticle by rolling circle amplification (RCA). We designed two mechanisms to achieve the loading of molecules onto the 3D superstructures. In one mechanism, ligands bound to target molecules are merged into the growing DNA during the RCA process (merging mechanism). In the other mechanism, target molecules are intercalated into the double‐stranded DNAs produced by origami folding (intercalating mechanism). We demonstrated that the as‐fabricated 3D superstructures have a high molecule‐loading capacity and that they enable the high‐efficiency transport of signal reporters and drugs for cellular imaging and drug delivery, respectively.     

2.4 A crystal structure of an oxaliplatin 1,2-d(GpG) intrastrand cross-link in a DNA dodecamer duplex

(1R,2R-Diaminocyclohexane)oxalatoplatinum(II) (oxaliplatin) is a third-generation platinum anticancer compound that produces the same type of inter- and intrastrand DNA cross-links as cisplatin. In combination with 5-fluorouracil, oxaliplatin has been recently approved in Europe, Asia, and Latin America for the treatment of metastatic colorectal cancer. We present here the crystal structure of an oxaliplatin adduct of a DNA dodecanucleotide duplex having the same sequence as that previously reported for cisplatin (Takahara, P. M.; Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J. Nature 1995, 377, 649-652). Pt-MAD data were used to solve this first X-ray structure of a platinated DNA duplex derived from an active platinum anticancer drug other than cisplatin. The overall geometry and crystal packing of the complex, refined to 2.4 A resolution, are similar to those of the cisplatin structure, despite the fact that the two molecules crystallize in different space groups. The platinum atom of the [Pt(R,R-DACH)](2+) moiety forms a 1,2-intrastrand cross-link between two adjacent guanosine residues in the sequence 5'-d(CCTCTGGTCTCC), bending the double helix by approximately 30 degrees toward the major groove. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice. The latter is positioned in the minor groove opposite the platinum cross-link. A novel feature of the present structure is the presence of a hydrogen bond between the pseudoequatorial NH hydrogen atom of the (R,R)-DACH ligand and the O6 atom of the 3'-G of the platinated d(GpG) lesion. This finding provides structural evidence for the importance of chirality in mediating the interaction between oxaliplatin and duplex DNA, calibrating previously published models used to explain the reactivity of enantiomerically pure vicinal diamine platinum complexes with DNA in solution. It also provides a new kind of chiral recognition between an enantiomerically pure metal complex and the DNA double helix.     

Label-free detection of DNA molecules on the dendron based self-assembled monolayer by electrochemical impedance spectroscopy

We report preparation of a novel platform for effective DNA hybridization and its application to the detection of single mismatched DNA. Cone-shaped dendrimer molecules have been immobilized on the gold surface at equidistance, 3.1 nm, from each other with a probe DNA molecule attached to the top of each dendrimer so that enough space would be secured for effective hybridization. This arrangement allows each probe DNA molecule to form a natural DNA double helix upon hybridization with a target DNA molecule. The single nucleotide polymorphism at either the central or end position of the 25-mer target DNA has been shown to be effectively discriminated against on this platform from each other as well as from a complementary DNA by electrochemical impedance measurements. We also report adverse effects exerted by probe ions, Fe(CN)₆^3−/4−, on DNA hybridization reactions. The significance of the results for the use in DNA analysis is discussed.     

Oligonucleotide Analogues with a Nucleobase‐Including Backbone,Part 7, Molecular Dynamics Simulation of a DNA Duplex Containing a 2′‐Deoxyadenosine 8‐(Hydroxymethyl)‐Derived Nucleotide

The structure and stability of a 14‐mer DNA duplex containing a nucleotide analog with a hydroxymethyl substituent at the C(8) of 2′‐deoxyadenosine has been investigated by molecular‐dynamics simulation. The DNA duplex studied has the sequence 5′‐d(CGTAAGCTCGATAG)‐3′⋅5′‐d(CTATCGA*GCTTACG)‐3′, where the O(3′) of the dG₆ nucleotide in the second strand is linked through a phosphinato group with the O(10) of the dA 2′‐deoxyadenosine‐derived nucleotide. Previous experimental results showed that the stability of this duplex in aqueous solution of 0.1M NaCl at pH 7 and room temperature is significantly lower than that of the corresponding unmodified DNA duplex. Comparison of molecular‐dynamics trajectories of the unmodified and modified B‐DNA duplexes in aqueous solution, at similar conditions than the experiment, shows that the substitution of the dA nucleotide by the dA* nucleotide in the second strand induces stretching of the double helix, which results in opening of the grooves and consequent exposure of the double‐helix core to the solvent.     

Scanning positional variations in single-nucleotide polymorphism of DNA: an electrochemical study

While there are a number of electrochemical methods reported that enable the detection of single nucleotide mismatches, the determination of mismatch position in a double stranded DNA remains an unsolved challenge. Using a model system, we systematically explored the electrochemical response of all possible positions of single nucleotide mismatches in a set of 25-mer DNA films. These ds-DNA sequences each with a single mismatch at one of the twenty-five positions were bound to gold surfaces through a Au-S linkage and analyzed by electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) in the absence and presence of Zn(2+). We expected a unique response from each mismatched sequence in order to discriminate the mismatch positions. A pattern emerges between the electrochemical signals and mismatch positions. The positions can be grouped broadly into positions that exhibit large differences between matched and mismatched DNA (around positions 5 and 9) and those that exhibit smaller differences (around positions 1, 13 and 23) in the charge transfer resistance ΔR(ct), evaluated by EIS, and the apparent rate constant k(0), evaluated by SECM. To the best of our knowledge, this is the first study evaluating the electrochemical response of a single nucleotide mismatch as a function of mismatch positions along an oligonucleotide sequence.     

Sequence-universal recognition of duplex DNA by oligonucleotides via pseudocomplementarity and helix invasion

The well-known Watson-Crick complementarity rules, which were discovered 50 years ago, elegantly direct the specific pairing of two DNA single strands. On the contrary, once formed, the double-stranded (ds) DNA lacks such a simple and sequence-universal recognition principle, since most of the characteristic chemical groups of nucleobases are now buried deep inside the double helix, the major DNA form. We report a promising versatile approach for highly selective recognition of designated sites within dsDNA featuring considerable practical potential for a variety of molecular-biological, biotechnological, gene-therapeutic, and diagnostic applications. It may also have implications for prebiotic evolution of genetic machinery at the primordial stages of the origin of life. Our design synergistically employs the robust helix-invasion ability of recently developed DNA mimics and analogs, pseudocomplementary peptide nucleic acids and pseudocomplementary oligonucleotides, thus enabling the sequence-unrestricted recognition of chosen DNA duplexes by nucleobase oligomers. Using this basically general approach, we selectively tagged a unique mixed-base site on the target dsDNA fragment with streptavidin and/or multiply labeled this site with fluorophores via the primer-extension reaction.     

Mode of binding of camptothecins to double helix oligonucleotides

We report an NMR study on the interaction of topotecan (Tpt) and other camptothecins (Cpts) with several double helix and single strand oligonucleotides. The results obtained by (31)P NMR spectroscopy, nuclear Overhauser experiments (NOE) and molecular dynamics (MD) simulations show that Cpt drugs do not intercalate into the double helix, as suggested by many authors. Phosphorus NMR spectra indicated that no deformation occurs at any level of the phosphodiester backbone, while 2D NOESY experiments allowed the detection of several contacts between the aromatic protons of Cpts and those of the double helix. Models of the drug/oligonucleotide complexes, built on the basis of NOE data, show that the drug is located at the end of the double helix, by stacking the A and B rings with the guanine or cytidine of the terminal CG base pairs, with a preference for the 3[prime or minute]-terminal end sites. Cpts interact with double strand, as well as with single strand oligomers, as can be seen from the NMR shift variation observed on the drug protons; but this shielding effect cannot be an evidence of intercalation, as it is largely due to external non-specific interactions of the positively charged drug with the negatively charged ionic surface of the oligonucleotide. The molecular weight of one of the complexes was obtained from the correlation time value. The conformational behaviour of the DNA fragment d(CGTACG)(2) was studied by MD simulations on a ns time scale in the presence of water molecules and Na(+) ions. Different models were examined and the deformations induced on the phosphodiester backbone by molecules that are known to intercalate, were monitored by MD simulations.     

Non‐hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single‐stranded DNA

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label‐free, and economical non‐hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single‐stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single‐base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84‐mer, in which guanine to adenine single‐base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.     

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.     

Iso-energy contour maps for the interaction of water with DNA double helix in the B conformation

The interaction energy between water with B-DNA double helix is computed for few cylindrical surfaces (enclosing the helix) using analytical pair potentials. The iso-energy contour maps indicate a strong attraction for water extending to three water layers surrounding DNA and very stable bridging structure of water molecules connecting two successive phosphate groups along a single helix in the innermost layer.     

Water clustering and percolation in low hydration DNA shells

The hydrogen-bonded networks of water at the surface of a model DNA molecule are analyzed. At low hydrations, only small water clusters are attached to the DNA surface, whereas, at high hydrations, it is homogeneously covered by a spanning water network. The spanning water network is formed via a percolation transition at an intermediate hydration number of about 15 water molecules per nucleotide, which is very close to the midpoint of polymorphic transitions between A- and B-forms of the double helix. The percolation transition can occur in both A- and B-DNA hydration shells with nearly identical percolation thresholds. However, the mechanism of the percolation transition in A- and B-DNA is qualitatively different in regard to the roles played by the two opposite grooves of the double helix. Free ions can shift the percolation threshold by preventing some water molecules from hydrogen bond networking. The results corroborate the suggested relationship between water percolation and the low hydration polymorphism in DNA.