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PubMed2005

Genotyping with hole-transporting DNA self-assembled monolayer.

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based on hole transport through DNA self-assembled monolayers. We prepared hole-transporting DNA self-assembled monolayers. Their photocurrent measurements were carried out using amperometory. Large photocurrent was observed when the DNA duplex which constituted a hole-transporting DNA self-assembled monolayer had a full-matched sequence, whereas the photocurrent for a DNA duplex containing a mismatched base pair was much smaller.

Aldehyde Dehydrogenase↗

PubMed1990

Sequence-specific interaction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the adenine-tract DNA duplex d(CTTTTGCAAAAG)2 has been studied in both D2O and H2O solutions by 1D and 2D 1H NMR spectroscopy. Thirty-one nuclear Overhauser effects between drug and nucleotide protons within the minor groove of the duplex, together with ring-current induced perturbations to the chemical shifts of basepair and deoxyribose protons, define the position and orientation of the bound dye molecules. Two drug molecules bind cooperatively and in symmetry related orientations at the centre of the 5'-TTTT and 5'-AAAA sequences with the binding interactions spanning only the four A-T basepairs. The positively charged N-methylpiperazine moieties point towards the centre of the duplex while the phenol groups are disposed towards the 3'-ends of the sequence. Resonance averaging is apparent for both the D2/D6 and D3/D5 phenol protons and D2"'/D6"' and D3"'/D5"' of the N-methylpiperazine ring and is consistent with these groups being involved in rapid rotation or ring-flipping motions in the bound state. Interstrand NOEs between adenine H2s and deoxyribose H1' are consistent with a high degree of propeller twisting of the A-T basepairs at the binding site of the aromatic benzimidazole and phenol rings of Hoechst. The data imply that the minor groove is particularly narrow with many contacts between the complementary curved surfaces of the drug and DNA indicating that strong van der Waals interactions, involving the floor and the walls of the minor groove, stabilize the complex. In our model the NH groups of the benzimidazole rings are positioned to make a pair of bifurcated hydrogen bonds with the adenine N3 and thymine O2 on the floor of the minor groove.

Adenine↗

PubMed1996

Solution conformation of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dC in a DNA duplex.

Combined NMR-molecular mechanics computational studies were undertaken on the C8-deoxyguanosine adduct formed by the carcinogen 1-nitropyrene embedded in the d(C5-[AP]G6-C7).d(G16-C17-G18) sequence context in a 11-mer duplex, with dC opposite the modified deoxyguanosine. The exchangeable and nonexchangeable protons of the aminopyrene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of several proton resonances for the three nucleotide d(G16-C17-G18) segment positioned opposite the [AP]dG6 lesion site resulting in weaker NOEs involving these protons in the adduct duplex. The solution conformation of the [AP]dG.dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine ring of [AP]dG6 is displaced into the major groove and stacks with the major groove edge of dC5 in the adduct duplex. Both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove as a consequence of the aminopyrene ring intercalation into the helix. This base-displaced intercalative structure of the [AP]dG.dC 11-mer duplex exhibits several unusually shifted proton resonances which can be accounted for by the ring current contributions of the deoxyguanosinyl and pyrenyl rings of the [AP]dG6 adduct. In summary, intercalation of the aminopyrene moiety is accompanied by displacement of both [AP]dG6 and the partner dC17 into the major groove in the [AP]dG.dC 11-mer duplex.

Base Composition↗

PubMed1994

Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.

Adenine↗

PubMed1997

Dam methyltransferase from Escherichia coli: kinetic studies using modified DNA oligomers: nonmethylated substrates.

Steady-state kinetics of the N6-adenine Dam methyltransferase have been measured using as substrates non-self-complementary tetradecanucleotide duplexes that contain the GATC target sequence. Modifications in the GATC target sequence of one or both of the strands included substitution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine). Thermodynamic parameters for the 14-mer duplexes were also determined. DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site was little perturbed compared with the canonical substrate. Replacement of dG residues by dl in both strands resulted in a decrease of the specificity constant. Substitution in both strands appears to be cumulative. Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation. Dam methyltransferase is rather tolerant to different substitutions. The results show much less spread than those for the analogous hemimethylated substrates studied previously (Marzabal et al., 1995). The absence of the methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the DNA substrate to the enzyme appears to be mostly determined by the thermodynamic stability of the DNA duplex.

2-Aminopurine↗

PubMed1996

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

The RecA protein of Escherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

Bacteriophage M13↗

PubMed2004

Structures of two RNA octamers containing tandem G.A base pairs.

The crystal structures of two RNA octamers, 5'-GGC(GA)GCC-3' and 5'-GIC(GA)GCC-3', have been determined from X-ray diffraction data to 2.8 and 2.7 A resolution, respectively. The RNA octamers crystallize in isomorphous unit cells containing two mispairs arranged in a self-complementary manner and one single strand in the asymmetric unit. The single strand pairs with another single strand related by crystallographic symmetry to form a third unique double helix. Tandem non-Watson-Crick G.A/A.G base pairs of the sheared type comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation between the six crystallographic duplexes and the solution structure. In the symmetric duplex of the octamer containing inosine, the sheared G.A pairs incorporate a bound water molecule. This duplex also binds one water molecule per strand in the minor groove adjacent to the G.A pairs.

Base Pairing↗

PubMed1995

Inhibition of gene expression by triple helix formation in hepatoma cells.

The aim of this study was to selectively inhibit human mitochondrial aldehyde dehydrogenase (ALDH2) gene expression by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to two purine-rich sequences in the 5'-flanking region of the human ALDH2 gene. Gel mobility shift assays showed that triplex formation is sequence-specific for the target duplex and the third strand oligonucleotide. In the presence of Mg2+, but absence of K+, triplex-forming oligonucleotides bind to their target sites with apparent dissociation constants (Kd) in the 10(-7) to 10(-9) M range. Potassium cation virtually suppressed the triplex formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent triplex formation with phosphorothioate-modified oligonucleotides. Phosphorothioate-modified oligonucleotides were delivered into human hepatoma Hep G2 cells by cationic liposomes. The reduction in ALDH2 mRNA levels in the cells was determined by the competitive reverse transcription-polymerase chain reaction. One of the phosphorothioate-modified oligonucleotides designed to forma an antiparallel triplex with a target in the 5'-flanking region of human ALDH2 gene (-105 to -125 from the translation initiation codon ATG) reduced by 80-90% the ALDH2 mRNA levels without affecting albumin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH2 gene expression for therapeutic use.

Aldehyde Dehydrogenase↗

PubMed2004

Retrovirus vector-mediated stable gene silencing in human cell.

RNA interference (RNAi) is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by short interfering RNAs (siRNAs) homologous to the gene being suppressed. siRNAs, which mediate sequence-specific mRNA degradation, are duplexes of about 21-23 nucleotides with 3'-overhangs synthesized in vitro or expressed by DNA-based vector. However, these systems rely on transfection for delivery and cannot generate long-term gene silencing in vivo. This obstacle may be circumvented by recently developed retrovirus- and lentivirus-delivered RNAi. Here, we describe a retroviral system for delivery of siRNA into cells, which can substantially down-regulate the expression of human p53 gene in human HepG2 cells. What's more, the G1 and S phases of cell cycle change dramatically in p53-down-regulated cells. These results indicate that retrovirus vector-delivered RNAi may be used in functional genomics and in gene therapy.

Base Sequence↗

PubMed2001

15-mer DNA duplexes containing an abasic site are thermodynamically more stable with adjacent purines than with pyrimidines.

Abasic site (AP)-containing duplexes, with flanking adenine (A) or cytosine (C) bases, were shown to be more stable with flanking A than with C bases [Sági, J., Hang, B., and Singer, B. (1999) Chem. Res. Toxicol. 12, 917-923]. We investigated whether the lower-magnitude destabilization by an AP site, with A neighbors, is a general effect of the purine versus the pyrimidine neighbors. Duplex stability, as compared to that of the corresponding control duplexes, was markedly decreased by the incorporation of the AP site (x) opposite any of the four bases. However, for the duplexes containing T, A, or C opposite the AP site, replacement of the symmetric doublet flanking pyrimidine bases with purines resulted in a smaller destabilization effect. The average stabilizing effect of the symmetric doublet purine neighbors of an AP site opposite T, A, or C bases was 3.2 degrees C (DeltaT(m)) and 1.3 kcal/mol (DeltaDeltaG degrees (37)) compared to those of pyrimidine neighbors. In contrast, a G.AP pair reduced or eliminated the differential effect of the neighbors. Using unrestrained molecular dynamics, it was shown that for the duplexes containing T opposite the AP site, with doublet pyrimidine neighbors, there was a larger magnitude of curvature around the lesion site than for the duplexes with the purines flanking the AP site. Purines flanking the AP site tend to shift toward each other, creating overlap, in contrast to the flanking pyrimidines. This indicates the possibility of stacking between purine bases at the AP site and can be the reason for the observed smaller thermodynamic destabilization of the duplexes with the AAxAA and GGxGG central sequences, as compared to those with TTxTT and CCxCC sequences. This work showed that for an AP site the GC content is not the only determinant of duplex stability, but rather is influenced more by whether purines or pyrimidines flank the AP site.

Adenine↗

PubMed1994

An asymmetric nucleotide in the parvoviral 3' hairpin directs segregation of a single active origin of DNA replication.

The 3' telomere of the linear single-stranded DNA genome of minute virus of mice (MVM), a murine parvovirus, can assume a complex hairpin structure. This contains a stem in which there is a mismatched 'bubble' sequence where a GA doublet opposes a GAA triplet. During replication, this hairpin is copied to form an imperfect palindrome which bridges adjacent genomes in a dimer duplex intermediate, leaving the two 'bubble' sequences embedded in potential replication origins on either side of the axis of symmetry. Such junctions are resolved asymmetrically in vitro in a DNA synthetic reaction which requires the viral initiator protein NS1. We show that the sequence surrounding the doublet is a potent origin, but the analogous region containing the triplet is completely inactive. The active origin is approximately 50 bp long, extending from an Activated Transcription Factor binding site at one end to a position some 7 bp beyond the major initiation site, to which NS1 ultimately becomes covalently attached. The actual sequence of the GA doublet is unimportant, but insertion of any third nucleotide here inactivates the origin, indicating that it represents a critical spacer element. Segregation of this asymmetry, therefore, allows the virus to confine replication initiation to one particular telomeric configuration.

Base Sequence↗

PubMed1975

A new method for mapping nucleic acid sequence homology by electron microscopy.

We describe here a new method for the electron microscopic mapping of sequence homology in nucleic acids. Specific segments of the T7 chromosome have been isolated following digestion with the restriction endonuclease from Hemophilus aegyptious (Haey). Denatured segments are annealed to the l-strand of T7 DNA; treatment of the hybrid with glyoxal allows only guanosine residues in the single-chain region to the reacted, producing an adduct which will no longer hydrogen bond with its complement on the r-strand. When the segment is displaced and the glyoxalated l-strand allowed to renature with the r-strand, "H" shaped structures are produced in which the duplex region corresponds to the position of sequence homology with the segment. The conditions employed for glyoxalation do not detectably disrupt duplex regions as small as 400 base pairs. This procedure should be generally useful for observing sequence homology in more complex DNA molecules containing duplex regions which can be specifically enriched for and their arrangement determined by electron microscopy.

Base Sequence↗

PubMed1991

Parallel-stranded DNA under topological stress: rearrangement of (dA)15.(dT)15 to a d(A.A.T)n triplex.

DNA oligonucleotides with appropriate sequences can form a stable duplex in which the two strands are paired in a parallel orientation instead of as the conventional antiparallel double helix of B-DNA. In parallel-stranded DNA (ps-DNA) base pairing is noncanonical with the glycosidic bonds in a trans orientation. The two grooves are equivalent. We have synthesized DNA duplexes consisting of a central parallel-stranded (dA)15.(dT)15 tract flanked by normal antiparallel regions, and ligated them into the pUC18 plasmid. The effect of negative supercoiling on the covalently closed circular molecules was studied by two-dimensional agarose gel electrophoresis and by chemical modification with OsO4-pyridine (Os,py) and diethylpyrocarbonate (DEPC). The following results were obtained: (i) The ps insert, and by inference ps-DNA in general, adopts a right handed helical form. (ii) Upon increasing the negative superhelix density (-sigma) to greater than 0.03 the 15 bp ps insert undergoes a major transition leading to a relaxation corresponding to a reduction in twist of approximately 2.5 helical turns. The transition free surgery is approximately kcal/mol. (iii) The chemical modification pattern of the resulting structure suggests that the purine strand folds back and associates with the pyrimidine strand, forming a novel intramolecular triplex structure consisting of d(A.A.T) base triplets. A model for the triplex conformation is proposed and its thermodynamic properties are analyzed by statistical mechanics.

Base Composition↗

PubMed2004

Initial bubble collapse plays a key role in the transition to elongation in T7 RNA polymerase.

RNA polymerases bind to specific sequences in DNA, melt open duplex DNA around the start site, and start transcription within the initially melted bubble. The initially transcribing complex is relatively unstable, releasing short abortive products. After synthesis of a minimal length of RNA (approximately 10-12 bases in the T7 system), RNA polymerases complete the transition to a processive (highly stable) elongation phase and lose the initial promoter contacts. The current study strongly supports a model for T7 RNA polymerase in which initial bubble collapse from position -4 to position +3 is responsible for initiating RNA displacement in the transition process. More specifically, collapse of the bubble from position -4 to position -1 indirectly and energetically facilitates the direct strand invasion offered by collapse at positions +1 to +3. Parallel work shows that promoter release, another key event occurring during this stage of transcription, begins after translocation to position +8 and is largely complete upon translocation to about position +12. The timing of promoter release agrees with the timing of initial bubble collapse determined by our previous fluorescence studies, suggesting that these two events are closely related.

Base Pair Mismatch↗

PubMed2001

Water and ion binding around r(UpA)12 and d(TpA)12 oligomers--comparison with RNA and DNA (CpG)12 duplexes.

The structural and dynamic properties of the water and ion first coordination shell of the r(A-U) and d(A-T) base-pairs embedded within the r(UpA)12 and d(TpA)12 duplexes are described on the basis of two 2.4 ns molecular dynamics simulations performed in a neutralizing aqueous environment with 0.25 M added KCl. The results are compared to previous molecular dynamics simulations of the r(CpG)12 and d(CpG)12 structures performed under similar conditions. It can be concluded that: (i) RNA helices are more rigid than DNA helices of identical sequence, as reflected by the fact that RNA duplexes keep their initial A-form shape while DNA duplexes adopt more sequence-specific shapes. (ii) Around these base-pairs, the water molecules occupy 21 to 22 well-defined hydration sites, some of which are partially occupied by potassium ions. (iii) These hydration sites are occupied by an average of 21.9, 21.0, 20.1, and 19.8 solvent molecules (water and ions) around the r(G=C), r(A-U), d(G=C), and d(A-T) pairs, respectively. (iv) From a dynamic point of view, the stability of the hydration shell is the strongest for the r(G=C) pairs and the weakest for the d(A-T) pairs. (v) For RNA, the observed long-lived hydration patterns are essentially non-sequence dependent and involve water bridges located in the deep groove and linking OR atoms of adjacent phosphate groups. Maximum lifetimes are close to 400 ps. (vi) In contrast, for DNA, long-lived hydration patterns are sequence dependent and located in the minor groove. For d(CpG)12, water bridges linking the (G)N3 and (C)O2 with the O4' atoms of adjacent nucleotides with 400 ps maximum lifetimes are characterized while no such bridges are observed for d(TpA)12. (vii) Potassium ions are observed to bind preferentially to deep/major groove atoms at RpY steps, essentially d(GpC), r(GpC), and r(ApU), by forming ion-bridges between electronegative atoms of adjacent base-pairs. On average, about half an ion is observed per base-pair. Positive ion-binding determinants are related to the proximity of two or more electronegative atoms. Negative binding determinants are associated with the electrostatic and steric hindrance due to the proximity of electropositive amino groups and neutral methyl groups. Potassium ions form only transient contacts with phosphate groups.

AT Rich Sequence↗

PubMed1985

Studies on transcription of 3'-extended templates by mammalian RNA polymerase II. Parameters that affect the initiation and elongation reactions.

Addition of short sequences of dCMP residues to the 3'-OH end of duplex linear DNAs allows rapid and efficient transcription to be initiated at these sites by purified mammalian RNA polymerase II [Kadesch, T. R., & Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295]. The use of such tailed DNA templates should allow biochemical studies on transcription elongation and termination with almost any desired DNA sequence. However, in vitro transcription with RNA polymerase II is aberrant in that the DNA template is not re-formed after transcription; rather, the DNA strands are separated, and most of the RNA product is found as a DNA-RNA hybrid. To better understand the factors that affect the process of transcription with these tailed DNA templates, we have varied a number of parameters that might be expected to play a role in the reaction. RNA polymerase II preparations from calf thymus, HeLa cells, and Drosophila all fail to displace the product RNA. However, RNA polymerase II from wheat germ gives only free RNA as a product, as does the Escherichia coli RNA polymerase. Hence, the displacement of the nascent RNA from a transcription complex seems to depend on some intrinsic property of the polymerase itself and not simply on the nature of the template. Variation of reaction conditions, or of the divalent metal ion, does not restore the renaturability of the DNA template. However, variation of the duplex 3'-terminal sequence of the template led to significant alterations. In general, GC-rich sites enhanced the displacement of the nascent RNA, while AT-rich sites enhanced formation of the DNA-RNA hybrid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Stabilization of triple-helical nucleic acids by basic oligopeptides.

Intermolecular triplex DNA is stabilized by metal cations and polyamines which reduce repulsion between the negatively charged phosphates of the three nucleic acid strands. We use a quantitative chemical-probing assay involving protection of duplex guanines in a homopyrimidine.homopurine (Py.Pu) sequence from dimethyl sulfate modification to study effects of basic oligopeptides on the stability of triplex DNA. An intermolecular protonated pyrimidine.purine.pyrimidine (Py.Pu*Py) triplex formed readily between a duplex DNA region and a 14-mer pyrimidine triplex-forming oligonucleotide (TFO) at pH 5. The triplex was stabilized at pH by the addition of magnesium ions. In the presence of spermine and lysine-rich peptides, the intermolecular triplex was stabilized up to pH 6.5-7.0. The effective peptide concentration required for stabilization was 10(-5)-10(-2) M. Of the basic peptides studied, pentalysine (Lys-Lys-Lys-Lys-Lys) was the most effective triplex stabilizer. It was effective at concentrations which are lower than those required for Lys-Gly-Lys-Gly-Lys and Lys-Ala-Lys-Ala-Lys and are similar to active concentrations of spermine. Basic peptides were more effective at stabilizing a Py.Pu*Py triplex than a pyrimidine.purine.purine (Py.Pu*Pu) triplex. At 1 mM, Lys-Lys-Lys-Lys-Lys stabilized the Py.Pu*Pu triplex at a level comparable to stabilization by Mn2+ and spermine, whereas Lys-Gly-Lys-Gly-Lys and Lys-Ala-Lys-Ala-Lys resulted in weaker TFO binding. The concentration of TFOs required to form triplex DNA were significantly reduced in the presence of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗

PubMed2006

The crystal structure at 1.5 angstroms resolution of an RNA octamer duplex containing tandem G.U basepairs.

The crystal structure of the RNA octamer, 5'-GGCGUGCC-3' has been determined from x-ray diffraction data to 1.5 angstroms resolution. In the crystal, this oligonucleotide forms five self-complementary double-helices in the asymmetric unit. Tandem 5'GU/3'UG basepairs comprise an internal loop in the middle of each duplex. The NMR structure of this octameric RNA sequence is also known, allowing comparison of the variation among the five crystallographic duplexes and the solution structure. The G.U pairs in the five duplexes of the crystal form two direct hydrogen bonds and are stabilized by water molecules that bridge between the base of guanine (N2) and the sugar (O2') of uracil. This contrasts with the NMR structure in which only one direct hydrogen bond is observed for the G.U pairs. The reduced stability of the r(CGUG)2 motif relative to the r(GGUC)2 motif may be explained by the lack of stacking of the uracil bases between the Watson-Crick and G.U pairs as observed in the crystal structure.

Base Pairing↗