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PubMed2004

Effects of a hairpin polyamide on DNA melting: comparison with distamycin and Hoechst 33258.

We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)H2Ngamma-Im-Py-Py-Py-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no interaction with the reverse sequence WCWWGW. Fluorescence melting studies with 11-mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences. At low concentrations, the polyamide produced biphasic melting curves with TGATCT, TGTACT and TGAACT, suggesting a strong interaction. In contrast, the melting profiles with TCTAGT were always monophasic and showed much smaller concentration dependent changes in Tm. The polyamide also showed weak binding to the sequence TGATCT when one of the central AT pairs was replaced with an AC mismatch. These melting profiles were compared with those produced by the AT-selective minor groove binding agents distamycin and Hoechst 33258 at the same sites and at similar sequences containing A5 and (AT)3, which are expected to bind distamycin in the 1:1 and 2:1 modes, respectively. These ligands produced simple monophasic melting curves in which the Tm steadily increased as the ligand concentration was raised.

Base Pair Mismatch↗
PubMed2001

Sequence-specific binding property of Arabidopsis thaliana telomeric DNA binding protein 1 (AtTBP1).

We have identified an Arabidopsis thaliana cDNA, designated as AtTBP1, encoding a protein with a predicted size of 70.6 kDa that specifically binds to the plant telomeric repeat sequence TTTAGGG. AtTBP1 is present as a single-copy gene in Arabidopsis genome and is expressed ubiquitously in various organs. AtTBP1 has a single Myb telomeric DNA binding domain at the C-terminus and an extensive homology with other known telomere-binding proteins. The isolated C-terminus of AtTBP1 is capable of sequence-specific DNA binding to plant duplex telomeric DNA. These results suggest that AtTBP1 may play important roles in plant telomere function in vivo.

Amino Acid Sequence↗
PubMedDate not supplied

DNA electrochemistry as a probe of base pair stacking in A-, B-, and Z-form DNA.

DNA-mediated charge transport (CT) chemistry is sensitive to DNA structure and base pair stacking. In an electrochemical assay based upon DNA CT, DNA-modified electrode surfaces are used to examine the electrochemical reduction of methylene blue (MB), a small molecule that binds to the DNA film by intercalation. Here electrochemically we probe CT in the three primary conformations of double-stranded nucleic acids, A-, B-, and Z-form DNA. The A-form is examined in the context of a DNA/RNA hybrid duplex and Z-DNA, in duplexes containing d((m)CG)(8) sequences at high Mg(2+) concentrations. We find that both A- and B-DNA support efficient DNA CT as measured by MB reduction in the DNA film; a lower level of reduction is evident with the Z-form film. Furthermore, mismatches incorporated into A-form duplexes, as in B-form duplexes, disrupt MB reduction, thus providing a strategy for mutation detection through testing of RNA transcripts at DNA electrodes.

Base Pair Mismatch↗
PubMed1993

5'-CGA sequence is a strong motif for homo base-paired parallel-stranded DNA duplex as revealed by NMR analysis.

The structure of the non-self-complementary DNA heptamer d(CGACGAC) at low pH has been determined by the quantitative NMR refinement procedure designated SPEDREF (SPEctral-Driven REFinement). Acid-base titration of the molecule indicated a prominent n = 2 pKa near 6.8. In the pH range up to 6.0, the heptamer forms a remarkably stable double helix, which was conclusively shown to be an unusual homobase-paired parallel-stranded double helix (termed II-DNA). In this II-DNA helix, the 5'-CGA trinucleotide is the structural motif that accounts for the stability, with the C+-C hemiprotonated base pair (in which C+ is N3-protonated cytosine) providing for the alignment site and the unusual interstrand G-A base stack in the GpA step furnishing the additional stabilizing forces. The exchangeable proton data from two-dimensional nuclear Overhauser effect spectroscopy are in total agreement with the refined structure. We conclude that the 5'-CGA or other related sequences (e.g., 5'-CCGA) are powerful motifs in promoting the II-DNA or II-RNA conformations that may play certain biological functions.

Base Sequence↗
PubMed1997

Reconstitution of human topoisomerase I by fragment complementation.

Human topoisomerase I (topo I, 91 kDa) is composed of four major domains; the unconserved and highly charged "N-terminal" domain (24 kDa), the conserved "core" domain (54 kDa), a poorly conserved and positively charged "linker" region (5 kDa), and the highly conserved "C-terminal" domain (8 kDa) which contains the active site tyrosine at position 723. Here we demonstrate that human topo I activity can be reconstituted by mixing a 58 kDa recombinant core domain (residues Lys175 to Ala659) with any one of a series of recombinant C-terminal fragments that range in size from 12 kDa (linker and C-terminal domains, residues Leu658 to Phe765) to 6.3 kDa (C-terminal domain residues Gln713 to Phe765). The C-terminal fragments bind tightly to the core domain, forming a 1:1 complex that is stable irrespective of ionic strength (0.01 to 1 M). The reconstituted enzymes are active only over a relatively narrow range of salt concentrations (25 to 200 mM KCl) as compared to the intact topo70 enzyme (missing the N-terminal domain). Under physiological conditions (150 mM KCl and 10 mM Mg2+) they are much more distributive in their mode of action than topo70. The reconstituted enzyme binds DNA with an affinity that is approximately 20-fold lower than that of the intact topo70. In addition, the cleavage/religation equilibrium of the reconstituted enzyme appears to be biased towards religation relative to that of the intact enzyme. Despite differences in the cleavage/religation equilibrium and affinity for DNA, the reconstituted and intact enzymes have identical sequence specificities for the cleavage of duplex DNA or suicide cleavage of oligonucleotide substrates.

Animals↗
PubMed1991

Melting of oligodeoxynucleotides with various structures.

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62-bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end-structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na+] less than 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30-40 bp-long conforms to the two state model. From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na+.

Base Sequence↗
PubMed1997

Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability.

In order to understand the effect of 2-thiouridine (s2U) substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through s2U-34 modification, a pentamer RNA sequence, Gs2UUUC, was synthesized and characterized by NMR spectroscopy. The single strand contains the UUU anticodon sequence of tRNALys with flanking GCs to increase duplex stability. Regiochemical effects of uridine thiolation were determined by comparing the structure and stability of the 2-thiouridine containing oligonucleotide with an identical sequence containing 4-thiouridine (s4U) and also the normal uridine nucleoside. Circular dichroism spectrum indicated an A-form helical conformation for Gs2UUUC which was further confirmed by 2D ROESY NMR experiments. The duplex stability of the three pentamers complexed with a 2'-O-methyl-ribonucleotide complementary strand, GmAmAmAmCm, was determined by UV thermal melting studies and by 1H NMR spectroscopy. The duplex containing s2U has a T m of 30.7 degrees C compared to 19. 0 degrees C for the unmodified control and 14.5 degrees C for the s4U containing duplex. The results from UV experiments were corroborated by imino proton NMR studies that show proton exchange rates, chemical shift differences, and NH proton linewidths indicative of the stability order s2U >U >s4U. The magnitude of the effect of s2U in our model system is comparable to the 20 degrees C stabilization observed by Grosjean and co-workers for 2-thiolation in a codon-anticodon model system composed of two tRNAs with complementary anticodon sequences [Houssier, C., Degee, P., Nicoghosian, K. and Grosjean, H. (1988) J. Biomol. Struct. Dyn., 5, 1259-1266].

Anticodon↗
PubMed1996

Genetically unstable CXG repeats are structurally dynamic and have a high propensity for folding. An NMR and UV spectroscopic study.

Recent molecular genetics studies have revealed a correlation between spontaneous, progressive expansion of several DNA trinucleotide repeats and certain hereditary neurodegenerative diseases. Triplet repeat (TR) sequences may be present in structured forms that can mediate the processes interrupting normal cellular replication, transcription, or repair activities, eventually leading to gene mutation. Using high resolution NMR spectroscopy and other biophysical methods, we probed the solution structures and properties of single-stranded TR sequences. These studies have led to the discovery of a new duplex motif (e-motif), present in CCG repeats, and to the elucidation of the structure of the (CTG)3 duplex. In this paper we provide a global picture of the solution behavior of the human disease-related CXG (X = A, C, G, or T) and the comparison GXC (X = A, or T) TR sequences. All six triplet repeats form antiparallel duplexes. The mismatched bases in CAG and CGG repeat duplexes are rather flexible and they do not appear to form stable, paired conformations. By comparison, GAC repeat duplexes and their mismatched A residues are well-structured. Most interestingly, the structures of the disease-related CXG repeats exhibit a propensity for folding at chain lengths as short as 12 residues. Furthermore, the energy barrier for the formation of homo-duplexes from the corresponding complementary hetero-duplexes are much lower for the CXG TR sequences than for the GAC or GTC TR sequences. These results provide insights into the conformation and physiochemical properties of TR sequences. Thus, a basis is provided for further studies of the behavior of long TR sequences in an effort to elucidate the molecular mechanisms of in vivo expansion and function of TR sequences.

Base Composition↗
PubMed1993

Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases.

There are numerous restriction endonucleases (ENases) which are known never to achieve total cleavage of certain unmethylated target DNAs. In addition to EcoRII we found seven ENases (AtuBI, Cfr9I, Eco57I, Ksp632I, NaeI, NarI, and SauBMKI) that were stimulated by oligodeoxyribonucleotide (oligo) duplexes containing enzyme-specific recognition sequences to cut the target DNAs much more efficiently and in most cases even to completion. These enzymes are class-II and class-IIS Enases isolated from different bacterial species and possess a varying number of specific sites in the refractory DNA substrates. For DNA analysis and large-scale preparation of certain restriction fragments where complete digestions are essential we recommend taking into account the fact that various ENases can be activated by specific oligo duplexes to drive restriction digestions to completion.

Base Sequence↗
PubMed1975

Recombination between endogenous and exogenous RNA tumor virus genes as analyzed by nucleic acid hybridization.

Certain chicken cells that do not spontaneously release virus particles have been shown to produce a subgroup E avian RNA tumor virus, Rous-associated virus 60 (RAV-60), after infection with viruses of other subgroups. The nucleic acids of RAV-60 were analyzed for sequence homologies with the viral nucleic acids contained in the uninfected cell and with those of RAV-2, the exogenous virus used for the preparation of this particular RAV-60 isolate. In addition, these nucleic acids were compared with those of RAV-0, an endogenous virus spontaneously released from line 100 chicken cells. RAV-60 appears to be intermediate between RAV-0 and RAV-2 in its genetic composition, based on the pattern of hybridization obtained with the nucleic acids of these viruses and on the melting profiles of the various hybrid combinations. Of the three viruses tested, RAV-0 appears to have the greatest sequence homology with the viral nucleic acids of the uninfected cell. Hybridization between RAV-60 3-H-labeled complementary DNA and either DNA or RNA from the uninfected cell indicates that RAV-60 contains some nucleic acid sequences which are not present in the cell. In addition, some RAV-60 sequences which hybridize with the cell nucleic acid contain significant amounts of mismatching, as indicated by the lower thermal stability of these hybrid duplexes. Hybrid formation between these partially homologous sequences was excluded under stringent annealing conditions. The data indicate that RAV-60 is a recombinant between exogenous and endogenous viral genes.

Animals↗
PubMed2003

Pausing of DNA polymerases on duplex DNA templates due to ligand binding in vitro.

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ø29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A(3) (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.

Base Sequence↗
PubMed2007

High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.

Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted.

Animals↗
PubMed1988

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity.

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.

Antigens, Polyomavirus Transforming↗
PubMed1998

DNA-mediated electron transfer from a modified base to ethidium: pi-stacking as modulator of reactivity.

BACKGROUND: The DNA double helix is composed of an array of aromatic heterocyclic base pairs and, as a molecular pi-stack, represents a novel system for studying long-range electron transfer. Because many base damage and repair processes result from electron-transfer reactions, the ability of DNA to mediate charge transport holds important biological implications. Seemingly contradictory conclusions have been drawn about electron transfer in DNA from the many different studies that have been carried out. These studies must be reconciled so that this phenomenon can be understood both at a fundamental level and in the context of biological systems. RESULTS: The photoinduced oxidation of a modified base, 7-deazaguanine, has been examined as a function of distance, sequence, and base stacking in DNA duplexes covalently modified with ethidium. Over ethidium/deazaguanine separations of 6-27 A, the photooxidation reaction proceeded on a subnanosecond time scale, and the quenching yield exhibited a shallow distance dependence. The efficiency of the reaction was highly sensitive to small changes in base composition. Moreover, the overall distance-dependence of the reaction is sensitive to sequence, despite the constancy of photoexcited ethidium as acceptor. CONCLUSIONS: The remarkable efficiency of deazaguanine photooxidation by intercalated ethidium over long distances provides new evidence for fast electron-transfer pathways through DNA. By varying sequence as well as reactant separation, this work provides the first experimental demonstration of the importance of reactant stacking in the modulation of long-range DNA mediated electron transfer.

DNA↗
PubMed1995

Single strand targeted triplex formation: strand displacement of duplex DNA by foldback triplex-forming oligonucleotides.

Linear oligonucleotides that bind to single-stranded nucleic acid targets by formation of both Watson-Crick (duplex) and Hoogsteen (triplex) hydrogen bonds simultaneously (foldback triplex-forming oligonucleotides; FTFOs) were studied for their ability to disrupt duplex DNA. Recently, we reported that FTFOs interfere with quadruplex forming ability of guanine rich RNA and DNA sequences and indicated that they might also disrupt duplex structures binding to the purine target strand by foldback triplex formation (Kandimalla and Agrawal, Nucleic Acids Res. (1995) 23, 1068-1074). We now obtained evidence for strand displacement of duplex DNA by FTFOs using nuclease assays and thermal melting studies. UV melting studies revealed that complementary strands of 16 to 31 bases long were completely displaced. Results of DNase I assays showed that the FTFOs bound to purine site by strand displacement probably by preassociating with the duplex DNA in the major groove via Hoogsteen hydrogen bonding and subsequently displacing the complementary strand. Experiments with S1 nuclease, an enzyme specific for single-stranded nucleic acids, confirmed the strand displacement ability of the FTFOs.

Base Sequence↗
PubMedDate not supplied

Optical pretargeting of tumor with fluorescent MORF oligomers.

OBJECTIVE: Pretargeting with radioactivity has significantly improved tumor to normal tissue radioactivity ratios over conventional antibody imaging in both animal studies and clinical trials. This laboratory is investigating DNA analogues such as phosphorodiamidate morpholinos (MORFs) for pretargeting using technetium-99m ((99m)Tc) for detection. However, the unique properties of fluorescence activation and quenching combined with oligomers with their unique properties of hybridization may be particularly useful when used together for pretargeting with optical detection. The use of linear fluorophore-conjugated oligomer duplexes have been little used in animals, and to our knowledge, have not previously been considered for pretargeting applications. METHODS: A MORF/cDNA pair was selected such that when hybridized, the fluorescence of the Cy5.5-conjugated 25 mer MORF (Cy5.5-MORF25) is inhibited with a BHQ3-conjugated 18 mer complementary DNA (BHQ3-cDNA18). The short BHQ3-cDNA18 was selected to dissociate in the presence of a long cMORF25 in the pretargeted tumor, thus releasing the inhibitor from the Cy5.5 emitter. In this manner, the Cy5.5 fluorescence will be inhibited everywhere but in the target. The dissociation was first examined in vitro by adding the duplex to the cMORF25 both in solution and immobilized on polystyrene microspheres and by surface plasmon resonance (SPR). Thereafter, biotinylated cMORF25 immobilized on streptavidin polystyrene microspheres were administered intramuscularly in one thigh of hairless SKH-1 mice as target while an identical weight of the identical microspheres but without the cMORF25 was administered in the contralateral thigh as control. The animals then received IV the Cy5.5-MORF25/BHQ3-cDNA18 duplex or equal molar dosage of single-chain Cy5.5-MORF25 and were imaged. RESULTS: The SPR studies showed that the immobilized cDNA18 rapidly captured the flowing MORF25 to provide a duplex with a slow dissociation rate constant. Furthermore, when cMORF25 was next allowed to flow over the now immobilized duplex, the cDNA18 was unable to prevent dissociation of the heteroduplex and the formation and release of the cMORF25-MORF25 homoduplex. Images of animals obtained soon after receiving the Cy5.5-MORF25 singlet showed intense whole body fluorescence obscuring the target thigh. However, only 5 minutes after receiving the Cy5.5-MORF25/BHQ3-cDNA18 duplex, the target thigh was clearly visible along with only the kidneys. CONCLUSIONS: This first study of optical pretargeting provides a proof of concept that oligomer pretargeting found to be useful with radioactivity detection is applicable with fluorescent detection as well. In addition, our results demonstrate that by using linear oligomers for optical pretargeting, chain lengths (and base sequences) may be manipulated to provide duplexes with stabilities and fluorescence inhibition optimized for pretargeting and other in vivo applications of optical imaging.

Animals↗
PubMed1992

Mode of action of the Spiroplasma CpG methylase M.SssI.

The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.

DNA (Cytosine-5-)-Methyltransferases↗