Search PubMed⌕ Search

Biomedical subjects

J Feigon

Publications and source records attributed to J Feigon.

At least 73 records · Page 4Linked to original sources
PubMed1993

Two- and three-dimensional HCN experiments for correlating base and sugar resonances in 15N,13C-labeled RNA oligonucleotides.

New 2D and 3D 1H-13C-15N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in 13C- and 15N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective 13C-refocusing and 15N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D 1H-15N and 3D 1H-13C-15N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra- and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.

Base Sequence↗
PubMed1992

Proton nuclear magnetic resonance assignments and structural characterization of an intramolecular DNA triplex.

Two-dimensional 1H n.m.r. spectroscopy has been used to study the 31-base DNA oligonucleotide 5'-dAGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3', which folds to form a stable intramolecular triplex in solution at acidic pH. This structure is considerably more difficult to assign than short B-DNA duplexes and requires new assignment methods. The assignment strategy and assignments of almost all of the exchangeable and nonexchangeable resonances are presented. Seven base triplets and one Watson-Crick base-pair form the core of the structure and are connected by a four C and four T loop at either end. The second pyrimidine "strand" (bases 24 to 31) in this intramolecular pyrimidine-purine-pyrimidine triplex binds via Hoogsteen base-pairs in the major groove and is parallel to the purine "strand" (bases 1 to 8). Analysis of the sugar puckers reveals that, contrary to widely accepted belief, the triplex sugars are not predominantly in the N-type (close to C3'-endo) conformation. Except for some of the C nucleotides, all sugars are predominantly S-type (close to C2'-endo). Thus, the duplex DNA does not assume N-type sugar conformations to accommodate a third strand in the major groove. A preliminary model of the triplex structure is presented.

Base Sequence↗
PubMed1992

Structure of a G.T.A triplet in an intramolecular DNA triplex.

A 32-base DNA oligonucleotide has been studied by one- and two-dimensional 1H NMR spectroscopy and is shown to form a stable, pyr.pur.pyr, intramolecular triple helical structure, with a four C loop and a TATA loop connecting the Watson-Crick- and Hoogsteen-paired strands, respectively. This triplex contains five T.A.T base triplets, two C+.G.C base triplets, and an unusual G.T.A base triplet which disrupts the pyr.pur.pyr motif. The G.T.A triplet consists of a Watson-Crick T.A base pair, with the T situated in the "purine strand" and the A situated in the "pyrimidine strand" and a G situated in the Hoogsteen-base-paired "pyrimidine strand" hydrogen bonded to the T. The base-pairing structure of the G.T.A triplet has been investigated and has been found to involve a single hydrogen bond from the guanine amino group to the O4 carbonyl of the thymine, leaving the guanine imino proton free. The specific amino proton involved in the hydrogen bond is the H2(2) proton. This orients the guanine such that its sugar is near the thymine methyl group. The guanine sugar adopts an N-type (C3'-endo) sugar pucker in this triplet. The stability of the G.T.A triplet within pyr.pur.pyr triplexes is discussed.

Base Composition↗
PubMed1992

Proton NMR study of the [d(ACGTATACGT)]2-2echinomycin complex: conformational changes between echinomycin binding sites.

The interactions of echinomycin and the DNA decamer [d(ACGTATACGT)]2 were studied by proton NMR. Echinomycin binds cooperatively as a bisintercalator at the CpG steps. The terminal A.T base pairs are Hoogsteen base paired, but none of the four central A.T base pairs are Hoogsteen base paired. However, binding of the drug induces unwinding of the DNA which is propagated to the central ApT step. All four central A.T base pairs are destabilized relative to those in the free DNA. Furthermore, based on these and other results from our laboratory, we conclude that the formation of stable Hoogsteen base pairs may not be the relevant structural change in vivo. The structural changes propagated between adjacent ACGT binding sites are the unwinding of the duplex and destabilization of the base pairing between binding sites.

Amino Acid Sequence↗
PubMed1992

Quadruplex structure of Oxytricha telomeric DNA oligonucleotides.

The telomeres of most eukaryotes contain a repeating G-rich sequence with the consensus d(T/A)1-4G1-8, of which 12-16 bases form a 3' single-strand overhang beyond the telomeric duplex. It has been proposed that these G-rich oligonucleotides associate to form four-stranded structures from one, two or four individual strands and that these structures may be relevant in vivo. The proposed structures contain Hoogsteen base-paired G-quartets, precedent for which has been in the literature for many years. Here we use 1H NMR spectroscopy to study the conformations of the DNA oligonucleotides d(G4T4G4) (Oxy-1.5) and d(G4T4G4T4G4T4G4) (Oxy-3.5) which contain the Oxytricha telomere repeat (T4G4). We find that these molecules fold to form a symmetrical bimolecular and an intramolecular quadruplex, respectively. Both structures have four G-quartets formed from nucleotides that are alternately syn and anti along each strand. This arrangement differs from earlier models in which the strands are alternately all syn or all anti. The T4 loops in Oxy-1.5 are on opposite ends of the quadruplex and loop diagonally across the G-quartet, resulting in adjacent strands being alternately parallel and antiparallel.

Animals↗
PubMed1992

Proton NMR studies of [N-MeCys3,N-MeCys7]TANDEM binding to DNA oligonucleotides: sequence-specific binding at the TpA site.

[N-MeCys3,N-MeCys7]TANDEM, an undermethylated analogue of Triostin A, contains two N-methyl groups on the cysteine residues only. Footprinting results showed that [N-MeCys3,N-MeCys7]TANDEM binds strongly to DNA rich in A.T residues [Low, C. M. L., Fox, K. R., Olsen, R. K., & Waring, M. J. (1986) Nucleic Acids Res. 14, 2015-2033]. However, it was not known whether specific binding of [N-MeCys3,N-MeCys7]TANDEM requires a TpA step or an ApT step. In 1:1 saturated complexes with the octamers [d(GGATATCC)]2 and [d(GGTTAACC)]2, [N-MeCys3,N-MeCys7]TANDEM binds to each octamer as a bis-intercalator bracketing the TpA step. The octadepsipeptide ring binds in the minor groove of the DNA. Analysis of sugar coupling constants from the phase-sensitive COSY data indicates that the sugar of the thymine in the TpA binding site adopts predominantly an N-type sugar conformation, while the remaining sugars on the DNA adopt an S-type conformation, as has been observed in other Triostin A and echinomycin complexes. The drug does not bind to the octamer [d(GGAATTCC)]2 as a bis-intercalator. Only weak nonintercalative binding is observed to this DNA octamer. These results show unambiguously that [N-MeCys3,N-MeCys7]TANDEM binds sequence specifically at TpA sites in DNA. The factors underlying the sequence specificity of [N-MeCys3,N-MeCys7]TANDEM binding to DNA are discussed.

Adenine Nucleotides↗
PubMed1991

Structure and stability of X.G.C mismatches in the third strand of intramolecular triplexes.

Intramolecular DNA triplexes that contain eight base triplets formed from the folding of a single DNA strand tolerate a single X.G.C mismatch in the third strand at acidic pH. The structure and relative stability of all four triplets that are possible involving a G.C Watson-Crick base pair were determined with one- and two-dimensional proton nuclear magnetic resonance techniques. Triplexes containing A.G.C, G.G.C, or T.G.C triplets were less stable than the corresponding parent molecule containing a C.G.C triplet. However, all mismatched bases formed specific hydrogen bonds in the major groove of the double helix. The relative effect of these mismatches on the stability of the triplex differs from the effect assayed (under different conditions) by two-dimensional gel electrophoresis and DNA cleavage with oligonucleotide EDTA.Fe(II).

Base Sequence↗
PubMed1991

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2.

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.

Base Sequence↗
PubMed1990

Formation of a stable triplex from a single DNA strand.

Homopurine.homopyrimidine DNA sequences have been shown to form triple-stranded structures readily under appropriate conditions. Interest in DNA triplexes arises from potential applications of intermolecular triplexes as antisense inhibitors of gene expression and from the possibility that intramolecular triplexes may have a role in gene expression and recombination. We recently presented NMR evidence for triplex formation from the DNA oligonucleotides d(GA)4 and d(TC)4, which showed unambiguously that the second pyrimidine strand is Hoogsteen base paired and the cytosines are protonated at N3 as required. To obtain a more well defined triplex, and to provide a model for in vivo triplex structures, we have designed and synthesized a 28-base DNA oligomer with a sequence that could potentially fold to form a triplex containing both T.A.T and C+.G.C triplets. Our NMR results indicate that the conformation at pH 5.5 is an intramolecular triplex and that a significant amount of triplex remains even at pH 8.0.

Base Sequence↗
PubMed1989

Non-contiguous regions of Z-DNA in a DNA dodecamer.

The conformation of the self-complimentary DNA dodecamer d(br5CGbr5CGAATTbr5CGbr5CG) has been investigated in a variety of salt and solvent conditions by one and two-dimensional 1H NMR. In low salt aqueous solutions, the molecule forms a regular B-DNA structure similar to the unmodified dodecamer. However, in aqueous solution containing high salt concentration and methanol, the dodecamer adopts a structure in which the br5CGbr5CG ends of the molecule are in a Z-DNA like conformation and the AATT region is neither standard B-DNA nor Z-DNA. The implications of these results for the structure of junctions between B and Z-DNA and the sequence specificity of Z-DNA are discussed.

Base Sequence↗
PubMed1989

NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4.

The complexes formed by the homopurine and homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 have been investigated by one- and two-dimensional 1H NMR. Under appropriate conditions [low pH, excess d(TC)4 strand] the oligonucleotides form a triplex containing one d(GA)4 and two d(TC)4 strands. The homopurine and one of the homopyrimidine strands are Watson-Crick base paired, and the second homopyrimidine strand is Hoogsteen base paired in the major groove to the d(GA)4 strand. Hoogsteen base pairing in GC base pairs requires hemiprotonation of C; we report direct observation of the C+ imino proton in these base pairs. Both homopyrimidine strands have C3'-endo sugar conformations, but the purine strand does not. The major triplex formed appears to have four TAT and three CGC+ triplets formed by binding of the second d(TC)4 strand parallel to the d(GA)4 strand with a 3' dangling end. In addition to the triplexes formed, at least one other heterocomplex is observed under some conditions.

Base Composition↗
PubMed1989

Triple-strand formation in the homopurine:homopyrimidine DNA oligonucleotides d(G-A)4 and d(T-C)4.

Interest in triple and quadruple DNA helices built from homopurine and homopyrimidine strands has recently intensified principally because such structures may occur in vivo but also because of the potential use of triplexes both in forming highly sequence-specific complexes for use in chromosome mapping and in repressing transcription. From fibre diffraction data, models for triplex structures with poly(U).poly(A).poly(U) and poly(dT).poly(dA).poly(dT) have been proposed, in which the purine and one pyrimidine strand are Watson-Crick paired in an A' helix, and the other pyrimidine strand is Hoogsteen base-paired parallel to the purine strand along the major groove. A similar base-pairing scheme involving G and C would require protonation of C for Hoogsteen base-pair formation, and models for such triplexes have been proposed by analogy to the single-sequence fibre diffraction data. To date, however, there have been no single crystal or NMR structural data on DNA triplexes, and no direct observation of the protonated C in such a context. We present here the first NMR evidence for triplex formation in DNA from the homopurine d(G-A) and homopyrimidine d(T-C) oligonucleotides, and report direct observation of imino protons from protonated cytosines in the triplex.

Base Sequence↗
PubMed1989

Unstable Hoogsteen base pairs adjacent to echinomycin binding sites within a DNA duplex.

The bisintercalation complex present between the DNA octamer [d(ACGTACGT)]2 and the cyclic octadepsipeptide antibiotic echinomycin has been studied by one- and two-dimensional proton NMR, and the results obtained have been compared with the crystal structures of related DNA-echinomycin complexes. Two echinomycins are found to bind cooperatively to each DNA duplex at the CpG steps, with the two quinoxaline rings of each echinomycin bisintercalating between the C.G and A.T base pairs. At low temperatures, the A.T base pairs on either side of the intercalation site adopt the Hoogsteen conformation, as observed in the crystal structures. However, as the temperature is raised, the Hoogsteen base pairs in the interior of the duplex are destabilized and are observed to be exchanging between the Hoogsteen base pair and either an open or a Watson-Crick base-paired state. The terminal A.T base pairs, which are not as constrained by the helix as the internal base pairs, remain stably Hoogsteen base-paired up to at least 45 degrees C. The implications of these results for the biological role of Hoogsteen base pairs in echinomycin-DNA complexes in vivo are discussed.

Base Composition↗
PubMed1988

'Petite' mutagenesis in Saccharomyces cerevisiae by a series of 2,7-di-alkyl-substituted derivatives of proflavine with differing DNA-binding properties.

The ability of proflavine (3,6-diaminoacridine) and its 2,7-dimethyl, 2,7-diethyl, 2,7-diisopropyl and 2,7-di-tert.-butyl derivatives to induce the 'petite' mutation in Saccharomyces cerevisiae has been studied in relation to the DNA-binding properties of the compounds. The nature of the binding has been investigated by nuclear magnetic resonance techniques, and the results support and clarify earlier suggestions that the first 3 members of the series intercalate into DNA while the diisopropyl and di-tert.-butyl compounds do not. Toxicity of the drugs was primarily associated with their mode of DNA binding, but lipophilicity had an important secondary effect. It seems likely that the toxic properties of the more lipophilic DNA-intercalating members of the series mask their potential for 'petite' mutagenesis.

Acridines↗
PubMed1986

An assessment of the Z-DNA forming potential of alternating dA-dT stretches in supercoiled plasmids.

The ability of a stretch of alternating dA-dT to adopt the left-handed Z form has been assessed by examining the behavior of the sequence d(CG)6(TA)4(CG)6 contained in the plasmid pBR322. The structural transition occurring within this sequence as a function of negative superhelicity was analyzed by several methods, including (1) the supercoiling-dependent unwinding of the insert as determined by two-dimensional gel electrophoresis, (2) the binding of anti-Z-DNA antibodies to the insert, (3) the sensitivity of the sequence to a single strand specific endonuclease, and (4) the sensitivity of the insert to digestion by a restriction endonuclease that cuts within the d(CG)6 segments when in the right-handed form. These studies have shown that in negatively supercoiled DNA the two d(CG)6 portions of the insert adopt the Z form, while the central d(TA)4 segment forms an underwound structure with a helical repeat that is best approximated as being intermediate between the B form and the Z form. A statistical mechanical treatment of the unwinding of the insert as a function of negative superhelicity provides an estimate of the minimum free energy required to convert an A-T bp from the B form to the Z form, as well as the free energy associated with the conversion of an A-T bp from the B form to the unwound form. These results strongly indicate that Z DNA is an unfavored structural alternative for stretches of d(AT)n in negatively supercoiled DNA.

Base Sequence↗
PubMed1985

Z-DNA forms without an alternating purine-pyrimidine sequence in solution.

Nuclear magnetic resonance spectra (proton and phosphorus-31) and ultraviolet absorption spectra of the DNA decamer d(br5CGbr5CGATbr5CGbr5CG), in which the central two adenine-thymine base pairs are out of order with the rest of the purine-pyrimidine alternation sequence, indicate that under appropriate solvent conditions (high salt and methanol) the molecule undergoes a structural transition from a right-handed B-DNA conformation to a left-handed Z-DNA conformation. Measurements of the two-dimensional nuclear Overhauser effect on the decamer indicate that all of the guanines as well as the two equivalent thymines adopt the syn conformation.

Base Sequence↗
PubMed1985

Substituent effects on the binding of ethidium and its derivatives to natural DNA.

The binding of eight ethidium derivatives to short (approximately 35 base-pair), random sequence DNA has been investigated using 1H-NMR. At 35 degrees C, all drugs cause upfield shifts of the DNA imino proton resonances characteristic of intercalative binding to DNA, but the line shapes vary significantly with the nature of the drug. The results confirm our previous proposal that removal of the amino group at position-3, but not at position-8, on the parent ethidium shortens the lifetime of the intercalative state (less than 1-2 ms at 35 degrees C). These results suggest that hydrogen-bonding interactions with the 3-NH2 group are involved in stabilization of the drug-DNA complex or that changes in charge distribution that accompany removal of the 3-NH2 group reduce the complex stability. The magnitude of the shift of the drug-DNA spectra indicates a slight preference for binding of the drugs adjacent to G X C base-pairs.

Animals↗