1,721,072 research outputs found
Targeting DNA with triplexes
No full textThe formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties which may be useful as antigene agents or tools in molecular biology. In these structures a third strand oligonucleotide binds in the DNA major groove, making specific contacts with substituents on the exposed faces of the base pairs. Although triplexes form with exquisite specificity their use suffers from several drawbacks. Two limitations of this approach, which are considered in this review are, firstly that conditions of low pH are necessary for formation of the C +l GC triplet, and secondly that these structures are often less stable than their duplex counterparts. This review outlines the strategies that have been employed to overcome these drawbacks. The pH problem is addressed by considering the various DNA base analogues that have been used to recognise GC base pairs in a pH independent fashion, and discusses the benefits and limitations of each analogue. Triplex stability can be increased by using novel base analogues, backbone modifications and the use of triplex-specific binding ligands
Preferred binding sites for [N-MeCys(3),N-MeCys(7)]TANDEM determined using a universal footprinting substrate
No full textWe have prepared a novel footprinting substrate which contains all 136 tetranucleotide sequences and have used this to determine the preferred binding sites for the synthetic quinoxaline antibiotic [N-MeCys3,N-MeCys7]TANDEM. We find that, although the ligand binds to all TpA steps, it binds best to the tetranucleotide sequence ATAT and shows only weak interaction with TTAA and GTAC. The best binding sites contain the sequences ATAX and XTAT. <br/
Influence of loop size on the stability of intramolecular DNA quadruplexes
No full textWe have determined the stability of intramolecular DNA quadruplexes in which the four G(3)-tracts are connected by non-nucleosidic linkers containing propanediol, octanediol or hexaethylene glycol, replacing the TTA loops in the human telomeric repeat sequence. We find that these sequences all fold to form intramolecular complexes, which are stabilized by lithium < sodium < potassium. Quadruplex stability increases in the order propanediol < hexaethylene glycol < octanediol. The shallower shape of the melting profile with propanediol linkers and its lower dependency on potassium concentration suggests that this complex contains fewer stacks of G-quartets. The sequence with octanediol linkers displays a biphasic melting profile, suggesting that it can adopt more than one stable structure. All these complexes display melting temperatures above 310 K in the presence of 10 mM lithium, without added potassium, in contrast to the telomeric repeat sequence. These complexes also fold much faster than the telomeric repeat and there is little or no hysteresis between their melting and annealing profiles. In contrast, the human telomeric repeat sequence and a complex containing two hexaethylene glycol groups in each loop, are less stable and fold more slowly. The melting and annealing profiles for the latter sequence show significant differences, even when heated at 0.2degreesC min(-1). CD spectra for the oligonucleotides containing non-nucleosidic linkers show positive maxima at 264 nm, with negative minima similar to244 nm, which are characteristic of parallel quadruplex structures. These results show that the structure and stability of intramolecular quadruplexes is profoundly influenced by the length and composition of the loops. <br/
The electrophoretic mobility of DNA three-way junctions is affected by the sequence of overhanging single-stranded ends
No full textThe folding of three- and four-way DNA junctions is often assessed by comparing the electrophoretic mobility of restriction enzyme fragments, using the long-short arm assay. We have compared the mobility of synthetic three-way junctions that contain identical branch point sequences, but different restriction sites in the arms. We show that the mobility of fragments is affected by the sequence of the overhanging ends. In general, GC-rich overhangs produce fragments with anomalous mobilities. These anomalies can be prevented by treating the cleaved junctions with mung bean endonuclease, elevating the electrophoresis temperature or using blunt cleaving restriction endonuclease
Inosine substitutions demonstrate that intramolecular DNA quadruplexes adopt different conformations in the presence of sodium and potassium
No full textWe have examined the stability of fluorescently-labelled oligonucleotides that are based on the human telomeric repeat [(GGGTTA)3GGG], in which one of the guanines in turn is substituted with inosine. We show that the relative stability of the substitutions is different in the presence of sodium and potassium. The data for potassium suggest a parallel arrangement of the strands, while the sodium form is mixed parallel and antiparallel.Graphical abstract
The relative stability of intramolecular quadruplexes containing inosine substitutions is different in sodium and potassium
Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes
No full textWe have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable
Interaction between synthetic analogues of quinoxaline antibiotics and nucleic acids: role of the disulphide cross‐bridge and d‐amino acid centres in des‐N‐tetramethyl‐triostin A
No full text1 [Ala3, Ala7] TANDEM is an analogue of des‐N‐tetramethyl‐triostin A (TANDEM) in which both l‐Cys residues of the octapeptide ring are replaced by l‐Ala; accordingly it lacks the disulphide cross‐bridge which limits the conformational flexibility of TANDEM.2 In [l‐Ser1] TANDEM the configuration of one of the serine residues is inverted, altering the disposition of one of the quinoxaline chromophores with respect to the peptide ring.3 Both compounds interact weakly but detectably with natural DNAs as judged by spectral shifts and increases in the thermal denaturation (‘melting’) temperature Tm. They also raise the Tm of poly rA.poly rU.4 Binding isotherms determined by solvent partition analysis with [Ala3, Ala7] TANDEM yield association constants of about 103 m−1 for its interaction with natural DNAs. A Scatchard plot for binding to poly(dA‐dT) determined by solvent partition and spectrophotometric methods shows marked evidence of cooperativity with an intrinsic association constant 1.9 times 104 m−1, 8.7 nucleotides per binding site, and cooperativity parameter 15.5 Binding of [Ala3, Ala7] TANDEM to short rod‐like fragments of poly(dA‐dT) increases their contour length by almost the theoretical amount expected for an ideal process of bifunctional intercalation.6 No effect of either compound on the winding of the DNA helix could be detected in sedimentation experiments with closed circular duplex PM2 DNA.7 It is concluded that the cross‐bridge of TANDEM greatly stabilizes its binding to DNA, most probably via entropic factors, but is not the only structural feature that influences its AT sequence‐selectivity. The consequences of epimerising one of the d‐Ser residues appear as disastrous as epimerising both.8 The experimental details for the synthesis of [Ala3, Ala7] TANDEM and [l‐Ser1] TANDEM are given in an appendix to this paper. 1980 British Pharmacological Society</p
Sequence selective binding of bis-daunorubicin WP631 to DNA
No full textWe have used footprinting techniques on a wide range of natural and synthetic footprinting substrates to examine the sequence-selective interaction of the bis-daunorubicin antibiotic WP631 with DNA. The ligand produces clear DNase I footprints that are very different from those seen with other anthracycline antibiotics such as daunorubicin and nogalamycin. Footprints are found in a diverse range of sequences, many of which are rich in GT (AC) or GA (TC) residues. As expected, the ligand binds well to the sequences CGTACG and CGATCG, but clear footprints are also found at hexanucleotide sequences such GCATGC and GCTAGC. The various footprints do not contain any particular unique di-, tri- or tetranucleotide sequences, but are frequently contain the sequence (G/C)(A/T)(A/T)(G/C). All sequences with this composition are protected by the ligand, though it can also bind to some sites that differ from this consensus by one base pair
Cleavage of fragments containing DNA mismatches by enzymic and chemical probes
No full textWe prepared synthetic 50-mer DNA duplexes, each containing four mismatched base-pairs in similar positions. We examined their cleavage by DNases I and II, micrococcal nuclease (MNase), methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] and hydroxyl radicals. We find that single mismatches only produce subtle changes in the DNase I-cleavage pattern, the most common of which is attenuated cleavage at locations 2–3 bases on the 3´-side of the mismatch. Subtle changes are also observed in most of the DNase II-cleavage patterns, although GT and GG inhibit the cleavage over longer regions and generate patterns that resemble footprints. MNase cleaves the heteroduplexes at the mismatches themselves (except for CC), and in some cases cleaves CpG and CpC steps. None of the mismatches causes any change in the cleavage patterns produced by hydroxyl radicals or MPE-Fe(II). We also examined the cleavage patterns of fragments containing tandem GA mismatches in the sequences RGAY/RGAY and YGAR/YGAR (R, purine; Y, pyrimidine). RGAY causes only subtle changes in the cleavage patterns, which are similar to those seen with single mismatches, except that there are no changes in MNase cleavage. However, YGAR inhibits DNases I and II cleavage over 4–6 bases, and attenuates MPE-Fe(II) and hydroxyl radical cleavage at 2 bases. These changes suggest that this mismatch has a more pronounced effect on the local DNA structure. These changes are discussed in terms of the structural and dynamic effects of each mismatch.<br/
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