Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs.

Buoyant density of DNA in CsCl gradients with Hoechst 33258 (bisbenzimide) was investigated as a function of guanine plus cytosine content of the DNA (%GC; in mole percent). A formula for calculating %GC from the refractive index (nD) of the isopycnic CsCl/Hoechst 33258 solution over the range of 0-75 %GC was established: %GC = 351762.28 X nD - 123778.66 X nD2 - 249789.47 (the coefficients must not be rounded off). The shape of this curve indicates that under these conditions, in contrast to dilute buffers, Hoechst 33258 binds to single AT base pairs on DNA. Resolution of DNA bands in CsCl/Hoechst 33258 gradients is 1.6 to 2.1 times better than comparative CsCl gradients without the dye. Potential application to %GC determination is discussed.     

Massive parallel analysis of DNA-Hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide microchip.

A generic oligodeoxyribonucleotide microchip was used to determine the sequence specificity of Hoechst 33258 binding to double-stranded DNA. The generic microchip contained 4096 oxctadeoxynucleo-tides in which all possible 4(6)= 4096 hexadeoxy-nucleotide sequences are flanked on both the 3'- and 5'-ends with equimolar mixtures of four bases. The microchip was manufactured by chemical immobilization of presynthesized 8mers within polyacrylamide gel pads. A selected set of immobilized 8mers was converted to double-stranded form by hybridization with a mixture of fluorescently labeled complementary 8mers. Massive parallel measurements of melting curves were carried out for the majority of 2080 6mer duplexes, in both the absence and presence of the Hoechst dye. The sequence-specific affinity for Hoechst 33258 was calculated as the increase in melting temperature caused by ligand binding. The dye exhibited specificity for A:T but not G:C base pairs. The affinity is low for two A:T base pairs, increases significantly for three, and reaches a plateau for four A:T base pairs. The relative ligand affinity for all trinucleotide and tetranucleotide sequences (A/T)(3)and (A/T)(4)was estimated. The free energy of dye binding to several duplexes was calculated from the equilibrium melting curves of the duplexes formed on the oligonucleotide microchips. This method can be used as a general approach for massive screening of the sequence specificity of DNA-binding compounds.     

Specific Recognition and Stabilization of an Abasic Site-Containing DNA Duplex by a Macrocyclic Bisacridine

Abstract

A strong and specific stabilization of a DNA undecamer containing an analog of the abasic site has been induced by the macrocyclic Bisacridine 1 . ¹H NMR analysis and molecular modeling of the structure of the complex showed that the drug was specifically docked into the apurinic pocket.     

Stabilization of DNA Triple Helices Using Conjugates of Oligonucleotides and Synthetic Ligands

The possibility is discussed of stabilizing a DNA triple helix by covalent conjugation to the third strand (through its terminal phosphate) of ligands that have affinity to double and triple helices. Two types of stabilizers are considered: minor groove binders based on oligopyrroles, and triplex-specific intercalators. As a target, a synthetic 29-mer duplex containing a natural polypurine sequence of the human immunodeficiency provirus was employed. The stabilization with minor groove binders requires several conditions to be respected: a sufficiently long linker capable of reaching the minor groove from the major groove, a specific double-stranded structure of the oligopyrrole fragment, and its in-phase fitness to the target sequence. The best stabilizers of a triplex were novel conjugates in which two parallel molecules containing six pyrrole units each are linked to the same 5"-phosphate of a 16-mer triplex-forming oligonucleotide. The stabilizing properties of these derivatives were comparable to those of benzoindoloquinoline (BIQ) intercalators attached to the terminal phosphate of triple-helix forming oligonucleotides.     

DNA Base Pair Binding Specificity of CC-1065

Abstract

Oligomer duplexes were prepared by a solid-phase phosphoramidite triester coupling approach in order to study the DNA base pair binding specificity of the antitumor antibiotic CC-1065 by CD spectroscopy.     

Recognition of B-DNA by neomycin--Hoechst 33258 conjugates

Recent developments have indicated that aminoglycoside binding is limited not to RNA but to nucleic acids that, like RNA, adopt conformations similar to the A-form. We have further sought to expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Described herein are novel neomycin-Hoechst 33258 conjugates developed for exploring B-DNA groove recognition. We have varied the two reported conjugates in linker length and composition in an effort to improve our understanding of the spatial differences that define B-DNA binding. Spectroscopic studies such as ultraviolet (UV) melting, isothermal fluorescence titrations, differential scanning calorimetry (DSC), and circular dichroism (CD) together illustrate the mode of binding by such conjugates. Both conjugates exhibit enhanced thermal stabilization of A.T rich duplexes when compared to Hoechst 33258.     

Theoretical ab Initio Study of the Effects of Methylation on Structure and Stability of G:C Watson-Crick Base Pair

Abstract

Methylation of DNA occurs most readily at N(3), N(7), and O(6) of purine bases and N(3) and O(2) of pyrimidines. Methylated bases are continuously formed through endogenous and exogenous mechanisms. The results of a theoretical ab initio study on the methylation of G:C base pair components are reported. The geometries of the local minima were optimized without symmetry restrictions by the gradient procedure at DFT level of theory and were verified by energy second derivative calculations. The standard 6–31G(d) basis set was used. The single-point calculations have been performed at the MP2/6–31G(d,p), MP2/6–31₊₊G(d,p), and MP2/6–311₊₊G(2d,2p) levels of theory. The geometrical parameters, relative stability and counterpoise corrected interaction energies are reported. Also, using a variation-perturbation energy decomposition scheme we have found the vital contributions to the total interaction energy.     

Novel Hoogsteen-like Bases for Recognition of the C-G Base Pair by DNA Triplex Formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides that bind duplex A-T or G-C base pair DNA sequences specifically at homopurine sites in the major groove as T·A-T and C⁺ ·G-C triplets. Here we report the successful modelling of novel unnatural nucleosides that recognize the C-G DNA base pair by Hoogsteen-like major groove interaction. These novel Hoogsteen nucleotides are examined within model A-type and B-type conformation triplex structures since the DNA triplex can be considered to incorporate A-type and/or B-type configurational properties. Using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration, a triplet comprised of a C-G base pair and the novel Hoogsteen nucleotide, Y2, replaces the central T·A-T triplet in the triplex. The presence of any structural or energetic perturbations due to the central triplet in the energy-minimized triplex is assessed with respect to the unmodified energy minimized (T·A-T)₁₁ starting structures. Incorporation of this novel triplet into both A-type and B-type natural triplex structures provokes minimal change in the configuration of the central and adjacent triplets.     

Molecular recognition of B-DNA by Hoechst 33258.

The binding sites of Hoechst 33258, netropsin and distamycin on three DNA restriction fragments from plasmid pBR322 were compared by footprinting with methidiumpropyl-EDTA X Fe(II) [MPE X Fe(II)]. Hoechst, netropsin and distamycin share common binding sites that are five +/- one bp in size and rich in A X T DNA base pairs. The five base pair protection patterns for Hoechst may result from a central three base pair recognition site bound by two bisbenzimidazole NHs forming a bridge on the floor of the minor groove between adjacent adenine N3 and thymine O2 atoms on opposite helix strands. Hydrophobic interaction of the flanking phenol and N-methylpiperazine rings would afford a steric blockade of one additional base pair on each side.     

Effects of Hoechst 33258 on human leukocytes in vitro.

The benzimidazole derivative Hoechst 33258 was added at various concentrations to human leukocyte cultures. After 16 or 24 h of treatment, with concentrations equal to or greater than 100 mug/ml of Hoechst 33258, a number of chromosomes showed regions in which the chromatin was undercontracted. The centromeric regions of chromosome 1 and, more rarely, of chromosomes 3 and 9 appeared to be decondensed. Short decondensed regions were also present on the long arms of chromosomes 1 and 2. The possible nature of these regions is discussed.     

In Search of a Hoogsteen Base Paired DNA Duplex in Aqueous Solution

Abstract

When the oligodeoxynucleotides d(A)₆ and d(T)₆ are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC ? H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)₆. We show that (i) addition of extra d(A)₆ does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix- drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, ³¹P NMR spectra, ¹H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded.

Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

Exploring a DNA Sequence for the Three-Dimensional Structure Determination of a Silver(I)-Mediated C-C Base Pair in a DNA Duplex By 1H NMR Spectroscopy

Recently, we discovered novel silver(I)-mediated cytosine–cytosine base pair (C–Ag^I–C) in DNA duplexes. To understand the properties of these base pairs, we searched for a DNA sequence that can be used in NMR structure determination. After extensive sequence optimizations, a non-symmetric 15-base-paired DNA duplex with a single C–Ag^I–C base pair flanked by 14 A–T base pairs was selected. In spite of its challenging length for NMR measurements (30 independent residues) with small sequence variation, we could assign most non-exchangeable protons (254 out of 270) and imino protons for structure determination.     

NMR Spectroscopic Study of a DNA Duplex with Mercury-Mediated T-T Base Pairs

Recently, we reported that T-T mismatches can specifically recognize Hg ^II (T-Hg ^II -T pair formation). In order to understand the properties of the T-Hg ^II -T pair, we recorded NMR spectra for a DNA duplex, d(CGCG TT GTCC) ? d(GGAC TT CGCG), with two successive T-T mismatches (Hg ^II -binding sites). We assigned ¹ H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg ^II by using 1D ¹ H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.     

Theoretical study of molecular recognition by Hoechst 33258 derivatives

The factors responsible for the binding of Hoechst 33258 with DNA residues have been investigated in this work using the AM1 method. First and foremost, it is found that, although all crystal structure determinations indicate a preference for binding at AT rich sites, the hydrogen bond strength is actually greater for complexes with cytosine and guanine. From this, it has been inferred that other factors such as electrostatic, van der Waals interactions and nonbonded contacts with the walls of the minor groove have a strong role to play in the binding process. The hydrogen bond is found to be stronger for complexation with the thymine O2 than with the adenine N3, in line with experimental observations. Combined QM/MM studies on the drug complexed with the Dickerson-Drew dodecamer reveal that binding induces structural changes in both the ligand as well as DNA. Electron donating substituents at the para position in the phenyl ring of Hoechst 33258 lead to stronger binding with DNA. A correlation with the octanol/water partition coefficients points to the importance of hydrophobic and electrostatic interactions.     

额外的错配碱基提高T4 DNA连接酶等位特异性连接

连接是一种主要的DNA处理过程。由于较低的商业成本以及核酸底物识别的灵活性,T4 DNA连接酶被广泛应用于生物分子工程,特别是特定核酸序列的等位特异性连接检测。本文评估了在T4 DNA连接酶介导的连接反应中,引入额外的错配碱基对所产生的影响。设计了超过150组DNA/DNA或DNA/RNA带有的额外错配碱基对的组合。结果发现,引入额外的错配碱基对后,T4 DNA 连接酶在DNA/DNA连接中特异性可提高60倍以上,而在DNA/RNA连接中特异性只能提高2倍。在等位特异性连接中,有的错配碱基对可使T4 DNA连接酶的特异性提高600多倍。     

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.     

Theoretical Design of Novel, 4 Base Pair Selective Derivatives of Mitoxantrone

Abstract

Mitoxantrone (MTX) is a recently synthesized antitumor intercalative molecule, currently in use in chemotherapy. Previous theoretical computations showed that the base pair selectivity of MTX is limited to the sole two base-pair sequence making up the intercalation site. In order to further extend the recognition site, we undertook, by means of theoretical computations, the design of novel MTX derivatives, in which the terminal hydroxyl group of each side chain is esterified with oligopeptides.

We compare in the present study the binding affinities of two derivatives, depsiGly-Lys(D) and depsiGly-Gly-Orn(L), for the palindromic sequences d(CCCGGG)₂, d(GCCGGC)₂, d(GGCGCC)₂, and d(CGCGCG)₂.

Major groove binding of the oligopeptide arms was shown to be significantly more favourable than either minor groove binding, or binding to the sole phosphate groups. With the two arms adopting two antiparallel directions, two distinct arrangements were investigated in the major groove: (a) the two oligopeptides are brought closer together by means of two hydrogen bonds involving the backbone of their second residue in a β-sheet like arrangement; (b) the two arms are remote from each other so as to reduce their mutual electrostatic repulsion. Whatever the disposition, the optimal binding configurations were invariably found to be those in which the cationic side chains of the terminal residues chelate N7/06 of two successive guanines, whenever present on a given strand. A distinct energetical preference for arrangement (a) was obtained with the depsiGly-Gly-Orn(L) derivative. Replacement of the central Gly residue by a Cys one, as in the sequence depsiGly-Cys-Orn(L), was proposed subsequently, so as to further stabilize such a β-sheet arrangement by means of a disulfide bridge between the two Cys residues.

The two investigated compounds were shown to preferentially bind sequences d(CCCGGG)₂ and d(GCCGGC)₂, with a tetrameric core CCGG rather than sequences d(GGCGCC)₂ and d(CGCGCG)₂, with a tetrameric core GCGC.     

Cooperative Non-enzymatic Base Recognition and the Stability of the G-U Wobble Pair

INVESTIGATIONS of paired complementary polynucleotides containing occasional mismatched bases have indicated that these bases are looped out of the double helical regions thereby making the helix unstable. But in a system containing mismatched G and U bases, no such definite conclusion could be drawn^1–4—stoichiometry and T_m values of such complexes can be interpreted in terms of the formation of a G-U wobble pair⁴. Recently the complexes formed by self-complementary block oligomers interrupted by mismatched bases, for example, A_nXU_n (X=G, C and U), have been studied in detail⁵·⁶. It seems that stacking interactions play a much more dominant role in the stabilization of double helices than was previously thought. Thus the extent of looping out and of non-Watson-Crick base pairing can hardly be assessed independently.     

Some Features of Base Pair Mismatch and Heterology Repair in Escherichia coli

We have used artificially constructed heteroallelic heteroduplex molecules of bacteriophage lambda DNA to transfect Escherichia coli, and E. coli mutants deficient in various functions involved in the adenine methylation-directed mismatch repair system, MutL, MutS, MutH, and UvrD (MutU). Analysis of the allele content of single infective centers shows that this repair system often acts on several mismatches, separated by as many as 2000 bp, on one of the strands of a heteroduplex molecule. When the methyl-directed mismatch repair system is disabled by mutH or uvrD mutations, localized mismatch repair becomes prominent. This prominent localized repair that can result in separation of very closely linked markers requires the functions MutL and MutS, is independent of adenine methylation, and appears to reflect another mechanism of mismatch repair. Heterology-containing heteroduplex molecules with a deletion in one strand often escape processing. However, when the heterology includes the stem and loop structure of a transposon, Tn10, the transposon is lost.