Groove Binding Ligands for the Interaction with Parallel-Stranded ps-Duplex DNA and Triplex DNA

DNA adopts different conformations not only based on novel base pairs, but also with different chain polarities. Besides several duplex structures (A, B, Z, parallel stranded (ps)-DNA, etc.), DNA also forms higher-order structures like triplex, tetraplex, and i-motif. Each of these structures has its own biological significance. The ps-duplexes have been found to be resistant to certain nucleases and endonucleases. Molecules that promote triple-helix formation have significant potential. These investigations have many therapeutic advantages which may be useful in the regulation of the expression of genes responsible for certain diseases by locking either their transcription (antigene) or translation (antisense). Each DNA minor groove binding ligand (MGBL) interacts with DNA through helical minor groove recognition in a sequence-specific manner, and this interferes with several DNA-associated processes. Incidentally, these ligands interact with some non-B-DNA and with higher-order DNA structures including ps-DNA and triplexes. While the design and recognition of minor grooves of duplex DNA by specific MGBLs have been a topic of many reports, limited information is available on the binding behavior of MGBLs with nonduplex DNA. In this review, we summarize various attempts of the interaction of MGBLs with ps-DNA and DNA triplexes.

Comparative studies on parallel and antiparallel duplex and triplex DNA

Parallel strand models for base sequences d(A)(10). d(T)(10), d(AT)(5) . d(TA)(5), d(G(5)C(5)). d(C(5)G(5)), d(GC)(5) . d(CG)(5) and d(CTATAGGGAT). d(GATATCCCTA), where reverse Watson-Crick A-T pairing with two H-bonds and reverse Watson-Crick G-C pairing with one H-bond or with two H-bonds were adopted, and three models of d(T)(14). d(A)(14). d(T)(14) triple helix with different strand orientations were built up by molecular architecture and energy minimization. Comparisons of parallel duplex models with their corresponding B-DNA models and comparisons among the three triple helices showed: (i) conformational energies of parallel AT duplex models were a little lower, while for GC duplex models they were about 8% higher than that of their corresponding B-DNA models; (ii) the energy differences between parallel and B-type duplex models and among the three triple helices arose mainly from base stacking energies, especially for GC base pairing; (iii) the parallel duplexes with one H-bond G-C pairs were less stable than those with two H-bonds G-C pairs. The present paper includes a brief discussion about the effect of base stacking and base sequences on DNA conformations. (C) 1997 Academic Press Limited.

Theoretical studies on conformation comparison of braid-like and triplex DNA

Based on the experimental data of scanning tunneling microscopy (STM), models of three-stranded braid-like DNAs composed by three kinds of base triplets AAA, TAT and GCA were constructed. We investigated the braid-like DNAs and their comparative tripler DNAs using a molecular mechanics method. The three strands of braid-like DNAs are proven equivalent, while those of tripler DNAs are not. The conformational energies for braid-like DNAs were found to be higher than that for tripler DNAs. Each period in one strand of braid-like DNA has 18 nucleotides, half of which are right-handed, while the other half are left-handed. Additional discussions concerning sugar puckering modes and the H-bonds are also included. (C) 1999 Elsevier Science B.V. All rights reserved.

Stability and molecular modeling of triplex DNA inhibiting DNA binding protein binding to the core promoter of hepatitis B virus.

Two three-dimensional structure models of the 21nt oligodeoxyribonucleotides, CPI (G3TG-2TGT2G5TG2TGT) and CP3 (TGTG2TGST2GTG2TG3), were constructed by InsightII (MSI) software in IRIS Indigo2 (SGI) workstation using the crystal structure of TAT tripler formation as the template. The initial structures subsequently were minimized by molecular mechanics. The final structures were believed as the dominant conformation. The results showed that the energy of CP1 is lower than that of CP3, and the former is more stable than the latter. Moreover, the results further proved that the 21nt oligodeoxyribo-nucleotide CP1 stably combines with the core promoter (Cp) fragment of hepatitis B virus (HBV) to form a tripler DNA, and CP1 specifically inhibits a specific cellular factor (DNA binding protein) binding to Cp fragment. These results indicated that specific repression of gene transcription of HBV DNA might be possible by tripler-formation DNA.

Interactions of mitoxantrone with duplex and triplex DNA studied by electrospray ionization mass spectrometry

We have examined interactions between mitoxantrone (MXT) and DNA duplexes or triplexes with different base compositions by using electrospray ionization mass spectrometry (ESI-MS), respectively. MXT interacts preferentially with DNA duplexes compared to the triplexes. In the mass spectrum of the duplex-MXT mixture, the complex peaks dominated in the ratios of duplex/MXT of 1:1, 1:2 and 1:3, and the 1:2 duplex/MXT peak was the most abundant. In contrast, only 1:1 triplex-MXT complexes were observed in the mass spectrum of the triplex-MXT mixture, and the most intensive peak was a free triplex ion without MXT.

Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B-A transition in solution

Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nano-device applications ranging from gene therapy and novel drug delivery to membrane separations. The miniaturization of DNA-nanotube devices for biological applications requires fully understanding DNA-nanotube interaction mechanism. We report here, for the first time, that DNA destabilization and conformational transition induced by SWNTs are sequence-dependent. Contrasting changes for SWNTs binding to poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were observed. For GC homopolymer, DNA melting temperature was decreased 40 degrees C by SWNTs but no change for AT-DNA. SWNTs can induce B-A transition for GC-DNA but AT-DNA resisted the transition. Our circular dichroism, competitive binding assay and triplex destabilization studies provide direct evidence that SWNTs induce DNA B-A transition in solution and they bind to the DNA major groove with GC preference.

Sequence specificity of triplex DNA formation: Analysis by a combinatorial approach, restriction endonuclease protection selection and amplification.

We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.

溴代胞嘧啶对三螺旋DNA 稳定性的影响

研究了三螺旋DNA 中胞嘧啶被5 - 溴胞嘧啶取代后的稳定性问题。通过建立CGC , CGBrC , BrCGC 和BrCGBrC 4 种模型, 并采用Insight Ⅱ软件包中的Discover 程序进行计算模拟, 发现用5 - 溴胞嘧啶取代三 螺旋DNA 分子中的胞嘧啶后, 整个分子的稳定性有所降低。同时, 当用5 - 溴胞嘧啶取代三螺旋DNA 不 同链上的胞嘧啶时, 被取代链的碱基堆积能显著增高, 导致分子链内稳定性降低, 但被取代链和相邻链间 相互作用能有所降低, 使得链间作用趋向稳定。

Triplex selective 2-(2-naphthyl)quinoline compounds: Origins of affinity and new design principles

A novel competition dialysis assay was used to investigate the structural selectivity of a series of substituted 2-(2-naphthyl)quinoline compounds designed to target triplex DNA. The interaction of 14 compounds with 13 different nucleic acid sequences and structures was studied. A striking selectivity for the triplex structure poly dA:[poly dT](2) was found for the majority of compounds studied. Quantitative analysis of the competition dialysis binding data using newly developed metrics revealed that these compounds are among the most selective triplex-binding agents synthesized to date. A quantitative structure-affinity relationship (QSAR) was derived using triplex binding data for all 14 compounds used in these studies. The QSAR revealed that the primary favorable determinant of triplex binding free energy is the solvent accessible surface area. Triplex binding affinity is negatively correlated with compound electron affinity and the number of hydrogen bond donors. The QSAR provides guidelines for the design of improved triplex-binding agents.

A molecular anchor for stabilizing triple-helical DNA.

Molecular modeling has been used to predict that 2,6-disubstituted amidoanthraquinones, and not the 1,4 series, should preferentially interact with and stabilize triple-stranded DNA structures over duplex DNA. This is due to marked differences in the nature of chromophore-base stacking and groove accessibility for the two series. A DNA foot-printing method that monitors the extent of protection from DNase I cleavage on triplex formation has been used to examine the effects of a number of synthetic isomer compounds in the 1,4 and 2,6 series. The experimental results are in accord with the predicted behavior and confirm that the 1,4 series bind preferentially to double- rather than triple-stranded DNA, whereas the isomeric 2,6 derivatives markedly favor binding to triplex DNA.

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged Lacl protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

米托蒽醌与双链及三链核酸相互作用的电喷雾质谱研究

The interaction of mitoxantrone (MXT) with duplex and triplex DNA, contain repeating sequence CTCT, CCTT and CTT were studied by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:3 specific complexes of mitoxantrone and duplex DNA and 1:2 specific complexes of mitoxantrone and triplex DNA were observed. The results show that mitoxantrone has no remarkable sequence selectivity, however it has distinct structure selectivity, and destabilization the triplex.

Preclinical development of cancer vaccines

Triplex cell vaccine is a cancer immunopreventive cell vaccine that can prevent almost completely mammary tumor onset in HER-2/neu transgenic mice. A future translation of cancer immunoprevention from preclinical to clinical studies should take into account several aspects. The work reported in this thesis deals with the study of three of these aspects: vaccine schedule, activity in a therapeutic set-up and second-generation DNA vaccines. An important element in determining human acceptance and compliance of a treatment protocol is the number of vaccinations. In order to improve the vaccination schedule a minimal protocol was searched, i.e. a schedule consisting of a lower number of administrations than standard protocol but with a similar efficacy. A candidate optimal protocol was identified by the use of an in silico model, SimTriplex simulator. The in vivo test of this schedule in HER-2/neu transgenic mice only partially confirmed in silico predictions. This result shows that in silico models have the potential ability to aid in searching of optimal treatment protocols, provided that they will be further tuned on experimental data. As a further result this preclinical study highlighted that kinetic of antibody response plays a major role in determining cancer prevention, leading to the hypothesis of a threshold that must be reached rapidly and maintained lifetime. Early clinical trials would be performed in a therapeutic, rather than preventive, setting. Thus, the activity of Triplex vaccine was investigated against experimental lung metastases in HER-2/neu transgenic mice in order to evaluate if the immunopreventive Triplex vaccine could be effective also against a pre-existing tumor mass. This preclinical model of aggressive metastatic development showed that the vaccine was an efficient treatment also 4 for the cure of micrometastases. However the immune mechanisms activated against tumor mass were not antibody dependent, i.e. different from those preventing the onset of primary mammary carcinoma. DNA vaccines could be more easily used than cellular ones. A second generation of Triplex vaccine based on DNA plasmids was evaluated in an aggressive preclinical model (BALBp53neu female mice) and compared with the preventive ability of cellular Triplex vaccine. It was observed that Triplex DNA vaccine was as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Molecular Dynamics Simulations of the d(TAT) Triple Helix

Molecular dynamics (MD) simulations have been used to study the dynamical and time-averaged characteristics of the DNA triple helix d(T)10âd(A)10âd(T)10. The structures sampled during the trajectory resemble closely the B-type model for the DNA triplex proposed on the basis of NMR data, although there are some subtle differences. Alternative P- and A-type conformations for the triplex, suggested from X-ray experiments, are not predicted to contribute significantly to the structure of the DNA triplex in solution. Comparison with the best available experimental data supports the correctnes of the MD-generated structures. The analysis of the collected data gives a detailed picture of the characteristics of triple-helix DNA. A new and interesting pattern of hydration, specific for triplex DNA, is an important observation. The results suggest that molecular dynamics can be useful for the study of novel nucleic acid structures.