Sequence-selective DNA Binding Research Articles

Targeted elimination of mutated mitochondrial DNA by a multi-functional conjugate capable of sequence-specific adenine alkylation

Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential to cure mitochondrial diseases, no chemical-based drugs in clinical trials are capable of selective modulation of mtDNA mutations. Here, we construct a class of compounds encompassing pyrrole-imidazole polyamides (PIPs), mitochondria-penetrating peptide, and chlorambucil, an adenine-specific DNA-alkylating reagent. The sequence-selective DNA binding of PIPs allows chlorambucil to alkylate mutant adenine more efficiently than other sites in mtDNA. Invitro DNA alkylation assay shows that our compound 8950A-Chb(Cl/OH) targeting a nonpathogenic point mutation in HeLa S3 cells (m.8950G>A) can specifically alkylate the mutant adenine. Furthermore, the compound reduces the mtDNA possessing the target mutation in cultured HeLa S3 cells. The programmability of PIPs to target different sequences could allow this class of compounds to be developed as designer drugs targeting pathogenic mutations associated with mitochondrial diseases in future studies.

Chemical Approaches to the Development of Artificial Transcription Factors Based on Pyrrole‐Imidazole Polyamides

To maintain the functions of living organisms, cells have developed complex gene regulatory networks. Transcription factors have a central role in spatiotemporal control of gene expression and this has motivated us to develop artificial transcription factors that mimic their function. We found that three functions could be mimicked by applying our chemical approaches: i) efficient delivery into organelles that contain target DNA, ii) specific DNA binding to the target genomic region, and iii) regulation of gene expression by interaction with other transcription coregulators. We chose pyrrole-imidazole polyamides (PIPs), sequence-selective DNA binding molecules, as DNA binding domains, and have achieved each of the required functions by introducing other functional moieties. The developed artificial transcription factors have potential as chemical tools that can be used to artificially modulate gene expression to enable cell fate control and to correct abnormal gene regulation for therapeutic purposes.

High-affinity sequence-selective DNA binding by iridium(III) polypyridyl organometallopeptides.

We demonstrate the application of solid-phase peptide synthesis methods for the straightforward assembly of polynuclear Ir(III) organometallopeptides, and show that their oligoarginine derivatives exhibit high DNA binding affinity, sequence selectivity, and high cytotoxicity towards a set of cancer cell lines.

Sequence-selective DNA binding with cell-permeable oligoguanidinium-peptide conjugates.

Conjugation of a short peptide fragment from a bZIP protein to an oligoguanidinium tail results in a DNA-binding miniprotein that selectively interacts with composite sequences containing the peptide-binding site next to an A/T-rich tract. In addition to stabilizing the complex with the target DNA, the oligoguanidinium unit also endows the conjugate with cell internalization properties.

Exploiting anthracene photodimerization within peptides: light induced sequence-selective DNA binding.

The unprecedented use of anthracene photodimerization within a protein or peptide system is explored through its incorporation into a DNA-binding peptide, derived from the GCN4 transcription factor. This study demonstrates an effective and dynamic interplay between a photoreaction and a peptide-DNA assembly, with each process able to exert control over the other.

Aromatic Interactions Modulate the 5′-Base Selectivity of the DNA-Binding Autoantibody ED-10

We present detailed computational analyses of the binding of four dinucleotides to a highly sequence-selective single-stranded DNA (ssDNA) binding antibody (ED-10) and selected point mutants. Anti-DNA antibodies are central to the pathogenesis of systemic lupus erythematosus (SLE), and a more complete understanding of the mode of binding of DNA and other ligands will be necessary to elucidate the role of anti-DNA antibodies in the kidney inflammation associated with SLE. Classical molecular mechanics based molecular dynamics simulations and density functional theory (DFT) computations were applied to pinpoint the origin of selectivity for the 5'-nucleotide. In particular, the strength of interactions between each nucleotide and the surrounding residues were computed using MMGBSA as well as DFT applied to a cluster model of the binding site. The results agree qualitatively with experimental binding free energies, and indicate that π-stacking, CH/π, NH/π, and hydrogen-bonding interactions all contribute to 5'-base selectivity in ED-10. Most importantly, the selectivity for dTdC over dAdC arises primarily from differences in the strength of π-stacking and XH/π interactions with the surrounding aromatic residues; hydrogen bonds play little role. These data suggest that a key Tyr residue, which is not present in other anti-DNA antibodies, plays a key role in the 5'-base selectivity, while we predict that the mutation of a single Trp residue can tune the selectivity for dTdC over dAdC.

Metal-Ion-Regulated Miniature DNA-Binding Proteins Based on GCN4 and Non-native Regulation Sites

The design of artificial peptide dimers containing polypyridine switching domains, for which metal-ion coordination is shown to regulate DNA binding, is reported. Short peptides, based on the basic domain of the GCN4 transcription factor (GCN4bd), dimerised with either 2,2'-bipyridine (bipy(GCN4bd)2 ) or 2,2':6',2''-terpyridine (terpy(GCN4bd)2 ) linker units, undergo a conformational rearrangement on Cu(II) and Zn(II) coordination. Depending on the linker substitution pattern, this is proposed to alter the relative alignment of the two peptide moieties, and in turn regulate DNA binding. Circular dichroism and UV-visible spectroscopy reveal that Cu(II) and Zn(II) coordination promotes binding to DNA containing the CRE target site, but to a differing and opposite degree for the two linkers, and that the metal-ion affinity for terpy(GCN4bd)2 is enhanced in the presence of CRE DNA. Binding to DNA containing the shorter AP1 target site, which lacks a single nucleobase pair compared to CRE, as well as half-CRE, which contains only half of the CRE target site, was also investigated. Cu(II) and Zn(II) coordination to terpy(GCN4bd)2 promotes binding to AP1 DNA, and to a lesser extent half-CRE DNA. Whereas, bipy(GCN4bd)2 , for which interpeptide distances are largely independent of metal-ion coordination and less suitable for binding to these shorter sites, displays allosteric ineffective behaviour in these cases. These findings for the first time demonstrate that biomolecular recognition, and specifically sequence-selective DNA binding, can be controlled by metal-ion coordination to designed switching units, non-native regulation sites, in artificial biomolecules. We believe that in the future these could find a wide range of applications in biotechnology.

Promoter scanning of the human COX-2 gene with 8-ring polyamides: Unexpected weakening of polyamide–DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine

Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methylpyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with β-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with β-alanine to generate a β/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that β-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.

Selective binding of small molecules to DNA: Application and perspectives

More and more researchers are interested in DNA binders with sequence selectivity which may be developed to potential drugs. In this paper, we mainly review the compounds that selectively bind to special bases or sequences such as AT and GC-rich sequences. The selective binding of them to DNA sequences could affect the interactions of transcription factors with their target sequences in DNA. Furthermore, the design and effectiveness of sequence-selective DNA binding drugs are discussed. At last we put forward the some current challenges and solution in developing targeting anticancer drugs binding to specific oncogene.

新規遺伝子発現制御化合物としてのピロールポリアミド-ヌクレオシドハイブリッド及びそのハイブリッドを組込んだオリゴマーの設計，合成及び評価

On the basis of reports that a minor groove binder pyrrolepolyamide can interfere with gene expression by the sequence-specific recognition of DNA, we expected that nucleoside bearing a pyrrolepolyamide would be able to regulate gene expression. Therefore, we designed and synthesized the pyrrolepolyamide-adenosine (Hybrid 1) and -2'-deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) as lead compounds for gene expression control compounds. The pyrrolepolyamide frame of Hybrid 2 and Hybrid 3 combines at the 2-exocyclic amino group of the 2'-deoxyguanosine by a linker and the 2-exocyclic amino group of guanine exists in the minor groove side of the duplex. Hybrid 2 is the 2'-deoxyguanosine-pyrrolepolyamide hybrid using the 3-aminopropionyl linker, while Hybrid 3 uses the 3-aminopropyl linker. An evaluation of the DNA binding sequence selectivity was performed by analysis of T(m) values and CD spectra, using distamycin A as a contrast. Hybrid 3 has provided more excellent sequence-distinguishable ability than other hybrids and Distamycin A. Moreover, on the basis of these results, we synthesized oligonucleotides conjugated to Hybrid 4, which is stable under conditions of DNA oligonucleotide solid phase synthesis, arranged from Hybrid 3. From T(m) values and CD spectral analysis, it was found that oligonucleotides conjugating Hybrid 4 possess high recognition ability and very high binding ability for the DNA that includes the pyrrolepolyamide binding sequence.

Design, synthesis, and analysis of minor groove binder pyrrolepolyamide-2'-deoxyguanosine hybrids.

Pyrrolepolyamide-2′-deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) incorporating the 3-aminopropionyl or 3-aminopropyl linker were designed and synthesized on the basis of previously reported results of a pyrrolepolyamide-adenosine hybrid (Hybrid 1). Evaluation of the DNA binding sequence selectivity of pyrrolepolyamide-2′-deoxyguanosine hybrids was performed by CD spectral and Tm analyses. It was shown that Hybrid 3 possessed greater binding specificity than distamycin A, Hybrid 1 and Hybrid 2.

Pyrrolo[2,1-c][1,4]benzodiazepine as a Scaffold for the Design and Synthesis of Anti- Tumour Drugs

Compounds that bind in the minor groove of DNA have found use in the experimental treatment of cancer and certain infectious diseases. Furthermore, agents which target and can recognize discrete sequences of DNA have the potential to offer selective therapies by modulating the activity of specific transcription factors or genes. For this reason, a number of sequence-selective DNA binding agents have been evaluated with a range of affinities and recognition fidelities. In this respect, the pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are of interest as they bind to guanine residues in the minor groove with a preference for Pu-G-Pu sequences. A dramatic increase in cytotoxicity and sequence selectivity has been achieved by linking two PBD units to form PBD dimers as cross-linking agents on opposite DNA strands (e.g., interstrand cross-links). SJG-136 is currently undergoing Phase I evaluation in both the United States (through the NCI) and United Kingdom (through Cancer Research United Kingdom). This review will focus on design, synthesis and structure activity relationship studies of pyrrolobenzodiazepines as anticancer therapeutics reported since 2003.

Progesterone regulation of RUSH/SMARCA3/HLTF includes DNA looping

RUSH/SMARCA3 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3) is capable of sequence-selective DNA binding and ATP-dependent DNA unwinding. In rabbit uterine epithelial cells, RUSH-1alpha (113 kDa) is the progesterone-dependent splice variant and RUSH-1beta (95 kDa) is the oestrogen-dependent splice variant. Rabbit RUSH/SMARCA3 mRNA is primarily regulated at the proximal promoter (-162/+90) via a PRE (progesterone-response element) half-site/overlapping Y-box domain (-38/-26) and two Sp (specificity protein) 3 sites centred at -128 and -58. We investigated hormone regulation by exploring binding of transcription factors to a putative RUSH/SMARCA3 site (-616/-611) and the distal Sp3 (-131/-126) site. In response to progesterone, RUSH-1alpha binds the RUSH site and the Sp3 site becomes a functional binding site for Egr-1 (early growth-response gene product 1)/Sp (specificity protein)1/3/MAZ (Myc-associated zinc-finger protein)/MZF1 (myeloid zinc finger 1)/c-Rel. TransSignal TF-TF Interaction Arrays, supershift assays and ChIP (chromatin immunoprecipitation) analyses confirmed strong physical interactions between RUSH and Egr-1/c-Rel. Higher-order long-range interactions between RUSH and the Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown with 3C (chromosome conformation capture) assays. Transient transfection assays with mutant constructs showed the co-operative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus DNA-bound RUSH/SMARCA3 communicates with its own proximal promoter by looping the intervening DNA. Moreover, progesterone-dependent DNA looping is an adjunct to progesterone induction of the RUSH/SMARCA3 gene because the availability of RUSH isoforms and relevant binding partners is progesterone-regulated.

Towards sequence selective DNA binding: design, synthesis and DNA binding studies of novel bis-porphyrin peptidic nanostructures

A new series of peptidic nanostructures bearing two intercalating moieties was designed and synthesized to achieve selective recognition of DNA sequences. A cationic porphyrin was attached to a glutamic acid side chain and the latter introduced into a peptidic sequence by standard solid-phase peptide synthesis methodology. Conformation of the hydrosoluble peptidic structures bearing two cationic porphyrins was studied by circular dichroism. Using UV-visible spectroscopy and induced circular dichroism, we demonstrate that the compounds are fully intercalated upon binding to double-stranded DNA and that the compounds exhibit a tremendous preference for GC over AT sequences for intercalation.

Forced Intercalation Probes (FIT Probes): Thiazole Orange as a Fluorescent Base in Peptide Nucleic Acids for Homogeneous Single‐Nucleotide‐Polymorphism Detection

Fluorescent base analogues in DNA are versatile probes of nucleic acid-nucleic acid and nucleic acid-protein interactions. New peptide nucleic acid (PNA) based probes are described in which the intercalator dye thiazole orange (TO) serves as a base surrogate. The investigation of six TO derivatives revealed that the linker length and the conjugation site decided whether a base surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. One TO derivative conferred universal PNA-DNA base pairing while maintaining duplex stability and hybridization selectivity. TO fluorescence increased up to 26-fold upon hybridization. In contrast to most other probes, in which fluorescence is invariant once hybridization had occurred, the emission of TO-containing PNA probes is attenuated when forced to intercalate next to a mismatched base pair. The specificity of DNA detection is therefore not limited by the selectivity of probe-target binding and a DNA target can be distinguished from its single-base mutant under nonstringent hybridization conditions. This property should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.

Design, synthesis and analysis of a pyrrolepolyamide-nucleoside hybrid

A pyrrolepolyamide-deoxyguanosine hybrid using the 3-aminopropyl linker (GAP) was designed and synthesized on the basis of previously reported results of a pyrrolepolyamide-adenosine hybrid (Apy) and a pyrrolepolyamide-deoxyguanoside hybrid using the 3-aminopropionyl linker (GBP). An evaluation of the DNA binding sequence selectivity of GAP was performed by analysis of CD spectra and Tm values using three DNA duplexes. It was shown that GAP possessed greater binding specificity than distamycin A, netropsin, Apy and GBP.

Comprehensive high-Resolution analysis of hairpin polyamides utilizing a fluorescent intercalator displacement (FID) assay

Four hairpin polyamides bearing subtle N- and C-terminal substitutions were examined in a fluorescent intercalator displacement (FID) assay enlisting a library of 512 DNA hairpins that contain all possible five base pair sequences in a challenging probe of its capabilities for establishing DNA binding sequence selectivity. Not only did the assay define the global sequence selectivity expected based on known structural interactions and Dervan's pairing rules establishing the utility of the method for characterizing such polyamides, but previously unappreciated subtle substituent effects on global sequence selectivity were also revealed. Thus, we report the discovery of a novel five base pair high affinity binding site of the form 5′-WWCWW (vs 5′-WGWWW) for the polyamide ImPyPy-γ-PyPyPy-β-Dp and its structural basis.

DNA Sequence Recognition by the Antitumor Drug Ditercalinium

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.

Sequence‐selective DNA binding drugs mithramycin A and chromomycin A3 are potent inhibitors of neuronal apoptosis induced by oxidative stress and DNA damage in cortical neurons

Global inhibitors of RNA or protein synthesis such as actinomycin D or cycloheximide abrogate neuronal apoptosis induced by numerous pathological stimuli in vitro and in vivo. The clinical application of actinomycin D or cycloheximide to human neurological disease has been limited by the toxicities of these agents. To overcome these toxicities, strategies must be developed to inhibit selectively the expression of deleterious proapoptotic proteins, while leaving the expression of antiapoptotic, proregeneration, and other critical homeostatic proteins unperturbed. Mithramycin A (trade name Plicamycin) is an aureolic acid antibiotic that has been used in humans to treat hypercalcemia and several types of cancers. This class of agents is believed to act, in part, by selectively inhibiting gene expression by displacing transcriptional activators that bind to G-C-rich regions of promoters. Here we demonstrate that mithramycin A and its structural analog chromomycin A3 are potent inhibitors of neuronal apoptosis induced by glutathione depletion-induced oxidative stress or the DNA-damaging agent camptothecin. We correlate the protective effects of mithramycin A with its ability to inhibit enhanced DNA binding of the transcription factors Sp1 and Sp3 to their cognate "G-C" box induced by oxidative stress or DNA damage. The protective effects of mithramycin A cannot be attributed to global inhibition of protein synthesis. Together, these results suggest that mithramycin A and its structural analogs may be effective agents for the treatment of neurological diseases associated with aberrant activation of apoptosis and highlight the potential use of sequence-selective DNA-binding drugs as neurological therapeutics.

Total Synthesis of Distamycin A and 2640 Analogues: A Solution-Phase Combinatorial Approach to the Discovery of New, Bioactive DNA Binding Agents and Development of a Rapid, High-Throughput Screen for Determining Relative DNA Binding Affinity or DNA Binding Sequence Selectivity

The development of a solution-phase synthesis of distamycin A and its extension to the preparation of 2640 analogues are described. Thus, solution-phase synthesis techniques with reaction workup and purification employing acid/base liquid−liquid extractions were used in the multistep preparation of distamycin A (8 steps, 40% overall yield) and a prototypical library of 2640 analogues providing intermediates and final products that are ≥95% pure on conventional reaction scales. The complementary development of a simple, rapid, and high-throughput screen for DNA binding affinity based on the loss of fluorescence derived from displacement of prebound ethidium bromide is disclosed which is applicable for assessing relative or absolute binding affinity to DNA homopolymers or specific sequences (hairpin oligonucleotides). Using hairpin oligonucleotides, this method permits the screening of a library of compounds against a single predefined sequence to identify high affinity binders, or the screening of a single compound against a full library of individual hairpin oligionucleotides to define its sequence selectivity. The combination permits the establishment of the complete DNA binding profile of each member of a library of compounds. Screening the prototypical library provided compounds that are 1000 times more potent than distamycin A in cytotoxic assays (67, IC50 = 29 nM, L1210), that bind to poly[dA]−poly[dT] with comparable affinity, and that exhibit an altered DNA binding sequence selectivity. Several candidates were identified which bound the five-base-pair AT-rich site of the PSA-ARE-3 sequence, and one (128, K = 3.2 × 106 M-1) maintained the high affinity binding (K = 4.5 × 106 M-1) to the ARE-consensus sequence containing a GC base-pair interrupted five-base-pair AT-rich site suitable for inhibition of gene transcription initiated by hormone insensitive androgen receptor dimerization and DNA binding characteristic of therapeutic resistant prostate cancer.