Sequence-specific DNA-binding Molecules Research Articles

Synthetic genome readers target clustered binding sites across diverse chromatin states

Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to "read" specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.

Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole-Imidazole Polyamide Probes.

Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole-imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5'-(TTAGGG)n-3') and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome.

ChemInform Abstract: Nature‐Inspired Design of Smart Biomaterials Using the Chemical Biology of Nucleic Acids

AbstractReview: [74 refs.

Nature-Inspired Design of Smart Biomaterials Using the Chemical Biology of Nucleic Acids

Abstract In the natural cellular environment, nucleic acid biomolecules like DNA have biological implications via structural modifications and through precise coordination with other biomolecules in the local environment. Here, we detail the design of nature-inspired smart biomaterials that are based on the chemical biology of nucleic acids. N-Methylpyrrole (P) and N-methylimidazole (I) polyamides (PIPs), sequence-specific DNA-binding molecules have been, developed to image specific DNA sequences and to alter gene expression inside the living cells. The self-assembling feature of DNA was harnessed to achieve the programmed assembly of nanostructures with different dimensions. Also, the advanced DNA architectures with well-defined properties allowed the real-time visualization of the complicated single-molecule interactions, which in-turn provided vital intracellular mechanistic information. The molecular recognition properties of DNA were exploited to design biologically inspired hybrid catalysts for sustainable organic synthesis. Our review could serve as a guidebook for researchers who aim to develop nucleic acid-based synthetic biomaterials.

Abstract 4773: Gene-targeted apoptosis as a novel therapeutic strategy for HER2-positive breast cancer

Abstract The ability to target HER2-driven breast cancers using anticancer agents with mechanisms of actions that are independent of the HER2 cellular growth function, represents a powerful new tool to circumvent drug resistance experienced with traditional HER2-targeted therapies. We have discovered that manipulation of the balance between the DNA repair response and apoptotic pathways provides an alternative strategy to induce targeted apoptosis in HER2-amplified breast cancers. Our recent finding that triplex-induced DNA damage can activate apoptosis in human cells has yielded the opportunity to develop a new class of HER2-targeted therapies. Here, we have uncovered the novel finding that triplex-induced apoptosis only occurs when multiple triplex structures are formed, while NER (nucleotide excision repair)-dependent repair prevails in the presence of one or two structures. With this in mind, we have designed sequence-specific DNA-binding molecules that create chromosomal triplex structures and activate tumor-specific apoptosis through the induction of excessive DNA damage at the HER2 locus. Importantly, this only occurs in malignant cells with multiple copies of the HER2 gene. We have evaluated the efficacy of the HER2-targeted triplex-forming molecules using cell growth and apoptosis assays and demonstrate that these molecules are particularly effective in eliciting tumor specific apoptosis in HER2-amplified breast cancer cell lines. Furthermore, these molecules have the ability to suppress the growth and metastasis of human HER2-positive breast cancers in a xenograft mouse model. Moreover, we demonstrate that the NER protein, XPD mediates the survival-death decision in response to triplex-induced DNA damage via a p53-independent mechanism that involves phosphorylation of the H2AX tyrosine 142 residue, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Our results establish an alternative and novel method to specifically target HER2-positive breast cancers by exploiting the cell's own DNA damage response machinery. This study introduces a new paradigm for gene-targeted drugs that can be used in the treatment of HER2-positive and drug-resistant breast cancers with minimal potential for toxicity to normal tissue. Citation Format: Faye A. Rogers, Meetu Kaushik. Gene-targeted apoptosis as a novel therapeutic strategy for HER2-positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4773.

Design of Sequence-Specific DNA Binding Molecules for DNA Methyltransferase Inhibition

The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5′-CGCG-3′ and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (1), previously identified as a high affinity 5′-CGCG-3′ binder, favors 5′-GCGC-3′ in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (3) restores the preference for 5′-CGCG-3′ binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation.

Abstract 5679: Automatic synthesis of efficient transcription inhibitors as anti-cancer agents designing sequence-specific DNA-binding molecules.

Abstract PURPOSE: Regulation of endogenous gene expression using synthetic chemicals, so called “Transcription therapy” is a unique and promising approach. Although we have reported in vitro and in vivo anti-tumor, anti-metastatic and various anti-disease efficacies of hairpin Pyrrole-Imidazole polyamide (hPIP), PIP still needs to improve on selectivity of target DNA sequences and sensitivity to bind specific sequences. Hence, we synthesized PIPs targeting transcription-factor-binding-sites with several different structures and tested their efficacy, selectivity and sensitivity in vitro as well as in vivo model. METHODS: We have developed an automatic system for PIP synthesis by using peptide synthesizer of PSSM8 (Shimazu) and synthesized hPIPs or cyclic PIPs targeting either the MMP9 AP1 site or NFkB site to evaluate sensitivity and specificity. Each PIP, which has a different beta-alanine position, was tested for target DNA binding by surface plasmon resonance (SPR) using BiaCore system. We also test cell invasion and migration in vitro and metastatic capability in an in vivo xenograft model. RESULTS: Each hPIPs targeting AP1 or NFkB site showed significant reduction of MMP9 expression in MDA-MB-231 cells and HeLa cells and metastatic capacity in vivo. hPIP with crossed beta-alanine position showed the almost same dissociation constant as those with parallel beta-alanine position. Cyclic PIPs also showed similar dissociation constant as hPIPs and showed better sequence specificity with reasonable efficacy at least in vitro analysis. CONCLUSION: The result suggested that various PIPs with different target sites or architectures give distinct efficacy, selectivity and sensitivity in at least in vitro cancer cell culture system. Depending on pharmacokinetic properties of PIP the most suitable PIP can be selected for the future trial as seen in combinatorial chemistry technique. Citation Format: Hiroki Nagase, Nobuko Koshikawa, Takayoshi Watanabe. Automatic synthesis of efficient transcription inhibitors as anti-cancer agents designing sequence-specific DNA-binding molecules. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5679. doi:10.1158/1538-7445.AM2013-5679

Oligomerization Route to Py−Im Polyamide Macrocycles

Cyclic eight-ring pyrrole-imidazole polyamides are sequence-specific DNA-binding small molecules that are cell permeable and can regulate endogenous gene expression. Syntheses of cyclic polyamides have been achieved by solid-phase and solution-phase methods. A rapid solution-phase oligomerization approach to eight-ring symmetrical cyclic polyamides yields 12- and 16-membered macrocycles as well. A preference for DNA binding by the 8- and 16-membered oligomers was observed over the 12-ring macrocycle, which we attributed to a conformational constraint not present in the smaller and larger systems.

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.

Fluorescent Sequence-Specific dsDNA Binding Oligomers

Sequence-specific detection methods for double-stranded DNA that obviate the need for denaturation would provide a powerful tool for bioorganic chemistry and genetics. As part of a sustained effort to develop sequence-specific fluorescent DNA detection methods, two programmable oligomers have been synthesized which target their respective sequences 5‘-WTACGW-3‘ and 5‘-WGGGGW-3‘ (W = A or T). The two oligomers were found to fluoresce weakly in the absence of DNA but showed significant fluorescence enhancement by the addition of match DNA. The fluorescence is shown to increase in a concentration-dependent manner, and the intensity varies depending on the number of mismatch sites incorporated into the DNA hairpins. This new class of oligomers provides a method to detect DNA sequences without denaturation and in the absence of conjugation to a dye molecule. This is a first step toward sequence-specific DNA-binding molecules containing a fluorescent switch integrated as part of the recognition modules.

Selective interaction between tylophorine B and bulged DNA

Tylophorine B exhibits pronounced cytotoxicity and antitumor activity. In order to survey the structure selectivity to DNA afforded by tylophorine B, we have synthesized a variety of duplex, bulge- and hairpin-containing oligodeoxyribonucleotides. Their binding to tylophorine B has been assayed by fluorescence spectroscopy and thermal melting experiments. The results indicate that oligonucleotides interact with tylophorine B at submicromolar concentration, and the affinity for DNA bulge is optimal (with Kd of 0.018μM). In addition, the bulged hairpin oligonucleotides are stabilized by binding to tylophorine B. These findings may shed some light on tylophorine B’s mode of action in biological systems and result in the rational design of sequence-specific DNA binding molecules.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands.

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

Synthesis of DNA-binding heteroaromatic oligoamides on liquid solid support

Heteroaromatic oligoamides as building blocks for the synthesis of sequence specific DNA binding molecules are obtained from nitro carboxylic acids on polyethylene glycol without the use of protecting groups

Identification of preferred distamycin-DNA binding sites by the combinatorial method REPSA.

The combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) was used to determine the preferred duplex DNA binding sites of the peptide N-methylpyrrolecarboxamide antibiotic distamycin A. After 12 rounds of REPSA, several sequences were identified that bound distamycin with an apparent affinity of 2-20 nM. Among these, the highest-affinity sites averaged 10 bp in length, suggesting that these sites may be occupied by multiple, cooperatively interacting distamycin molecules. Presently, REPSA is the only combinatorial approach that allows the identification of preferred DNA targets for small molecule ligands at physiologically relevant concentrations in solution. As such, it should prove useful in the design and screening of sequence-specific DNA-binding molecules.

Base Changes and Triple-Helix Hybridization Properties of GT Containing Third Strands: A Systematical Study

Triple-helix recognition of the DNA major grove offers a powerful approach for the design of sequence specific DNA binding molecules. Although the triple-helix binding of pyrimidine containing third strands is well characterized now, the factors governing the recognition of double stranded DNA by GT containing third strands are poorly understood. Oligonucleotides with a variable amount of G and GpT or TpG steps have been synthesized here. A systematical study was performed to determine the influence of these changes on the triple-helix hybridization properties of these oligonucleotides. Large difference in the hybridization properties have been observed by band shift experiments.

Second Structural Motif for Recognition of DNA by Oligonucleotide-Directed Triple-Helix Formation

Relative orientations of the DNA strands within a purine.purine.pyrimidine triple helix have been determined by affinity cleaving. A purine-rich oligonucleotide bound in the major groove of double-helical DNA antiparallel to the Watson-Crick purine strand. Binding depended upon the concentration of multivalent cations such as spermine or Mg2+, and appeared to be relatively independent of pH. Two models with specific hydrogen-bonding patterns for base triplets (G.GC, A.AT, and T.AT) are proposed to explain the sequence specificity of binding. The two models differ in the conformation about the glycosyl bond (syn or anti) and the location of the phosphate-deoxyribose backbone in the major groove of DNA. This motif broadens the structural frameworks available as a basis for the design of sequence-specific DNA binding molecules.

A synthetic peptide binds 16 base pairs of A,T double helical DNA

One approach to the design of synthetic sequence specific DNA binding molecules that bind large sequences of double helical DNA is to couple together DNA binding domains of similar or diverse base pairs specificities. The DNA binding domains should be linked together in a way that allows simultaneous binding of the units to contiguous DNA sequences.

Specific sequence-specific recognition of right-handed double-helical DNA by (2S,3S)- and (2R,3R)-dihydroxybis(netropsin)succinamide

We report a class of chiral sequence-specific DNA binding molecules based on two netropsin analogues and the enantiomers of dl-tartaric acid: (2R,3R)- and (2S,3S)-dihydroxybis(netropsin)succinamide-EDTA [(R,R)- and (S,S)-1, respectively] (Figures 1 and 2). The enantiomeric threo-(R,R)1 and -(SS)-1 should afford diastereomeric complexes at common binding sites on double-helical DNA (Figure 1). Attachment of EDTA to one terminus of the crescent-shaped hexamides allows use of the affinity cleaving method to determine the differences between (R,R)-1•Fe^(II) and (S,S)-1•Fe^(II) with regard to relative binding affinities, sequence specificities, and orientations by analysis of the DNA-cleavage patterns on a 32P end-labeled DNA restriction fragment using high-resolution gel electrophoresis. For comparison to the enantiomers of 1, bis(netropsin)succinamide-EDTA (2) was synthesized (Figure 2).

Design of Sequence‐specific DNA Cleaving Molecules

The design of sequence-specific cleaving molecules for double-helical DNA requires the attachment of DNA cleaving moieties to sequence-specific DNA binding molecules. The site of attachment and choice of cleaving strategy will be dependent on the availability of molecular models for the sequence-specific binding of small molecules (i.e., 1000 MW) to right-handed double-helical DNA. Productive model building is possible because of recent progress in the development of analytical methods, such as footprinting and affinity cleaving, to determine the preferred binding locations, binding site sizes, and orientations of natural or synthetic sequence-specific DNA binding molecules on large DNA such as DNA restriction fragments (100-500 bp). The natural product distamycin is a crescent-shaped tripeptide containing three N-methylpyrrolecarboxamides which binds in the minor groove of right-handed double-helical DNA with a strong perference for A•T rich sequences (FIG. I). The preferred sequences, binding site size, and orientation preference for tris-N-methylpyrrolecarboxamide are known from footprinting and affinity cleaving data. Moreover, from recent X-ray analysis of the complex of the dipeptide, netropsin, with the B-DNA dodecamer, S-CGCGAATTCGCG-3', Dickerson and co-workers have provided a molecular basis for the recognition of DNA by netropsin, and by extension, distamycin. They find that netropsin sits symmetrically in the center of the minor groove of A•T rich right-handed DNA and displaces the water molecules of the spine of hydration. Each of its three amide NH groups forms a bridge between adjacent adenine N3 or thymine O2 atoms on opposite helix strands. Dickerson and coworkers suggest that the base specificity of netropsin for contiguous sequences of A •T base pairs in B-DNA is provided not by hydrogen bonding but by close van der Waals contacts between adenine C2 hydrogens and CH groups on the pyrrole rings of the oligopeptide molecules.