Modulation of dynamic DNA G-quadruplex structures in the hTERT promoter region by ligands.

Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5-12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5-12 in presence of PDS, PA, or PA-PDS conjugate are observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5-12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5-12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

Substitution to hydrophobic linker and formation of host-guest complex enhanced the effect of synthetic transcription factor made of pyrrole-imidazole polyamide.

GAA repeat expansion in the first intron of the frataxin (FXN) gene represses the transcription of FXN, and that induces Friedreich's ataxia (FRDA). Pyrrole-imidazole polyamides (PIPs) are the class of oligopeptide that targets double-stranded DNA with sequence selectivity. Previously, bromodomain inhibitors such as JQ1 conjugated with PIPs were reported to selectively increase transcription. Here, we report the synthesis of a compound that increases the transcription of FXN in cells derived from an FRDA patient. The compound was effective in lower (one tenth) concentration than the compound that previously reported. High concentration of the compound is toxic, but toxicity was reduced with a host-guest complex.

Twelve Colors of Streptavidin-Fluorescent Proteins (SA-FPs): A Versatile Tool to Visualize Genetic Information in Single-Molecule DNA.

Streptavidin-fluorescent proteins (SA-FPs) are a versatile tool to visualize a broad range of biochemical applications on a fluorescence microscope. Although the avidin-biotin interaction is widely used, the use of SA-FPs has not been applied to single-molecule DNA visualization. Here, we constructed 12 bright SA-FPs for DNA staining or labeling reagents. To date, 810 FPs are available, many of which are brighter than organic dyes. In this study, 12 bright FPs were selected to construct SA-FP plasmids covering green to red colors. Their brightness ranges from 40 to 165 mM-1 cm-1. Moreover, SA-FP is brighter than FP itself because streptavidin forms a tetramer complex; thus, four FPs are in a single complex. In addition, FPs often form a dimer or a tetramer, resulting in multiple FPs in a single spot on a microscopic image. This feature is advantageous because multiple fluorescent ß-barrels on a single biotin tag provide enough brightness to be easily visualized by epifluorescence microscopy. Using SA-FPs, we visualized DNA backbones, nickase-based optical mapping, and AT-frequency profiling. Finally, we demonstrated the combination of nickase-based optical mapping using SA-FP and AT-frequency profiling.

N-terminal Cationic Modification of Linear Pyrrole-Imidazole Polyamide Improves Its Binding to DNA.

Pyrrole-imidazole polyamides (PIPs) bind to double-stranded DNA (dsDNA) with varied sequence selectivity. We synthesized linear PIPs that can bind to narrow minor grooves of polypurine/polypyrimidine sequences and target long recognition sequences but have lower molecular weights than commonly used hairpin PIPs. We modified the N-terminus of linear PIPs using several groups, including ß-alanine extension and acetyl capping. Melting curve analysis of dsDNA demonstrated that cationic modifications improved the binding affinity of the PIPs to the targeted dsDNA. In addition, circular dichroism assays revealed the characteristic spectra depending on the binding stoichiometry of the N-cationic linear PIP and dsDNA (1 : 1, monomeric; 2 : 1, dimeric). Surface plasmon resonance assays confirmed the high binding affinities of linear PIPs. These findings may aid in the design of effective linear PIPs.

Strong and Specific Recognition of CAG/CTG Repeat DNA (5'-dWGCWGCW-3') by a Cyclic Pyrrole-Imidazole Polyamide.

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (Tm ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.

Evaluation of the DNA Alkylation Properties of a Chlorambucil-Conjugated Cyclic Pyrrole-Imidazole Polyamide.

Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP-Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP-Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP-Chb conjugates 1 and 2. Subsequent HPLC analysis revealed that the alkylation site of conjugate 3, which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP-Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2. These results suggest that cPIP-Chbs could be novel DNA alkylating anticancer drugs.

Submolecular dissection reveals strong and specific binding of polyamide-pyridostatin conjugates to human telomere interface.

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA-ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant (Kd) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found Kd of 0.8 nM for PA-PDS, which is much lower than PDS (Kd ∼ 450 nM) or PA (Kd ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.

A Near-Infrared Fluorogenic Pyrrole-Imidazole Polyamide Probe for Live-Cell Imaging of Telomeres.

Telomeres are closely associated with cellular senescence and cancer. Although some techniques have been developed to label telomeres in living cells for study of telomere dynamics, few biocompatible near-infrared probes based on synthetic molecules have been reported. In this study, we developed a near-infrared fluorogenic pyrrole-imidazole polyamide probe (SiR-TTet59B) to visualize telomeres by conjugating a silicon-rhodamine (SiR) fluorophore with a tandem tetramer pyrrole-imidazole polyamide targeting 24 bp in the telomeric double-stranded (ds) DNA. SiR-TTet59B was almost nonfluorescent in water but increased its fluorescence dramatically on binding to telomeric dsDNA. Using a peptide-based delivery reagent, we demonstrated the specific and effective visualization of telomeres in living U2OS cells. Moreover, SiR-TTet59B could be used to observe the dynamic movements of telomeres during interphase and mitosis. This simple imaging method using a synthetic near-infrared probe could be a powerful tool for studies of telomeres and for diagnosis.

DNA Alkylation of the RUNX-Binding Sequence by CBI-PI Polyamide Conjugates*.

Many types of molecular targeted drugs that inhibit cancer growth by acting on specific molecules have been developed. The runt-related transcription factor (RUNX) family, which induces cancer development by binding to a specific DNA sequence, has attracted attention as a new target for cancer treatment. We have developed Chb-M', which targets the RUNX-binding sequence. Chb-M' was developed by conjugating pyrrole-imidazole (PI) polyamides and chlorambucil as an anticancer agent. It was recently reported that Chb-M' had a remarkable anticancer effect in vivo. In this study, to explore the possibility of an alternative structure, we designed a new series of CBI-PI polyamides, in which seco-CBI was applied as a DNA-alkylating agent. We examined the characteristics of the CBI-PI polyamides targeting the RUNX-binding sequence and found that these conjugates have great potential for cancer treatment.

Molecular Characteristics of DNA-Alkylating PI Polyamides Targeting RUNX Transcription Factors.

The runt-related transcription factor (RUNX) family has been associated with cancer development. The binding of RUNX family members to specific DNA sequences is hypothesized to promote the expression of downstream genes and cause cancer proliferation. On the basis of this proposed mechanism of cancer growth, we developed conjugate 1, which inhibits the binding of RUNX to its target DNA. Conjugate 1 is a DNA-alkylating pyrrole-imidazole (PI) polyamide conjugate containing chlorambucil as an anticancer agent. Conjugate 1 was reported to have a marked anticancer effect in mouse models of acute myeloid leukemia. Although the effectiveness of 1 has been demonstrated in vivo, the detailed mechanism by which it alkylates DNA is unknown. Here, we chemically elucidated the molecular characteristics of conjugate 1 to confirm its potential as a RUNX-inhibiting drug. We also generated an alternative conjugate 2, which targets the same DNA sequence, by replacing one pyrrole with ß-alanine. Comparison of the characteristics of conjugates 1 and 2 suggested that reaction selectivity and binding affinity to the RUNX-binding sequence were improved by the introduction of ß-alanine. These findings indicate the possibility of DNA-alkylating PI polyamides as candidates for cancer chemotherapeutics.

A synthetic DNA-binding inhibitor of SOX2 guides human induced pluripotent stem cells to differentiate into mesoderm.

Targeted differentiation of human induced pluripotent stem cells (hiPSCs) using only chemicals would have value-added clinical potential in the regeneration of complex cell types including cardiomyocytes. Despite the availability of several chemical inhibitors targeting proteins involved in signaling pathways, no bioactive synthetic DNA-binding inhibitors, targeting key cell fate-controlling genes such as SOX2, are yet available. Here, we demonstrate a novel DNA-based chemical approach to guide the differentiation of hiPSCs using pyrrole-imidazole polyamides (PIPs), which are sequence-selective DNA-binding synthetic molecules. Harnessing knowledge about key transcriptional changes during the induction of cardiomyocyte, we developed a DNA-binding inhibitor termed PIP-S2, targeting the 5'-CTTTGTT-3' and demonstrated that inhibition of SOX2-DNA interaction by PIP-S2 triggers the mesoderm induction in hiPSCs. Genome-wide gene expression analyses revealed that PIP-S2 induced mesoderm by targeted alterations in SOX2-associated gene regulatory networks. Also, employment of PIP-S2 along with a Wnt/ß-catenin inhibitor successfully generated spontaneously contracting cardiomyocytes, validating our concept that DNA-binding inhibitors could drive the directed differentiation of hiPSCs. Because PIPs can be fine-tuned to target specific DNA sequences, our DNA-based approach could be expanded to target and regulate key transcription factors specifically associated with desired cell types.

G-Quadruplex Induction by the Hairpin Pyrrole-Imidazole Polyamide Dimer.

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole-imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy.

Pip-HoGu: An Artificial Assembly with Cooperative DNA Recognition Capable of Mimicking Transcription Factor Pairs.

Cooperation between pairs of transcription factors (TFs) has been widely demonstrated to play a pivotal role in the spatiotemporal regulation of gene expression, but blocking cooperative TF pair-DNA interactions synergistically has been challenging. To achieve this, we designed programmable DNA binder pyrrole-imidazole polyamides conjugated to host-guest assemblies (Pip-HoGu) to mimic the cooperation between natural TF pairs. By incorporating cyclodextrin (Cyd)-adamantane (Ada), we synthesized Ada1 (PIP1-Ada) and Cyd1 (PIP2-Cyd), which were evaluated using Tm, EMSA, competitive, and SPR assays and molecular dynamics studies. The results consistently demonstrated that Pip-HoGu system formed stable noncovalent cooperative complexes, thereby meeting key criteria for mimicking a TF pair. The system also had a longer recognition sequence (two-PIP binding length plus gap distance), favorable sequence selectivity, higher binding affinity, and in particular, a flexible gap distance (0-5 bp). For example, Ada1-Cyd1 showed thermal stability of 7.2 °C and a minimum free energy of interaction of -2.32 kcal·mol-1 with a targeting length of 14 bp. Furthermore, cell-based evaluation validated the capability of Pip-HoGu to exhibit potent cooperative inhibitory effects on gene expression under physiological conditions by disrupting TF pair-DNA function. In conclusion, the modular design of Pip-HoGu defines a general framework for mimicking naturally occurring cooperative TF pair-DNA interactions that offers a promising strategy for applications in the precise manipulation of cell fate.

Biomimetic Artificial Epigenetic Code for Targeted Acetylation of Histones.

While the central role of locus-specific acetylation of histone proteins in eukaryotic gene expression is well established, the availability of designer tools to regulate acetylation at particular nucleosome sites remains limited. Here, we develop a unique strategy to introduce acetylation by constructing a bifunctional molecule designated Bi-PIP. Bi-PIP has a P300/CBP-selective bromodomain inhibitor (Bi) as a P300/CBP recruiter and a pyrrole-imidazole polyamide (PIP) as a sequence-selective DNA binder. Biochemical assays verified that Bi-PIPs recruit P300 to the nucleosomes having their target DNA sequences and extensively accelerate acetylation. Bi-PIPs also activated transcription of genes that have corresponding cognate DNA sequences inside living cells. Our results demonstrate that Bi-PIPs could act as a synthetic programmable histone code of acetylation, which emulates the bromodomain-mediated natural propagation system of histone acetylation to activate gene expression in a sequence-selective manner.

Simultaneous Binding of Hybrid Molecules Constructed with Dual DNA-Binding Components to a G-Quadruplex and Its Proximal Duplex.

A G-quadruplex (quadruplex) is a nucleic acid secondary structure adopted by guanine-rich sequences and is considered to be relevant to various pharmacological and biological contexts. Although a number of researchers have endeavored to discover and develop quadruplex-interactive molecules, poor ligand designability originating from topological similarity of the skeleton of diverse quadruplexes has remained a bottleneck for gaining specificity for individual quadruplexes. This work reports on hybrid molecules that were constructed with dual DNA-binding components, a cyclic imidazole/lysine polyamide (cIKP), and a hairpin pyrrole/imidazole polyamide (hPIP), with the aim toward specific quadruplex targeting by reading out the local duplex DNA sequence adjacent to designated quadruplexes in the genome. By means of circular dichroism (CD), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and NMR techniques, we showed the dual and simultaneous recognition of the respective segment via hybrid molecules, and the synergistic and mutual effect of each binding component that was appropriately linked on higher binding affinity and modest sequence specificity. Monitoring quadruplex and duplex imino protons of the quadruplex/duplex motif titrated with hybrid molecules clearly revealed distinct features of the binding of hybrid molecules to the respective segments upon their simultaneous recognition. A series of the systematic and detailed binding assays described here showed that the concept of simultaneous recognition of quadruplex and its proximal duplex by hybrid molecules constructed with the dual DNA-binding components may provide a new strategy for ligand design, enabling targeting of a large variety of designated quadruplexes at specific genome locations.

Orthogonal γPNA Dimerization Domains Empower DNA Binders with Cooperativity and Versatility Mimicking that of Transcription Factor Pairs.

Synthetic molecules capable of DNA binding and mimicking cooperation of transcription factor (TF) pairs have long been considered a promising tool for manipulating gene expression. Our previously reported Pip-HoGu system, a programmable DNA binder pyrrole-imidazole polyamides (PIPs) conjugated to host-guest moiety, defined a general framework for mimicking cooperative TF pair-DNA interactions. Here, we supplanted the cooperation modules with left-handed (LH) γPNA modules: i.e., PIPs conjugated with nucleic acid-based cooperation system (Pip-NaCo). LH γPNA was chosen because of its bioorthogonality, sequence-specific interaction, and high binding affinity toward the partner strand. From the results of the Pip-NaCo system, cooperativity is highly comparable to the natural TF pair-DNA system, with a minimum energetics of cooperation of -3.27 kcal mol-1 . Moreover, through changing the linker conjugation site, binding mode, and the length of γPNAs sequence, the cooperative energetics of Pip-NaCo can be tuned independently and rationally. The current Pip-NaCo platform might also have the potential for precise manipulation of biological processes through the construction of triple to multiple heterobinding systems.

Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer.

N-Methylpyrrole-N-methylimidazole (PI) polyamides are a class of DNA minor groove binders with DNA sequence-specificity. DNA-alkylating PI polyamide conjugates are attractive candidates as anticancer drugs acting through DNA damage and its subsequent inhibition of cell proliferation. One example is a chlorambucil-PI polyamide conjugate targeting the runt-related transcription factor (RUNX) family. RUNX1 has pro-oncogenic properties in acute myeloid leukemia, and recently the chlorambucil-PI polyamide conjugate was demonstrated to have anticancer effects. Herein, we apply another DNA-alkylating agent, seco-CBI, to target the consensus sequence of the RUNX family. Two types of CBI conjugates were prepared and their binding properties were characterized by Bind-n-Seq analysis using a high-throughput sequencer. The sequencing data were analyzed by two methods, MERMADE and our new MR (motif identification with a reference sequence), and the resultant binding motif logos were as predicted from the pairing rules proposed by Dervan et al. This is the first report to employ the MR method on alkylating PI polyamide conjugates. Moreover, cytotoxicity of conjugates 3 and 4 against a human non-small cell lung cancer, A549, were examined to show promising IC50s of 120 nm and 63 nm, respectively. These findings suggest seco-CBI-PI polyamide conjugates are candidates for oncological therapy.

Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing.

Chemically engineered small molecules targeting specific genomic sequences play an important role in drug development research. Pyrrole-imidazole polyamides (PIPs) are a group of molecules that can bind to the DNA minor-groove and can be engineered to target specific sequences. Their biological effects rely primarily on their selective DNA binding. However, the binding mechanism of PIPs at the chromatinized genome level is poorly understood. Herein, we report a method using high-throughput sequencing to identify the DNA-alkylating sites of PIP-indole-seco-CBI conjugates. High-throughput sequencing analysis of conjugate 2: showed highly similar DNA-alkylating sites on synthetic oligos (histone-free DNA) and on human genomes (chromatinized DNA context). To our knowledge, this is the first report identifying alkylation sites across genomic DNA by alkylating PIP conjugates using high-throughput sequencing.

Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression.

Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs' properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations.