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1.
Trends Genet ; 39(1): 15-30, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36414480

RESUMO

G-quadruplexes (G4s) are non-canonical structures formed in guanine (G)-rich sequences through stacked G tetrads by Hoogsteen hydrogen bonding. Several studies have demonstrated the existence of G4s in the genome of various organisms, including humans, and have proposed that G4s have a regulatory role in various cellular functions. However, little is known regarding the dissemination of G4s in mitochondria. In this review, we report the observation that the number of potential G4-forming sequences in the mitochondrial genome increases with the evolutionary complexity of different species, suggesting that G4s have a beneficial role in higher-order organisms. We also discuss the possible function of G4s in mitochondrial (mt)DNA and long noncoding (lnc)RNA and their role in various biological processes.


Assuntos
Quadruplex G , Humanos , Mitocôndrias/genética
2.
Nano Lett ; 23(5): 2046-2055, 2023 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-36688839

RESUMO

The growing knowledge of the links between aberrant mitochondrial gene transcription and human diseases necessitates both an effective and dynamic approach to control mitochondrial DNA (mtDNA) transcription. To address this challenge, we developed a nanoparticle-based synthetic mitochondrial transcription regulator (MitoScript). MitoScript provides great colloidal stability, excellent biocompatibility, efficient cell uptake, and selective mitochondria targeting and can be monitored in live cells using near-infrared fluorescence. Notably, MitoScript controlled mtDNA transcription in a human cell line in an effective and selective manner. MitoScript targeting the light strand promoter region of mtDNA resulted in the downregulation of ND6 gene silencing, which eventually affected cell redox status, with considerably increased reactive oxygen species (ROS) generation. In summary, we developed MitoScript for the efficient, nonviral modification of mitochondrial DNA transcription. Our platform technology can potentially contribute to understanding the fundamental mechanisms of mitochondrial disorders and developing effective treatments for mitochondrial diseases.


Assuntos
DNA Mitocondrial , Nanopartículas , Humanos , DNA Mitocondrial/genética , Mitocôndrias/genética , Transcrição Gênica , Transporte Biológico
3.
Bioconjug Chem ; 32(10): 2129-2133, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34498851

RESUMO

Phase transition is a promising aspect of DNA as biopolymers. Anionic DNA oligonucleotides easily form complexes with cationic polypeptides such as polylysine, and duplex formation significantly influences their complexation and resulting microcompartments. In this study, phase transition of microcompartments containing DNA and polylysine was systematically induced by modulating duplex formation of arylazopyrazole-conjugated oligonucleotides with light. We demonstrated that UV irradiation destabilized DNA duplex and generated isotropic coacervates, while duplex stabilization by visible light irradiation caused the formation of liquid crystalline coacervates. This photocontrol of phase transition was highly repeatable, and similar changes were observed even after ten cycles of light irradiation. Our approach would provide a robust control layer to the development of tailor-made microcompartments.


Assuntos
Oligonucleotídeos , DNA , Fotoquímica
4.
Chem Rec ; 21(6): 1374-1384, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33332727

RESUMO

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.


Assuntos
DNA/química , Imidazóis/química , Nylons/síntese química , Pirróis/química , Fatores de Transcrição/síntese química , DNA/genética , Humanos , Nylons/química , Fatores de Transcrição/química
5.
J Am Chem Soc ; 142(41): 17356-17363, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32955878

RESUMO

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.


Assuntos
DNA/análise , Corantes Fluorescentes/química , Imidazóis/química , Nylons/química , Pirróis/química , Telômero/química , Linhagem Celular Tumoral , Humanos , Substâncias Intercalantes/química , Mitose , Imagem Óptica , Rodaminas/química , Silício/química , Espectroscopia de Luz Próxima ao Infravermelho
6.
Bioorg Med Chem ; 28(3): 115248, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31879179

RESUMO

Metastasis, a deadly feature of cancer, compromises the prognosis and accounts for mortality in the majority of cancer patients. SOX2, a well-known pluripotency transcription factor, plays a central role in cell fate determination and has an overlapping role as a regulatory factor in tumorigenesis and metastasis. The demand is increasing for clinically useful strategies for artificial control of SOX2 expression and its complex transcription machinery in cancer cells. N-Methylpyrrole (Py) and N-methylimidazole (Im) polyamides are small programmable designer ligands that can be pre-programmed to selectively recognize DNA sequence and control endogenous gene expression. Herein, we evaluated the anticancer activity of a designer ligand (SOX2i). SOX2i remarkably altered the expression of SOX2 at the mRNA and protein level in human cancer cell lines such as SW620 (colorectal adenocarcinoma), MKN45 (gastric adenocarcinoma), MCF7 (breast carcinoma), U2OS (osteosarcoma) and other cancer cell lines of different origin and type. Genome-wide transcriptome analysis and cell-based assays showed SOX2 to be a downregulated upstream regulator that alters cell proliferation, cell cycle progression, metabolism and apoptotic pathway. Studies in the mouse model confirmed the anti-metastatic property of SOX2i. SOX2i inhibited the expression of genes associated with EMT and stemness. Moreover, Wnt-canonical signaling was found to be downregulated in the SOX2i-treated group. Our proof-of-concept study supports the potential of DNA-based programmable small molecules for controlling the key regulatory factors associated with tumorigenesis and metastasis.


Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Nylons/farmacologia , Pirróis/farmacologia , Fatores de Transcrição SOXB1/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/química , Camundongos , Estrutura Molecular , Nylons/síntese química , Nylons/química , Pirróis/química , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
7.
Nucleic Acids Res ; 45(16): 9219-9228, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934500

RESUMO

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.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Mesoderma/citologia , Miócitos Cardíacos/citologia , Nylons/farmacologia , Pirróis/farmacologia , Fatores de Transcrição SOXB1/antagonistas & inibidores , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Consenso , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Miócitos Cardíacos/metabolismo , Nylons/química , Pirróis/química , Fatores de Transcrição SOXB1/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos
8.
J Am Chem Soc ; 140(23): 7108-7115, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29792694

RESUMO

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.

9.
J Am Chem Soc ; 139(25): 8444-8447, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28614654

RESUMO

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.


Assuntos
DNA Mitocondrial/efeitos dos fármacos , Regiões Promotoras Genéticas , Sítios de Ligação , Células HeLa , Humanos , Ligantes , Modelos Biológicos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética
10.
Cell Chem Biol ; 29(4): 690-695.e5, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-34450110

RESUMO

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. In vitro 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.


Assuntos
Adenina , DNA Mitocondrial , Alquilação , Clorambucila/química , DNA Mitocondrial/genética , Humanos , Mitocôndrias , Mutação , Nylons/química
11.
ACS Chem Biol ; 17(10): 2704-2709, 2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-36190780

RESUMO

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios.


Assuntos
Acrilonitrila , Sequenciamento por Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inosina , RNA/genética
12.
Methods Mol Biol ; 2275: 217-225, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118040

RESUMO

Mitochondria possess multiple copies of mitochondrial DNA (mtDNA) that encode 37 genes and their transcription and replication get controlled by unique molecular codes different from that in the nuclear DNA. The mtDNA has been gaining increased attention as one of the critical therapeutic targets as mutations in them impair the function of mitochondria and cause mitochondrial diseases like MELAS. In this chapter, we describe artificial control of mitochondrial transcription based on mtDNA sequence information with a new type of compounds termed MITO-PIPs, which encompasses two domains: pyrrole-imidazole polyamide as DNA recognition domain and mitochondrial penetrating peptide as the mitochondria-targeting domain. Because MITO-PIPs are amenable to tunability, they can be expanded as a synthetic strategy to modulate mitochondrial gene(s) on demand.


Assuntos
Azóis/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Cristalografia por Raios X , Genes Sintéticos , Células HeLa , Humanos , Conformação Molecular , Transcrição Gênica
13.
Chem Commun (Camb) ; 56(82): 12371-12374, 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-32930253

RESUMO

The tri-arginine moiety enhanced nuclear accumulation of a 12-ring pyrrole-imidazole polyamide (PIP) without compromising sequence-selectivity and achieved efficient repression of SOX2-downstream genes and HER2 transcription in live cells. This simple vector expands the application of long PIPs in live cells by overcoming the compound delivery problems associated with them.

14.
Adv Drug Deliv Rev ; 147: 66-85, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30742856

RESUMO

Recent innovations in cutting-edge sequencing platforms have allowed the rapid identification of genes associated with communicable, noncommunicable and rare diseases. Exploitation of this collected biological information has facilitated the development of nonviral gene therapy strategies and the design of several proteins capable of editing specific DNA sequences for disease control. Small molecule-based targeted therapeutic approaches have gained increasing attention because of their suggested clinical benefits, ease of control and lower costs. Pyrrole-imidazole polyamides (PIPs) are a major class of DNA minor groove-binding small molecules that can be predesigned to recognize specific DNA sequences. This programmability of PIPs allows the on-demand design of artificial genetic switches and fluorescent probes. In this review, we detail the progress in the development of PIP-based designer ligands and their prospects as advanced DNA-based small-molecule drugs for therapeutic gene modulation.


Assuntos
Corantes Fluorescentes/farmacologia , Imidazóis/farmacologia , Nylons/farmacologia , Pirróis/farmacologia , Animais , DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes
15.
ChemistryOpen ; 5(6): 517-521, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-28032018

RESUMO

An integrated multi-target small molecule capable of altering dynamic epigenetic and transcription programs associated with the brain and nervous system has versatile applications in the regulation of therapeutic and cell-fate genes. Recently, we have been constructing targeted epigenetic ON switches by integrating sequence-specific DNA binding pyrrole-imidazole polyamides with a potent histone deacetylase inhibitor SAHA. Here, we identified a DNA-based epigenetic ON switch termed SAHA-L as the first-ever multi-target small molecule capable of inducing transcription programs associated with the human neural system and brain synapses networks in BJ human foreskin fibroblasts and 201B7-iPS cells. Ingenuity pathway analysis showed that SAHA-L activates the signaling of synaptic receptors like glutamate and γ-aminobutyric acid, which are key components of autism spectrum disorders. The long-term incubation of SAHA-L in 201B7-iPS cells induced morphology changes and promoted a neural progenitor state. Our finding suggests that the tunable SAHA-L could be advanced as a cell-type-independent multi-target small molecule for therapeutic and/or cell-fate gene modulation.

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