Mitochondria and G-quadruplex evolution: an intertwined relationship.

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.

Nucleic acid-based small molecules as targeted transcription therapeutics for immunoregulation.

Transcription therapy is an emerging approach that centers on identifying the factors associated with the malfunctioning gene transcription machinery that causes diseases and controlling them with designer agents. Until now, the primary research focus in therapeutic gene modulation has been on small-molecule drugs that target epigenetic enzymes and critical signaling pathways. However, nucleic acid-based small molecules have gained popularity in recent years for their amenability to be pre-designed and realize operative control over the dynamic transcription machinery that governs how the immune system responds to diseases. Pyrrole-imidazole polyamides (PIPs) are well-established DNA-based small-molecule gene regulators that overcome the limitations of their conventional counterparts owing to their sequence-targeted specificity, versatile regulatory efficiency, and biocompatibility. Here, we emphasize the rational design of PIPs, their functional mechanisms, and their potential as targeted transcription therapeutics for disease treatment by regulating the immune response. Furthermore, we also discuss the challenges and foresight of this approach in personalized immunotherapy in precision medicine.

Nanoparticle-Based Artificial Mitochondrial DNA Transcription Regulator: MitoScript.

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.

Food Intake and Colorectal Cancer.

Colorectal cancer (CRC) accounts for considerable mortalities worldwide. Several modifiable risk factors, including a high intake of certain foods and beverages can cause CRC. This review summarized the latest findings on the intake of various foods, nutrients, ingredients, and beverages on CRC development, with the objective of classifying them as a risk or protective factor. High-risk food items include red meat, processed meat, eggs, high alcohol consumption, sugar-sweetened beverages, and chocolate candy. Food items that are protective include milk, cheese and other dairy products, fruits, vegetables (particularly cruciferous), whole grains, legumes (particularly soy beans), fish, tea (particularly green tea), coffee (particularly among Asians), chocolate, and moderate alcohol consumption (particularly wine). High-risk nutrients/ingredients include dietary fat from animal sources and industrial trans-fatty acids (semisolid/solid hydrogenated oils), synthetic food coloring, monosodium glutamate, titanium dioxide, and high-fructose corn sirup. Nutrients/ingredients that are protective include dietary fiber (particularly from cereals), fatty acids (medium-chain and odd-chain saturated fatty acids and highly unsaturated fatty acids, including omega-3 polyunsaturated fatty acids), calcium, polyphenols, curcumin, selenium, zinc, magnesium, and vitamins A, C, D, E, and B (particularly B6, B9, and B2). A combination of micronutrients and multi-vitamins also appears to be beneficial in reducing recurrent adenoma incidence.

An informatics approach to distinguish RNA modifications in nanopore direct RNA sequencing.

Modifications in RNA can influence their structure, function, and stability and play essential roles in gene expression and regulation. Methods to detect RNA modifications rely on biophysical techniques such as chromatography or mass spectrometry, which are low throughput, or on high throughput short-read sequencing techniques based on selectively reactive chemical probes. Recent studies have utilized nanopore-based fourth-generation sequencing methods to detect modifications by directly sequencing RNA in its native state. However, these approaches are based on modification-associated mismatch errors that are liable to be confounded by SNPs. Also, there is a need to generate matched knockout controls for reference, which is laborious. In this work, we introduce an internal comparison strategy termed "IndoC," where features such as 'trace' and 'current signal intensity' of potentially modified sites are compared to similar sequence contexts on the same RNA molecule within the sample, alleviating the need for matched knockout controls. We first show that in an IVT model, 'trace' is able to distinguish between artificially generated SNPs and true pseudouridine (Ψ) modifications, both of which display highly similar mismatch profiles. We then apply IndoC on yeast and human ribosomal RNA to demonstrate that previously reported Ψ sites show marked changes in their trace and signal intensity profiles compared with their unmodified counterparts in the same dataset. Finally, we perform direct RNA sequencing of RNA containing Ψ intact with a chemical probe adduct (N-cyclohexyl-N'-ß-(4-methylmorpholinium) ethylcarbodiimide [CMC]) and show that CMC reactivity also induces changes in trace and signal intensity distributions in a Ψ specific manner, allowing their separation from high mismatch sites that display SNP-like behavior.

Artificial intelligence in microbial natural product drug discovery: current and emerging role.

Covering: up to the end of 2022Microorganisms are exceptional sources of a wide array of unique natural products and play a significant role in drug discovery. During the golden era, several life-saving antibiotics and anticancer agents were isolated from microbes; moreover, they are still widely used. However, difficulties in the isolation methods and repeated discoveries of the same molecules have caused a setback in the past. Artificial intelligence (AI) has had a profound impact on various research fields, and its application allows the effective performance of data analyses and predictions. With the advances in omics, it is possible to obtain a wealth of information for the identification, isolation, and target prediction of secondary metabolites. In this review, we discuss drug discovery based on natural products from microorganisms with the help of AI and machine learning.

Targeted epigenetic modulation using a DNA-based histone deacetylase inhibitor enhances cardiomyogenesis in mouse embryonic stem cells.

The epigenome has an essential role in orchestrating transcriptional activation and modulating key developmental processes. Previously, we developed a library of pyrrole-imidazole polyamides (PIPs) conjugated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, for the purpose of sequence-specific modification of epigenetics. Based on the gene expression profile of SAHA-PIPs and screening studies using the α-myosin heavy chain promoter-driven reporter and SAHA-PIP library, we identified that SAHA-PIP G activates cardiac-related genes. Studies in mouse ES cells showed that SAHA-PIP G could enhance the generation of spontaneous beating cells, which is consistent with upregulation of several cardiac-related genes. Moreover, ChIP-seq results confirmed that the upregulation of cardiac-related genes is highly correlated with epigenetic activation, relevant to the sequence-specific binding of SAHA-PIP G. This proof-of-concept study demonstrating the applicability of SAHA-PIP not only improves our understanding of epigenetic alterations involved in cardiomyogenesis but also provides a novel chemical-based strategy for stem cell differentiation.

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.

Discovery of 3-(benzofuran-2-ylmethyl)-1H-indole derivatives as potential autophagy inducers in cervical cancer cells.

In this manuscript we have documented the identification of a novel anticancer scaffold 3-(benzofuran-2-ylmethyl)-1H-indole. This scaffold has been designed by tweaking the known bisindolylmethane scaffold of natural products that display a wide range of biological activities. A series of 24 new conjugates have been synthesized and among them 5 derivatives exhibited IC50 values less than 40 µM against two cervical cancer cell lines SiHa and C33a. Further mechanistic studies of two compounds 3eb and 3ec revealed that the toxicity of these compounds was due to the effective induction of autophagy mediated cell death. The autophagy induction was confirmed by the progressive conversion of LC3I to LC3II and downregulation of p62 in cervical cancer cells.

Targeted suppression of metastasis regulatory transcription factor SOX2 in various cancer cell lines using a sequence-specific designer pyrrole-imidazole polyamide.

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.

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.

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.

Direct Observation of H3-H4 Octasome by High-Speed AFM.

Despite evidence that histone H3 and H4 proteins may act as the precursor for orientating the DNA sequence to form nucleosome structures, there is no direct evidence of the formed compact structure. Here, it is demonstrated that a histone H3-H4 octasome could be constructed without the involvement of histone H2A-H2B under in vitro reconstitution conditions. Atomic force microscopy was used to obtain the first direct observation of the octasome structure, which exhibited a similar core-protein size as that of a nucleosome but with a shorter core histone-binding DNA region. The octasome also displayed a one-step histone-dissociation pattern under heat treatment, distinct micrococcal nuclease and peplomycin accessibility, which suggests a different wrapping pattern to that in nucleosomes.

Chemical Control System of Epigenetics.

Epigenetics represents the inheritable changes to the chemical control system governing the gene expression with no ensuing changes to the underlying DNA sequence. Environment-mediated modification of the natural epigenetic interactions can perturb the cellular homeostasis and drive cells to a diseased state by switching therapeutically essential genes ON and OFF. Contemporary bioinformatics tools have revealed the structural chemical modifications of the epigenetic enzymes associated with several complex diseases, including cancers, immune disorders, and neurodegenerative disorders at the fundamental level. The amenable nature of the epigenetic enzymes to chemical modifications aided the screening and identification of synthetic small-molecule inhibitors. Continuing the current steady progress in the development of these small-molecule inhibitors as 'epi-drugs' in preclinical studies requires further advances to enable existing clinical barriers to be overcome. Recently, an epigenetic modifier complemented with selective DNA-binding small molecules was shown to function as an artificial biomimetic epigenetic code. Herein, we summarize the chemical aspects of the natural epigenetic control system and detail the recent advances in the synthetic strategies to mimic the genetic and epigenetic control system.

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.

Nucleosome Assembly Alters the Accessibility of the Antitumor Agent Duocarmycin B2 to Duplex DNA.

To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B2 on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B2 were significantly decreased in core DNA and increased at the histone-free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B2 to duplex DNA could advance its design as an antitumor agent.

Integrating epigenetic modulators into NanoScript for enhanced chondrogenesis of stem cells.

N-(4-Chloro-3-(trifluoromethyl)phenyl)-2-ethoxybenzamide (CTB) is a small molecule that functions by altering the chromatin architecture to modulate gene expression. We report a new CTB derivative with increased solubility and demonstrate CTB's functionality by conjugating it on the recently established NanoScript platform to enhance gene expression and induce stem cell differentiation. NanoScript is a nanoparticle-based artificial transcription factor that emulates the structure and function of transcription factor proteins (TFs) to effectively regulate endogenous gene expression. Modifying NanoScript with CTB will more closely replicate the TF structure and enhance CTB functionality and gene expression. To this end, we first conjugated CTB onto NanoScript and initiated a time-dependent increase in histone acetyltransferase activity. Next, because CTB is known to trigger the pathway involved in regulating Sox9, a master regulator of chondrogenic differentiation, we modifed a Sox9-specific NanoScript with CTB to enhance chondrogenic gene activity and differentiation. Because NanoScript is a tunable and robust platform, it has potential for various gene-regulating applications, such as stem cell differentiation.

Advancing small-molecule-based chemical biology with next-generation sequencing technologies.

Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening.

A Synthetic Transcriptional Activator of Genes Associated with the Retina in Human Dermal Fibroblasts.

Small molecules capable of modulating epigenetic signatures can activate the transcription of tissue-restricted genes in a totally unrelated cell type and have potential use in epigenetic therapy. To provide an example for an initial approach, we report here on one synthetic small-molecule compound-termed "SAHA-PIP X"-from our library of conjugates. This compound triggered histone acetylation accompanied by the transcription of retinal-tissue-related genes in human dermal fibroblasts (HDFs).

A Synthetic DNA-Binding Domain Guides Distinct Chromatin-Modifying Small Molecules to Activate an Identical Gene Network.

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands.