RESUMEN

Interactions of RNA with DNA are principles of gene expression control that have recently gained considerable attention. Among RNA-DNA interactions are R-loops and RNA-DNA hybrid G-quadruplexes, as well as RNA-DNA triplexes. It is proposed that RNA-DNA triplexes guide RNA-associated regulatory proteins to specific genomic locations, influencing transcription and epigenetic decision making. Although triplex formation initially was considered solely an in vitro event, recent progress in computational, biochemical, and biophysical methods support in vivo functionality with relevance for gene expression control. Here, we review the central methodology and biology of triplexes, outline paradigms required for triplex function, and provide examples of physiologically important triplex-forming long non-coding RNAs.

Asunto(s)

RESUMEN

Over the past decade, long non-coding RNAs (lncRNAs) have been recognized as key players in gene regulation, influencing genome organization and expression. The locus-specific binding of these non-coding RNAs (ncRNAs) to DNA involves either a non-covalent interaction with DNA-bound proteins or a direct sequence-specific interaction through the formation of RNA:DNA triplexes. In an effort to develop a novel strategy for characterizing a triple-helix formation, we employed atomic force microscopy (AFM) to visualize and study a regulatory RNA:DNA triplex formed between the Khps1 lncRNA and the enhancer of the proto-oncogene SPHK1. The analysis demonstrates the successful formation of RNA:DNA triplexes under various conditions of pH and temperature, indicating the effectiveness of the AFM strategy. Despite challenges in discriminating between the triple-helix and R-loop configurations, this approach opens new perspectives for investigating the role of lncRNAs in gene regulation at the single-molecule level.

Asunto(s)

RESUMEN

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.

Asunto(s)

RESUMEN

The use of DNA triplex association is advantageous for the reconfiguration of dynamic DNA nanostructures through pH alteration and can provide environmental control for both structural changes and molecular signaling. The combination of pH-induced triplex-forming oligonucleotide (TFOs) binding with toehold-mediated strand displacement has recently garnered significant attention in the field of structural DNA nanotechnology. While most previous studies use single-stranded DNA to displace or replace TFOs within the triplex, here we demonstrate that pH alteration allows a DNA duplex, with a toehold assistance, to displace TFOs from the components of another DNA duplex. We examined the dependence of this process on toehold length and show that the pH changes allow for cyclic oscillations between two molecular formations. We implemented the duplex/triplex design onto the surface of 2D DNA origami in the form outlining binary digits 0 or 1 and verified the oscillatory conformational changes between the two formations with atomic force microscopy.

Asunto(s)

RESUMEN

This study aimed to construct a novel DNA triplex molecular switch modified with DNA tetrahedron (DTMS-DT) with sensitive response to extracellular pH using a DNA tetrahedron as the anchoring unit and DNA triplex as the response unit. The results showed that the DTMS-DT had desirable pH sensitivity, excellent reversibility, outstanding anti-interference ability, and good biocompatibility. Confocal laser scanning microscopy suggested that the DTMS-DT could not only be stably anchored on the cell membrane but also be employed to dynamically monitor the change in extracellular pH. Compared with the reported probes for extracellular pH monitoring, the designed DNA tetrahedron-mediated triplex molecular switch exhibited higher cell surface stability and brought the pH-responsive unit closer to the cell membrane surface, making the results more reliable. In general, developing the DNA tetrahedron-based DNA triplex molecular switch is helpful for understanding and illustrating the pH dependent cell behaviors and disease diagnostics.

Asunto(s)

RESUMEN

Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex-triplex DNA motifs as pH-dependent building blocks in DNA origami. We investigate design rules for incorporating triplex forming oligonucleotides and noncanonical duplex-triplex crossovers in multilayer DNA origami objects. We use single-particle cryoelectron microscopy to elucidate the structural basis of triplex domains and of duplex-triplex crossovers. We find that duplex-triplex crossovers can complement and fully replace the canonical duplex-duplex crossovers within DNA origami objects, for example, to increase the crossover density for potentially greater rigidity and reduced interhelical spacing, and to create connections at sites where conventional crossovers may be undesirable. We also show the pH-induced formation of a DNA origami object stabilized entirely by triplex-mediated strand crossovers.

Asunto(s)

RESUMEN

DNA nanotechnology enables straightforward fabrication of user-defined and nanometer-precise templates for a cornucopia of different uses. To date, most of these DNA assemblies have been static, but dynamic structures are increasingly coming into view. The programmability of DNA not only allows for encoding of the DNA object shape but also it may be equally used in defining the mechanism of action and the type of stimuli-responsiveness of the dynamic structures. However, these "robotic" features of DNA nanostructures are usually demonstrated for only small, discrete, and device-like objects rather than for collectively behaving higher-order systems. Here, we show how a large-scale, two-dimensional (2D) and pH-responsive DNA origami-based lattice can be assembled into two different configurations ("open" and "closed" states) on a mica substrate and further switched from one to the other distinct state upon a pH change of the surrounding solution. The control over these two configurations is achieved by equipping the arms of the lattice-forming DNA origami units with "pH-latches" that form Hoogsteen-type triplexes at low pH. In short, we demonstrate how the electrostatic control over the adhesion and mobility of the DNA origami units on the surface can be used both in the large lattice formation (with the help of directed polymerization) and in the conformational switching of the whole lattice. To further emphasize the feasibility of the method, we also demonstrate the formation of pH-responsive 2D gold nanoparticle lattices. We believe this work can bridge the nanometer-precise DNA origami templates and higher-order large-scale systems with the stimuli-induced dynamicity.

Asunto(s)

RESUMEN

Long noncoding RNAs (lncRNAs) are involved in transcriptional regulation, and their deregulation is associated with the development of various human cancers, including prostate cancer (PCa). However, their underlying mechanisms remain unclear. In this study, lncRNAs that interact with DNA and regulate mRNA transcription in PCa were screened and identified to promote PCa development. First, 4195 protein-coding genes (PCGs, mRNAs) were obtained from the The Cancer Genome Atlas (TCGA) database, in which 1148 lncRNAs were differentially expressed in PCa. Then, 44,270 pairs of co-expression relationships were calculated between 612 lncRNAs and 2742 mRNAs, of which 42,596 (96%) were positively correlated. Among the 612 lncRNAs, 392 had the potential to interact with the promoter region to form DNA:DNA:RNA triplexes, from which lncRNA AD000684.2(AC002128.1) was selected for further validation. AC002128.1 was highly expressed in PCa. Furthermore, AD000684.2 positively regulated the expression of the correlated genes. In addition, AD000684.2 formed RNA-DNA triplexes with the promoter region of the regulated genes. Functional assays also demonstrated that lncRNA AD000684.2 promotes cell proliferation and motility, as well as inhibits apoptosis, in PCa cell lines. The results suggest that AD000684.2 could positively regulate the transcription of target genes via triplex structures and serve as a candidate prognostic biomarker and target for new therapies in human PCa.

Asunto(s)

RESUMEN

DNA switches that can change conformation in response to certain wavelengths of light could enable rapid and noninvasive control of chemical processes for a wide range of applications. However, most current photoresponsive DNA switches are limited by either irreversible switching or reversible switching with impractically slow kinetics. Here, we report the design of an intramolecular triplex photoswitch (TPS) design based on single-stranded DNA that undergoes rapid and reversible photoswitching between folded and unfolded states through isomerization of internal azobenzene modifications. After optimizing the performance of our photoswitch design, we used molecular dynamics simulations to reveal how individual azobenzenes contribute to the stabilization or destabilization of the triplex depending on their photoisomerization state. By coupling our TPS to an existing aptamer, we can reversibly modulate its binding affinity with less than 15 s of UV light exposure. We further demonstrate reproducible shifting in affinity over multiple cycles of UV and blue light irradiation without substantial photobleaching.

Asunto(s)

RESUMEN

Trinucleotide repeat instability is a driver of human disease. Large expansions of (GAA)n repeats in the first intron of the FXN gene are the cause Friedreich's ataxia (FRDA), a progressive degenerative disorder which cannot yet be prevented or treated. (GAA)n repeat instability arises during both replication-dependent processes, such as cell division and intergenerational transmission, as well as in terminally differentiated somatic tissues. Here, we provide a brief historical overview on the discovery of (GAA)n repeat expansions and their association to FRDA, followed by recent advances in the identification of triplex H-DNA formation and replication fork stalling. The main body of this review focuses on the last decade of progress in understanding the mechanism of (GAA)n repeat instability during DNA replication and/or DNA repair. We propose that the discovery of additional mechanisms of (GAA)n repeat instability can be achieved via both comparative approaches to other repeat expansion diseases and genome-wide association studies. Finally, we discuss the advances towards FRDA prevention or amelioration that specifically target (GAA)n repeat expansions.

Asunto(s)

RESUMEN

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5mC with 8oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5mC with 8oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5mC with 8oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.

RESUMEN

This study explored the relationship between 3D genome organization and RNA-DNA triplex-forming sites of long noncoding RNAs (lncRNAs), a group of RNAs that do not code for proteins but are important factors regulating different aspects of genome activity. The triplex-forming sites of anti-sense cardiac lncRNA GATA6-AS1 derived from DBD-Capture-Seq were examined and compared to modular features of 3D genome organization called topologically associated domains (TADs) obtained from Hi-C data. It was found that GATA6-AS1 triplex-forming sites are positioned non-randomly in TADs and their boundaries. The triplex sites showed a preference for TAD boundaries over internal regions of TADs. Computational prediction analysis indicated that CTCF, the key protein involved in TAD specification, may interact with GATA6-AS1, and their binding sites correlate with each other. Examining locations of repeat elements in the genome suggests that the ability of lncRNA GATA6-AS1 to form triplex sites with many genomic locations may be achieved by the rapid expansion of different repeat elements. Some of the triplex-forming sites were found to be positioned in regions that undergo dynamic chromatin organization events such as loss/gain of TAD boundaries during cardiac differentiation. These observed associations suggest that lncRNA-DNA triplex formation may contribute to the specification of TADs in 3D genome organization.

RESUMEN

G-quadruplex (G4)-prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites. Furthermore, we show that DNA double-strand breaks at the ectopic site are dependent on the nuclease Mus81. Hypermutagenesis during sister chromatid repair extends several kilobases from the G4 site and breaks at the G4 site resulting in microhomology-mediated translocations. To determine whether H3 or G4 structures are responsible for homopurine/homopyrimidine tract instability, we derived constructs and cell lines from the PKD1 repeat, which can only form H3 or G4 structures. Under normal growth conditions, we found that G4 cell lines lost the G4 consensus sequence early during clonal outgrowth, whereas H3 cells showed DNA instability early during outgrowth but only lost reporter gene expression after prolonged growth. Thus, both the H3 and G4 non-B conformation DNAs exhibit genomic instability, but they respond differently to endogenous replication stress. Our results show that the outcomes of replication-dependent double-strand breaks at non-B-DNAs model the instability observed in microhomology-mediated break-induced replication (BIR). Marked variability in the frequency of mutagenesis during BIR suggests possible dynamic heterogeneity in the BIR replisome.

Asunto(s)

RESUMEN

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer with a poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in human cancers. Krüppel-like Factor 5 (KLF5) is a key oncogenic transcription factor in BLBC. However, the underlying mechanism of mutual regulation between KLF5 and lncRNA remains largely unknown. Here, we demonstrate that lncRNA KPRT4 promotes BLBC cell proliferation in vitro and in vivo. Mechanistically, KLF5 directly binds to the promoter of KPRT4 to promote KPRT4 transcription. Reciprocally, KPRT4 recruits the YB-1 transcription factor to the KLF5 promoter by interacting with YB-1 at its 5' domain and forming an RNA-DNA-DNA triplex structure at its 3' domain, resulting in enhanced transcription of KLF5 and ultimately establishing a feedforward circuit to promote cell proliferation. Moreover, the antisense oligonucleotide (ASO)-based therapy targeting KPRT4 substantially attenuated tumor growth in vivo. Clinically, the expression levels of YB-1, KLF5 and KPRT4 are positively correlated in clinical breast specimens. Together, our data suggest that KPRT4 is a major molecule for BLBC progression and that the feedforward circuit between KLF5 and KPRT4 may represent a potential therapeutic target in BLBC.

Asunto(s)

RESUMEN

Herein, we present a class of potent triplex DNA binding ligands derived from the natural product quercetin, which is the first of its kind that has ever been reported in the literature. The binding of 5-substituted quercetin derivatives (3, 3', 4', 7-tetramethoxyflavonoids) to triplex and duplex DNA was investigated using several biophysical tools, including thermal denaturation monitored by UV, circular dichroism, differential scanning calorimetry, and isothermal titration calorimetry. Experimental data reveal that several 5-substituted 3, 3', 4', 7-tetramethoxyflavonoids have remarkable effects on binding to DNA triple helices, and they do not influence the double-helical DNA structures. A few derivatives such as compounds 5 and 7 have comparable (if not better) binding affinities to neomycin, a well-known DNA triplex binding ligand, under the same conditions. The amino-containing side chains at the 5-position of 3, 3', 4', 7-tetramethoxyflavonoids are crucial for the observed binding affinity and specificity.

Asunto(s)

RESUMEN

Limitations of clinical platinum(II) therapeutics include systemic toxicity and inherent resistance. Modern approaches, therefore, seek new ways to deliver active platinum(II) to discrete nucleic acid targets. In the field of antigene therapy, triplex-forming oligonucleotides (TFOs) have attracted interest for their ability to specifically recognise extended duplex DNA targets. Here, we report a click chemistry based approach that combines alkyne-modified TFOs with azide-bearing cis-platinum(II) complexes-based on cisplatin, oxaliplatin, and carboplatin motifs-to generate a library of PtII -TFO hybrids. These constructs can be assembled modularly and enable directed platinum(II) crosslinking to purine nucleobases on the target sequence under the guidance of the TFO. By covalently incorporating modifications of thiazole orange-a known DNA-intercalating fluorophore-into PtII -TFOs constructs, enhanced target binding and discrimination between target and off-target sequences was achieved.

Asunto(s)

RESUMEN

BACKGROUND: Emerging evidence suggests that long noncoding RNAs (lncRNAs) play crucial roles in various cancers. In the present study, we aim to investigate the function and molecular mechanism of an up-regulated and survival-associated lncRNA, LINC00525, in lung adenocarcinoma (LUAD). METHODS: The expression level of LINC00525 in tissues was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and in situ hybridization (ISH). The functional role of LINC00525 in LUAD was investigated using gain-and loss-of-function approaches, both in vivo and in vitro. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), triplex-capture assay, dual-luciferase assay, gene expression microarray, and bioinformatics analysis were used to investigate the potential underlying mechanisms involved. RESULTS: LINC00525 is highly expressed in LUAD cells and tissues. Survival analysis indicated that upregulation of LINC00525 was associated with poor prognosis in patients with LUAD patients. Knockdown of LINC00525 inhibited cell proliferation and cell cycle progression in vitro. In xenograft models, LINC00525 knockdown suppressed tumor growth and tumorigenesis of tumor-bearing mice. Mechanistically, LINC00525 epigenetically suppressed p21 transcription by guiding Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) to the p21 promoter through an formation of RNA-DNA triplex with the p21 promoter, leading to increased trimethylation of lysine 27 on histone 3 (H3K27me3) of the p21 promoter. In addition, LINC00525 repressed p21 expression post-transcriptionally by enhancing p21 mRNA decay. LINC00525 promoted p21 mRNA decay by competitively binding to RNA Binding Motif Single Stranded Interacting Protein 2 (RBMS2). CONCLUSION: Our findings demonstrate that LINC00525 promotes the progression of LUAD by reducing the transcription and stability of p21 mRNA in concert with EZH2 and RBMS2, thus suggesting that LINC00525 may be a potential therapeutic target for clinical intervention in LUAD.

Asunto(s)

RESUMEN

BACKGROUND: Chromosomes are organized into units called topologically associated domains (TADs). TADs dictate regulatory landscapes and other DNA-dependent processes. Even though various factors that contribute to the specification of TADs have been proposed, the mechanism is not fully understood. Understanding the process for specification and maintenance of these units is essential in dissecting cellular processes and disease mechanisms. RESULTS: In this study, we report a genome-wide study that considers the idea of long noncoding RNAs (lncRNAs) mediating chromatin organization using lncRNA:DNA triplex-forming sites (TFSs). By analyzing the TFSs of expressed lncRNAs in multiple cell lines, we find that they are enriched in TADs, their boundaries, and loop anchors. However, they are evenly distributed across different regions of a TAD showing no preference for any specific portions within TADs. No relationship is observed between the locations of these TFSs and CTCF binding sites. However, TFSs are located not just in promoter regions but also in intronic, intergenic, and 3'UTR regions. We also show these triplex-forming sites can be used as predictors in machine learning models to discriminate TADs from other genomic regions. Finally, we compile a list of important "TAD-lncRNAs" which are top predictors for TADs identification. CONCLUSIONS: Our observations advocate the idea that lncRNA:DNA TFSs are positioned at specific areas of the genome organization and are important predictors for TADs. LncRNA:DNA triplex formation most likely is a general mechanism of action exhibited by some lncRNAs, not just for direct gene regulation but also to mediate 3D chromatin organization.

Asunto(s)

RESUMEN

Eukaryotic genomes contain a large number of pyrimidine-purine rich regions and such regions can assume varied DNA conformations, including triple-stranded structures. These structures have fascinated scientists because of their considerable therapeutic applications. These structures have also profound implications in the field of nanotechnology as they can be used to develop DNA-based nanostructures and materials. Therefore, for any application, it is important to understand the formation of triplex structures, both in quantitative and qualitative terms. A combination of gel electrophoresis, UV-thermal denaturation and circular dichroism (CD) spectroscopy was used to investigate the formation of inter- as well as intramolecular triplex, in pyrimidine motif at BOLF1 gene of human herpesvirus 4 (HH4) genome. This gene codes for inner tegument protein, which plays crucial roles in viral replication. The said oligopurine•oligopyrimidine duplex was targeted via a designed triple helix forming oligopyrimidine nucleotide (TFO) in intermolecular as well as intramolecular fashion. Our studies revealed that intramolecular triplex formation takes place at acidic as well as at neutral pH; whereas low pH is required for its intermolecular version. This comparative study between inter- and intramolecular triplex allowed us to demonstrate that intramolecular structure is more stable to its intermolecular counterpart. Numerous models for mono-, bi- and trimolecular structures adopted by these DNA sequences have been suggested. This report adds to our existing knowledge about DNA triple helical structures.

Asunto(s)

RESUMEN

miRNAs, a class of endogenous noncoding RNAs, are involved in many crucial biological processes, which have emerged as a new set of biomarkers for disease theranostics. Exploring efficient signal amplification strategy is highly desired to pursue a highly sensitive miRNA biosensing platform. DNA nanotechnology shows great promise in the fabrication of amplified miRNA biosensors. In this work, a novel DNA walking and rolling nanomachine is developed for highly sensitive and selective detection of miRNA. Particularly, this approach programs two forms of dynamic DNA nanomachines powered by corresponding enzymes, which are well integrated. It is able to achieve a limit of detection as low as 39 × 10-18 m, along with excellent anti-interfering performance and clinical applications. In addition, by designing pH-controlled detachable intermolecular DNA triplex, the main sensing elements can be conveniently reset, which fulfills the requirements of point-of-care profiling of miRNA. The high consistency between the proposed approach and quantitative real-time polymerase chain reaction validates the robustness and reliability. Therefore, it is anticipated that the DNA walking and rolling nanomachine has attractive application prospects in miRNA assay for biological researches and clinical diagnosis.

Asunto(s)