Triplex Structure Research Articles

Modification of the aminopyridine unit of 2′-deoxyaminopyridinyl-pseudocytidine allowing triplex formation at CG interruptions in homopurine sequences

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.

LncRNA MIR100HG promotes cell proliferation in triple-negative breast cancer through triplex formation with p27 loci

Triple-negative breast cancer (TNBC) exhibits poor prognosis, with high metastasis and low survival. Long non-coding RNAs (lncRNAs) play critical roles in tumor progression. Here, we identified lncRNA MIR100HG as a pro-oncogene for TNBC progression. Knockdown of MIR100HG decreased cell proliferation and induced cell arrest in the G1 phase, whereas overexpression of MIR100HG significantly increased cell proliferation. Furthermore, MIR100HG regulated the p27 gene to control the cell cycle, and subsequently impacted the progression of TNBC. In analyzing its underlying mechanism, bioinformatics prediction and experimental data demonstrated that MIR100HG participated in the formation of RNA–DNA triplex structures. MIR100HG in The Cancer Genome Atlas (TCGA) and breast cancer cell lines showed higher expression in TNBC than in other tumor types with poor prognosis. In conclusion, our data indicated a novel working pattern of lncRNA in TNBC progression, which may be a potential therapeutic target in such cancers.

A label-free light-up fluorescent sensing platform based upon hybridization chain reaction amplification and DNA triplex assembly

A label-free light-up fluorescent sensing strategy using hybridization chain reaction (HCR) amplification and DNA triplex assembly has been developed. Remarkably, the proposed fluorescence assay is successfully applied to the determination of avian influenza A (H7N9) virus DNA and thrombin. Herein, in the presence of targets, the target DNA/initiator triggers a cascade of hybridization events between H1 and H2 that yields nicked double helices analogous to alternating copolymers. With the additions of triplex-forming oligonucleotide (TFO) and berberine, the triplex structures form between HCR products and TFO. Then, a large amount of berberine can bind to the triplex structures and the sensing system exhibits a dramatic increase in the fluorescence intensity at 530 nm. Under optimal conditions, the label-free fluorescent sensing platform shows sensitive responses to H7N9 virus DNA and thrombin in the range of 0.2–100 nM and 0.5–200 nM, respectively. The detection limits of H7N9 virus DNA and thrombin are as low as 0.14 nM and 0.32 nM, respectively. Owing to the simplicity and low cost, the proposed strategy can be used in other biomarkers assays, providing a promising tool for clinical diagnosis and biomedical detection.

Bifacial Nucleobases for Hexaplex Formation in Aqueous Solution.

Although DNA can form triplex and quadruplex structures through hydrogen bonds, design and preparation of structures with more than five strands is difficult even when artificial nucleic acids are used. Herein we report a hexaplex formed by oligomers of artificial nucleic acids bearing bifacial molecules on d-threoninol. Aminopyrimidine and cyanuric acid derivatives were selected as bases because they have complementary hydrogen bonding patterns. The complex formed by aminopyrimidine and cyanuric acid decamers melted with large hysteresis. Hexaplex formation was indicated by gel electrophoresis, size exclusion chromatography and atomic force microscopy imaging, and proven directly through native mass spectrometry. CD measurements and molecular dynamics simulations indicated that the hexaplex adopts a helical structure. The hexaplex formation was highly dependent on pH and the presence of divalent cations. The hexaplex was stable in aqueous solution, and its unique structure and properties may lead to novel nanostructures, molecular assemblies, metal sensors, and ion channels.

LncRNA PAPAS tethered to the rDNA enhancer recruits hypophosphorylated CHD4/NuRD to repress rRNA synthesis at elevated temperatures.

Attenuation of pre-rRNA synthesis in response to elevated temperature is accompanied by increased levels of PAPAS ("promoter and pre-rRNA antisense"), a long noncoding RNA (lncRNA) that is transcribed in an orientation antisense to pre-rRNA. Here we show that PAPAS interacts directly with DNA, forming a DNA-RNA triplex structure that tethers PAPAS to a stretch of purines within the enhancer region, thereby guiding associated CHD4/NuRD (nucleosome remodeling and deacetylation) to the rDNA promoter. Protein-RNA interaction experiments combined with RNA secondary structure mapping revealed that the N-terminal part of CHD4 interacts with an unstructured A-rich region in PAPAS. Deletion or mutation of this sequence abolishes the interaction with CHD4. Stress-dependent up-regulation of PAPAS is accompanied by dephosphorylation of CHD4 at three serine residues, which enhances the interaction of CHD4/NuRD with RNA and reinforces repression of rDNA transcription. The results emphasize the function of lncRNAs in guiding chromatin remodeling complexes to specific genomic loci and uncover a phosphorylation-dependent mechanism of CHD4/NuRD-mediated transcriptional regulation.

Improved Force Fields for Peptide Nucleic Acids with Optimized Backbone Torsion Parameters.

Peptide nucleic acids are promising nucleic acid analogs for antisense therapies as they can form stable duplex and triplex structures with DNA and RNA. Computational studies of PNA-containing duplexes and triplexes are an important component for guiding their design, yet existing force fields have not been well validated and parametrized with modern computational capabilities. We present updated CHARMM and Amber force fields for PNA that greatly improve the stability of simulated PNA-containing duplexes and triplexes in comparison with experimental structures and allow such systems to be studied on microsecond time scales. The force field modifications focus on reparametrized PNA backbone torsion angles to match high-level quantum mechanics reference energies for a model compound. The microsecond simulations of PNA-PNA, PNA-DNA, PNA-RNA, and PNA-DNA-PNA complexes also allowed a comprehensive analysis of hydration and ion interactions with such systems.

Exonuclease III-aided recycling amplification of proximity ligation assay using thymine-melamine-thymine triplex structure for ultrasensitive fluorometric determination of melamine

A fluorescence sensing strategy was described for the determination of melamine with ultrasensitivity and high specificity. This strategy relies on the combination of proximity ligation assay (PLA)-modulated thymine–melamine–thymine (T–melamine–T) triplex structure along with exonuclease III (Exo III)-aided recycling amplification. The reaction system involves three DNA probes (poly A, tDNA and molecular beacon (MB)). Probe ploy A is an adenine enriched sequence containing an apurinic/apyrimidinic site (AP site). It can provide a hydrophobic vacancy for specially recognizing melamine to bind the nucleobase through pseudo-base pairing and participate in PLA modulated T-melamine-T triplex structure. Probe tDNA can form T-melamine-T with probe poly A via PLA only in the presence of melamine. Probe MB is modified with a fluorophore at its 5′-terminus and a quencher at an internal position, which can self-hybridize to form a hairpin structure, positioning the fluorophore in close proximity to the quencher. Therefore, the binding of MB did not yield any fluorescence signal. However, in the presence of melamine, a T-melamine-T triplex structure is formed through PLA. Probe MB can hybridize with t T-melamine-T triplex structure to yield a substrate for interaction with Exo III. As a result, the T-melamine-T triplex structure is released. Next, the released T-melamine-T triplex structure will hybridize with another MB probe to achieve the Exo III-aided recycle amplification. This is the first example of PLA-modulated T-melamine-T triplex structure coupled with Exo III-aided recycling amplification. Under optimal conditions, the assay has a detection range over 4 orders of magnitude and a limit of detection of 4.4 nM. Moreover, the assay is rapid, simple and inexpensive. Hence, the strategy may open new avenues for ultrasensitive and highly specific detection of melamine encounted in food analysis.

Biological and nanotechnological applications using interactions between ionic liquids and nucleic acids.

Nucleic acids have emerged as powerful biological and nanotechnological tools. In biological and nanotechnological experiments, methods of extracting and purifying nucleic acids from various types of cells and their storage are critical for obtaining reproducible experimental results. In nanotechnological experiments, methods for regulating the conformational polymorphism of nucleic acids and increasing sequence selectivity for base pairing of nucleic acids are important for developing nucleic acid-based nanomaterials. However, dearth of media that foster favourable behaviour of nucleic acids has been a bottleneck for promoting the biology and nanotechnology using the nucleic acids. Ionic liquids (ILs) are solvents that may be potentially used for controlling the properties of the nucleic acids. Here, we review researches regarding the behaviour of nucleic acids in ILs. The efficiency of extraction and purification of nucleic acids from biological samples is increased by IL addition. Moreover, nucleic acids in ILs show long-term stability, which maintains their structures and enhances nuclease resistance. Nucleic acids in ILs can be used directly in polymerase chain reaction and gene expression analysis with high efficiency. Moreover, the stabilities of the nucleic acids for duplex, triplex, and quadruplex (G-quadruplex and i-motif) structures change drastically with IL cation-nucleic acid interactions. Highly sensitive DNA sensors have been developed based on the unique changes in the stability of nucleic acids in ILs. The behaviours of nucleic acids in ILs detailed here should be useful in the design of nucleic acids to use as biological and nanotechnological tools.

Thermal stability and conformation of DNA and proteins under the confined condition in the matrix of hydrogels.

Spatially confined environments are seen in biological systems and in the fields of biotechnology and nanotechnology. The confinement restricts the conformational space of polymeric molecules and increasing the degree of molecular crowding. Here, we developed preparation methods for agarose and polyacrylamide gels applicable to UV spectroscopy that can evaluate the confinement effects on DNA and protein structures. Measurements of UV absorbance and CD spectra showed no significant effect of the confinement in the porous media of agarose gels on the base-pair stability of DNA polynucleotides [poly(dA)/poly(dT)] and oligonucleotides (hairpin, duplex, and triplex structures). On the other hand, a highly confined environment created by polyacrylamide gels at high concentrations increased the stability of polynucleotides while leaving that of oligonucleotides unaffected. The changes in the base-pair stability of the polynucleotides were accompanied by the perturbation of the helical conformation. The polyacrylamide gels prepared in this study were also used for the studies on proteins (lysozyme, bovine serum albumin, and myoglobin). The effects on the proteins were different from the effects on DNA structures, suggesting different nature of interactions within the gel. The experimental methods and results are useful to understand the physical properties of nucleic acids and proteins under confined conditions.

Peptide Nucleic Acids as a Tool for Site-Specific Gene Editing.

Peptide nucleic acids (PNAs) can bind duplex DNA in a sequence-targeted manner, forming a triplex structure capable of inducing DNA repair and producing specific genome modifications. Since the first description of PNA-mediated gene editing in cell free extracts, PNAs have been used to successfully correct human disease-causing mutations in cell culture and in vivo in preclinical mouse models. Gene correction via PNAs has resulted in clinically-relevant functional protein restoration and disease improvement, with low off-target genome effects, indicating a strong therapeutic potential for PNAs in the treatment or cure of genetic disorders. This review discusses the progress that has been made in developing PNAs as an effective, targeted agent for gene editing, with an emphasis on recent in vivo, nanoparticle-based strategies.

Single step production of Cas9 mRNA for zygote injection.

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Highly sensitive electrochemical assay for Nosema bombycis gene DNA PTP1 via conformational switch of DNA nanostructures regulated by H+ from LAMP

The portable and rapid detection of biomolecules via pH meters to monitor the concentration of hydrogen ions (H+) from biological reactions (e.g. loop-mediated isothermal amplification, LAMP) has attracted research interest. However, this assay strategy suffered from inherent drawback of low sensitivity, resulting in great limitations in practical applications. Herein, a novel electrochemical biosensor was constructed for highly sensitive detection of Nosema bombycis gene DNA (PTP1) through transducing chemical stimuli H+ from PTP1-based LAMP into electrochemical output signal of electroactive ferrocene (Fc). With use of target PTP1 as the template, the H+ from LAMP induced the conformational switch of pH-responsive DNA nanostructures (DNA NSs, Fc-Sp@Ts) that was assembled by the hybridization of Fc-labeled signal probe (Fc-Sp) with DNA-based receptor (Ts). Due to the folding of Ts into stable triplex structure at decreased pH, the configuration change of Fc-Sp@Ts led to the releasing of Fc-Sp, which was subsequently immobilized in the electrode interface through the hybridization with the capture probe modified with -SH (SH-Cp), generating amplified electrochemical signal from Fc. The developed biosensor for PTP1 exhibited a reliable linear range of 1 fg µL−1 to 50 ng µL−1 with the limit of detection of 0.31 fg µL−1. Thus, by the regulation of H+ from LAMP reaction on DNA NSs allostery, this novel and simple transduction scheme would be interesting and promising to open up a novel analytical route for sensitive monitoring of different target DNAs in related disease diagnosis.

Triplex target sites of MEG3 RNA-chromatin interactions

Many long noncoding RNAs are bound to chromatin. MEG3 binds to multiple different genomic locations, containing GA-rich motifs, and form RNA-DNA triplex structures. In this work, we test whether the MEG3 binding sites are specific enough to be regulated by a particular lncRNA. We show that at least in the case of MEG3, a subset of the triplex target sites (TTS) is able to hybridize with various different RNAs almost irrespectively of their sequences. Nowadays, the role of chromatin bound RNAs in the formation of 3D chromatin structure is actively discussed. We speculate that such universal TTSs may contribute to establishing long-distance chromosomal contacts.

A pH-controlled bidirectionally pure DNA hydrogel: reversible self-assembly and fluorescence monitoring.

A concept for a pure DNA hydrogel has been demonstrated in which triplex structures act as the core components and are assisted by single strands and artfully designed Y-shaped structures. Typically, the triplex structure which is based on protonated cytosine-guanine-cytosine (C-G·C+) is formed at pH 5.0 and disassembles at pH 7.0, whereas the thymine-adenine-thymine (T-A·T)-based triplex structure is formed at pH 7.0 and disassembles at pH 10.0. Therefore, such triplex DNA structure-based pure DNA hydrogels provide pH-controlled reversible self-assembly of DNA structures with a transition between gel and liquid states. More significantly, the introduction of both a fluorophore (FAM) and a quencher (BHQ1) to the hydrogels provides an innovative method for monitoring the self-assembly and disassembly processes through a fluorescence technology.

DNA triplex structure, thermodynamics, and destabilisation: insight from molecular simulations.

Molecular dynamics simulations are used to elucidate the structure and thermodynamics of DNA triplexes associated with the neurodegenerative disease Friedreich's ataxia (FRDA), as well as complexes of these triplexes with the small molecule netropsin, which is known to destabilise triplexes. The ability of molecular simulations in explicit solvent to accurately capture triplex thermodynamics is verified for the first time, with the free energy to dissociate a 15-base antiparallel purine triplex-forming oligomer (TFO) from the duplex found to be slightly higher than reported experimentally. The presence of netropsin in the minor groove destabilises the triplex as expected, reducing the dissociation free energy by approximately 50%. Netropsin binding is associated with localised narrowing of the minor groove near netropsin, an effect that has previously been under contention. This leads to localised widening of the major groove, weakening hydrogen bonds between the TFO and duplex. Consequently, destabilisation is found to be highly localised, occurring only when netropsin is bound directly opposite the TFO. The simulations also suggest that near saturation of the minor groove with ligand is required for complete triplex dissociation. A structural analysis of the DNA triplexes that can form with the FRDA-related duplex sequence indicates that the triplex with a parallel homopyrimidine TFO is likely to be more stable than the antiparallel homopurine-TFO triplex, which may have implications for disease onset and treatment.

Design and Characterization of pH-Triggered DNA Nanoswitches and Nanodevices Based on DNA Triplex Structures.

Triplex DNA is becoming a very useful domain to design pH-triggered DNA nanoswitches and nanodevices. The high versatility and programmability of triplex DNA interactions allows the integration of pH-controllable modules into DNA-based reactions and self-assembly processes. Here, we describe the procedure to characterize DNA-based triplex nanoswitches and more in general pH-triggered structure-switching mechanisms. Procedures to characterize pH-triggered DNA nanodevices will be useful for many applications in the field of biosensing, drug delivery systems and smart nanomaterials.

Purine- and pyrimidine-triple-helix-forming oligonucleotides recognize qualitatively different target sites at the ribosomal DNA locus.

Triplexes are noncanonical DNA structures, which are functionally associated with regulation of gene expression through ncRNA targeting to chromatin. Based on the rules of Hoogsteen base-pairing, polypurine sequences of a duplex can potentially form triplex structures with single-stranded oligonucleotides. Prediction of triplex-forming sequences by bioinformatics analyses have revealed enrichment of potential triplex targeting sites (TTS) at regulatory elements, mainly in promoters and enhancers, suggesting a potential function of RNA–DNA triplexes in transcriptional regulation. Here, we have quantitatively evaluated the potential of different sequences of human and mouse ribosomal RNA genes (rDNA) to form triplexes at different salt and pH conditions. We show by biochemical and biophysical approaches that some of these predicted sequences form triplexes with high affinity, following the canonical rules for triplex formation. We further show that RNA triplex-forming oligos (TFOs) are more stable than their DNA counterpart, and point mutations strongly affect triplex formation. We further show differential sequence requirements of pyrimidine and purine TFO sequences for efficient binding, depending on the G–C content of the TTS. The unexpected sequence specificity, revealing distinct sequence requirements for purine and pyrimidine TFOs, shows that in addition to the Hoogsteen pairing rules, a sequence code and mutations have to be taken into account to predict genomic TTS.

Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA

DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA) offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures

The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.

Microfluidic Lithography of Bioinspired Helical Micromotors.

Considerable efforts have been devoted to developing artificial micro/nanomotors that can convert energy into movement. A flow lithography integrated microfluidic spinning and spiraling system is developed for the continuous generation of bioinspired helical micromotors. Because the generation processes could be precisely tuned by adjusting the flow rates and the illuminating frequency, the length, diameter, and pitch of the helical micromotors were highly controllable. Benefiting from the fast online gelation and polymerization, the resultant helical micromotors could be imparted with Janus, triplex, and core-shell cross-sectional structures that have never been achieved by other methods. Owing to the spatially controlled encapsulation of functional nanoparticles in the microstructures, the helical micromotors can perform locomotion not only by magnetically actuated rotation or corkscrew motion but also through chemically powered catalytic reaction.