Hairpin Form Research Articles

Impact of Single Acyclic Phosphonate Nucleotide (ZNA) Modifications on DNA Duplex Stability.

An acyclic phosphonate-linked nucleic acid backbone (ZNA) demonstrated the capability to support duplex formation and propagate genetic information in vivo, unveiling its potential for evolution into a synthetic genetic system (XNA). To determine the structural impact of such modification, modified Dickerson Drew DNA dodecamers (DDDs) were prepared by solid phase synthesis, each containing either an (R) or (S) isomeric form of a cytosine ZNA nucleotide. While the DDD is known to adopt a stable duplex, both duplex and hairpin forms were simultaneously observed for both modified oligonucleotides by NMR spectroscopy over a broad temperature range (5-65 °C). Diffusion-ordered spectroscopy (DOSY) experiments allowed to separate duplex and hairpin signals based on the different diffusion constants of both conformational states. For the oligomer containing (R)-ZNA, only the duplex form occurred at 5 °C, while it was not possible to determine by NMR a single hairpin conformation at higher temperatures. In the case of the (S)-ZNA nucleoside modified oligomer, both hairpin and duplex forms were observable at 0 °C, while a single hairpin conformation was detected at 37 °C, suggesting a higher destabilizing effect on dsDNA.

DNA minicircles capable of forming a variety of non-canonical structural motifs.

Although more than 10% of the human genome has the potential to fold into non-B DNA, the formation of non-canonical structural motifs as part of long dsDNA chains are usually considered as unfavorable from a thermodynamic point of view. However, recent experiments have confirmed that non-canonical motifs do exist and are non-randomly distributed in genomic DNA. This distribution is highly dependent not only on the DNA sequence but also on various other factors such as environmental conditions, DNA topology and the expression of specific cellular factors in different cell types. In this study, we describe a new strategy used in the preparation of DNA minicircles containing different non-canonical motifs which arise as a result of imperfect base pairing between complementary strands. The approach exploits the fact that imperfections in the pairing of complementary strands thermodynamically weaken the dsDNA structure at the expense of enhancing the formation of non-canonical motifs. In this study, a completely different concept of stable integration of a non-canonical motif into dsDNA is presented. Our approach allows the integration of various types of non-canonical motifs into the dsDNA structure such as hairpin, cruciform, G-quadruplex and i-motif forms but also combinations of these forms. Small DNA minicircles have recently become the subject of considerable interest in both fundamental research and in terms of their potential therapeutic applications.

Synthesis and properties of RNA constrained by a 2'-O-disulfide bridge.

We recently reported the properties of RNA hairpins constrained by a dimethylene (DME) disulfide (S-S) linker incorporated between two adjacent nucleosides in the loop and showed that this linker locked the hairpin conformation thus disturbing the duplex/hairpin equilibrium. We have now investigated the influence of the length of the linker and synthesized oligoribonucleotides containing diethylene (DEE) and dipropylene (DPE) S-S bridges. This was achieved via the preparation of building blocks, namely 2'-O-acetylthioethyl (2'-O-AcSE) and 2'-O-acetylthiopropyl (2'-O-AcSP) uridine phosphoramidites, which were successfully incorporated into RNA sequences. Thermal denaturation analysis revealed that the DEE and DPE disulfide bridges destabilize RNA duplexes but do not disrupt the hairpin conformation. Furthermore, our investigation of the duplex/hairpin equilibrium indicated that sequences modified with DME and DEE S-S linkers predominantly lock the hairpin form, whereas the DPE S-S linker provides flexibility. These findings highlight the potential of S-S linkers to study RNA interactions.

Recycle Pekat (Perca Kain Tenunan) Flores Dalam Meningkatkan Kreativitas Mahasiswa Dan Kepedulian Lingkungan

Woven fabric is a decorative variety in Indonesia that is widespread in every region of the archipelago from Sabang to Merauke, characterized by various motifs, patterns, colors and meanings. Woven cloth developed in East Nusa Tenggara is a craft art that has been taught to children and grandchildren from generation to generation. However, it is a shame because currently most of the younger generation cannot weave. This will have an impact on the extinction of ikat fabrics. Efforts made to ensure that woven fabric does not become extinct are by re-preserving its existence and re-introducing its use. One way to preserve it is by recycling woven fabric patchwork waste into products in the form of earrings, necklaces, bracelets, hairpins, brooches, bags and hats which have several benefits such as preserving local cultural products, namely typical Flores woven fabrics. In an effort to produce products, of course there are stages, namely the preparation stage of materials and tools, the production stage, packaging to marketing and then continuing with product marketing techniques.

In Silico Evaluation of the PCR Performance of Different Tests for Detection of WSSV

In this study, the primers of different protocols for the detection of White Spot Syndrome Virus (WSSV) were analyzed in silico to evaluate their potential performance in PCR. As with any biological entity, this virus evolves constantly. Thus, this analysis showed that a few primers, including those recommended by the World Organization for Animal Health (WOAH), might mismatch with some isolates of WSSV, specially with isolates more recently sequenced. Furthermore, a set of primers recommended by WOAH, showed the potential to self-dimer and form hairpin loop structures, which could affect the efficiency of PCR, resulting in an inaccurate diagnostic result. From our perspective, and considering the evolutionary trajectory of this virus, it may be time for the WOAH to update the PCR protocols recommended for WSSV detection, which remains as a highly prevalent and lethal virus.

A cyclic pyrrole-imidazole polyamide reduces pathogenic RNA in CAG/CTG triplet repeat neurological disease models.

Expansion of CAG and CTG (CWG) triplet repeats causes several inherited neurological diseases. The CWG repeat diseases are thought to involve complex pathogenic mechanisms through expanded CWG repeat-derived RNAs in a noncoding region and polypeptides in a coding region, respectively. However, an effective therapeutic approach has not been established for the CWG repeat diseases. Here, we show that a CWG repeat DNA-targeting compound, cyclic pyrrole-imidazole polyamide (CWG-cPIP), suppressed the pathogenesis of coding and noncoding CWG repeat diseases. CWG-cPIP bound to the hairpin form of mismatched CWG DNA, interfering with transcription elongation by RNA polymerase through a preferential activity toward repeat-expanded DNA. We found that CWG-cPIP selectively inhibited pathogenic mRNA transcripts from expanded CWG repeats, reducing CUG RNA foci and polyglutamine accumulation in cells from patients with myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Treatment with CWG-cPIP ameliorated behavioral deficits in adeno-associated virus-mediated CWG repeat-expressing mice and in a genetic mouse model of HD, without cytotoxicity or off-target effects. Together, we present a candidate compound that targets expanded CWG repeat DNA independently of its genomic location and reduces both pathogenic RNA and protein levels. CWG-cPIP may be used for the treatment of CWG repeat diseases and improvement of clinical outcomes.

Molecular machine powered catalytic hairpin assembly for signal-on electrochemical detection of Alzheimer’s amyloid-β oligomer

We have developed a bipedal driven catalytic hairpin assembly for the electrochemical detection of highly sensitive amyloid-β oligomers. Aptamers specifically recognize of amyloid-βoligomers can activate catalytic hairpin assembly and form bipedal molecular machines. Catalytic hairpin assembly hybridizes with surface-fixed hairpin DNA1 (HP1) through proximity effect, exposing the pre-locking foot domain of HP1 for fusion with ferrocene labeled hairpin DNA2 (HP2-Fc). The electrochemical signal molecule Fc approaches the electrode under the action of fulcrum mediated chain shift reaction, and the bipedal molecular machines generated by catalytic hairpin assembly crosses the surface of the sensor, where a large number of ferrocenes HP1-HP2 bipedal structures towards the electrode are formed, which realizes the amplification of the electrochemical signal. The linear detection range of this component is 0.1 pM∼1 nM, and the detection limit is 38 fM. As an enzyme-free detection method, it can realize the high sensitivity and specificity detection of multiple target proteins through the appropriate DNA-based affinity probe, which has great reference significance and application value in the field of biological detection.

Primitive Oligomeric RNAs at the Origins of Life on Earth.

There are several theories on the origin of life, which differ by choosing the preponderant factor of emergence: main function (autocatalysis versus replication), initial location (black smokers versus ponds) or first molecule (RNA versus DNA). Among the two last ones, the first assumes that an RNA world involving a collaboration of small RNAs with amino-acids pre-existed and the second that DNA-enzyme-lipid complexes existed first. The debate between these classic theories is not closed and the arguments for one or the other of these theories have recently fueled a debate in which the two have a high degree of likelihood. It therefore seems interesting to propose a third intermediate way, based on the existence of an RNA that may have existed before the latter stages postulated by these theories, and therefore may be the missing link towards a common origin of them. To search for a possible ancestral structure, we propose as candidate a small RNA existing in ring or hairpin form in the early stages of life, which could have acted as a "proto-ribosome" by favoring the synthesis of the first peptides. Remnants of this putative candidate RNA exist in molecules nowadays involved in the ribosomal factory, the concentrations of these relics depending on the seniority of these molecules within the translation process.

Development of a selective ligand for G-G mismatches of CGG repeat RNA inducing the RNA structural conversion from the G-quadruplex into a hairpin-like structure.

The trinucleotide CGG repeat is located in the 5'-UTR of FMR1 and its abnormal expansion and formation of a noncanonical RNA structure causes fetal genetic diseases. In this study, a small molecular dimer-type ligand consisting of dual G-clamp units for the recognition of two neighboring guanines was synthesized, and the binding properties for the r(CGG) repeats were investigated. Compound 2 was confirmed to bind to the mismatch guanines in the stem region of the r(CGG) repeat hairpin. In addition, the RNase T1 assay demonstrated that 2 induced the structural conversion of the r(CGG)8 repeat from the G-quadruplex into a hairpin-like structure.

A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks.

The hexanucleotide repeat expansion GGGGCC [r(G4C2)exp] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(G4C2)exp RNA gain- and loss-of-function mechanisms. The r(G4C2)exp RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(G4C2)exp. Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.

Use of anion-exchange HPLC to study DNA conformational polymorphism

Anion-exchange chromatography carried out under non-denaturing conditions is a versatile tool to differentiate DNA conformations. In this work, the utility of this form of HPLC was demonstrated in four examples. The hairpin and duplex forms of d(CG)9 were readily resolved, which allowed for the studies of the influence of salt on the equilibrium of these two forms of secondary structures. Similarly, the minimum size of Tn in the loop region required for the sequence 5′-d(CCCAA-(T)n-TTGGG)-3′ to form hairpin was established to be two nucleotides using anion-exchange HPLC and fluorescence resonance energy transfer. Furthermore, the efficiency of hybridization of partially self-complementary sequences d[(CG)6Nx] was readily monitored by non-denaturing anion-exchange HPLC. Finally, different structures adopted by quadruplex-forming sequences were resolved in the same manner.

Ultrasensitive DNA Detection Based on Inorganic-Organic Nanocomposite Cosensitization and G-Quadruplex/Hemin Catalysis for Signal Amplification.

A novel photoelectrochemical (PEC) aptasensor was fabricated for DNA detection based on the coupling of cosensitization and peroxidase-like catalytic activity. Specifically, the surfaces of branched-TiO2 nanorods (B-TiO2 NRs) were modified with Cd2+ and S2+ to obtain B-TiO2 NRs/CdS hybrid structures, which were subsequently used as matrices to immobilize hairpin DNA (hDNA) probes. CdTe/TCPP (TCPP = meso-tetra(4-carboxyphenyl)-porphine) used for signal amplification was labeled on the terminal of the hDNA probe. Without the target DNA (tDNA) presence, the immobilized hDNA probe with CdTe/TCPP possessed a hairpin form and was located near the B-TiO2 NRs/CdS electrode surface, forming a cosensitized structure formation and then generating strong photocurrent with H2O2 as the electron donor. During detection, the specific recognition of tDNA by the sensing hDNA probe triggered the formation of the G-quadruplex/hemin DNAzyme, which effectively catalyzed the decomposition of H2O2. Meanwhile, cosensitization disappeared when the hDNA probe hybridized with tDNA, further reducing the photocurrent. With a double-signal amplification strategy, the sensing platform designed in this work demonstrated a linear detection ability in the 0.5 fM-5 nM range with a detection limit equal to 0.14 fM. Notably, through encoding in the base sequences of the hDNA and marking it, a versatile PEC platform could be structured for the detection of various DNA targets, which could promise applications in point-of-care diagnostic fields.

Development of fluorescent aptasensing system for ultrasensitive analysis of kanamycin

The analysis of residual kanamycin (KANA) has drawn great attentions based on its potential threat to human health and environment caused by its residual in aquatic and soil system. Herein, an ultrasensitive fluorescent aptasensing system was proposed based on the fluorescence change resulting from the conformation switch of kanamycin-aptamer (KANA-aptamer) with or without kanamycin in the sensing system, in which berberine was employed as the fluorescence reporter and KANA-aptamer as the recognition unit. Free berberine emits weak fluorescence at 535 nm in aqueous solution, and its fluorescence enhanced remarkably after it intercalated to KANA-aptamer. Once kanamycin was introduced into the sensing system, the randomly coiling structure of KANA-aptamer switched to a hairpin form, which made the dissociation of berberine from the KANA-aptamer, thus the fluorescence was quenched. Various experimental conditions including the concentration of berberine, KANA-aptamer, pH, ion strength and equilibrium time were studied in detail. Under the optimal experimental condition, a good correlation was obtained between the fluorescence of the sensing system and the concentration of kanamycin ranged from 5.0 nM to 71.0 nM with LOD at 2.3 nM. The presented fluorescent aptasensor also showed a delightful recovery of 96.50%–101.20% in human serum and milk. The excellent analytical performance of kanamycin shows a promising prospect in the practical application.

Specific interaction of zinc finger protein Com with RNA and the crystal structure of a self-complementary RNA duplex recognized by Com.

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com–RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.

The Hairpin Form of r(G4C2)exp in c9ALS/FTD Is Repeat-Associated Non-ATG Translated and a Target for Bioactive Small Molecules

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G4C2 repeat [(G4C2)exp] in C9ORF72. ALS/FTD-associated toxicity has been traced to the RNA transcribed from the repeat expansion [r(G4C2)exp], which sequesters RNA-binding proteins (RBPs) and undergoes repeat-associated non-ATG (RAN) translation to generate toxic dipeptide repeats. Using invitro and cell-based assays, we identified a small molecule (4) that selectively bound r(G4C2)exp, prevented sequestration of an RBP, and inhibited RAN translation. Indeed, biophysical characterization showed that 4 selectively bound the hairpin form of r(G4C2)exp, and nuclear magnetic resonance spectroscopy studies and molecular dynamics simulations defined this molecular recognition event. Cellular imaging revealed that 4 localized to r(G4C2)exp cytoplasmic foci, the putative sites of RAN translation. Collectively, these studies highlight that the hairpin structure of r(G4C2)exp is a therapeutically relevant target and small molecules that bind it can ameliorate c9ALS/FTD-associated toxicity.

Capturing intracellular oncogenic microRNAs with self-assembled DNA nanostructures for microRNA-based cancer therapy.

Aberrantly overexpressed oncogenic microRNAs (miRNAs, miRs) are excellent targets for therapeutic interventions. Nevertheless, thus far, little progress has been made in developing miRNA-based drugs and techniques for clinical applications, especially for overexpressed miRNAs. In this study, we demonstrate that self-assembled DNA nanostructures bearing multiple DNA sequences that are complementary to a target miRNA can effectively capture the overexpressed oncogenic miRNA and subsequently inhibit cancer cell proliferation. Specifically, a DNA nanotube structure that carries functional DNA segments (single-stranded, duplex and hairpin forms) was designed and synthesized to capture two well-known overexpressed miRNAs, miR-21 and miR-155. It was found that all three DNA nanotubes significantly reduced both miRNA levels and inhibited cancer cell growth. Moreover, the capture efficiency was highly concentration dependent and was associated with the structural design of the DNA nanotube. These results demonstrate that through careful design, programmable DNA nanostructures can hijack the natural cellular machinery and can serve as nucleic acid drugs themselves. The concept of using self-assembled DNA nanostructures to disrupt the intracellular machinery for therapeutic purposes opens a new paradigm for exploiting self-assembled DNA nanostructures for miRNA-based anticancer therapy.

Quenching effect of exciton energy transfer from CdS:Mn to Au nanoparticles: A highly efficient photoelectrochemical strategy for microRNA-21 detection

A novel and simple photoelectrochemical (PEC) biosensing method for microRNA-21 (miRNA-21) detection is reported based on energy transfer (ET) between CdS:Mn doped structure (CdS:Mn) and Au nanoparticles (AuNPs). In this protocol, TiO2-CdS:Mn hybrid structure was used as a sensing platform for hairpin DNA immobilization. In the absence of miRNA-21, the immobilized DNA was in the hairpin form. In this state, the photocurrent of the electrode was greatly depressed, due to the effective ET effect produced by short interparticle distance between CdS:Mn and AuNPs. In the presence of miRNA-21, the hairpin DNA hybridized with miRNA-21 and changed into a more rigid, rodlike double helix, which forced the AuNPs away from the electrode surface, leading to obvious recover of photocurrent because of the vanished damping effect. Integrating the fine PEC performance of TiO2-CdS:Mn hybrid structure with the significant ET effect between CdS:Mn and AuNPs, the sensitive detection of miRNA-21 was realized in a linear range of 1.0fM to 10.0pM with a low detection limit of 0.5 fM. This method might be aussichtsreich for the detection of miRNAs and other biomarkers.

Turbulent flow over an array of boulders placed on a rough, permeable bed

Large eddy simulations (LES) of flow over wall-mounted boulders placed on a rough, permeable bed, composed of one layer of densely-packed spheres, are performed. This configuration is based on the experimental configuration reported in Papanicolaou et al. (2012) [14] the data of which are used to validate the LES. A fairly good agreement between calculated and measured time-averaged streamwise velocity and turbulence intensity profiles is found confirming the accuracy of the simulation. Details of the time-averaged and instantaneous flow are revealed using various visualisation methods. The time-averaged flow is characterised by a symmetric pair of funnel vortices comprising the recirculation zone in the lee of the boulder. The funnel vortex is bounded by significant shear layers caused by high-momentum fluid filling the low momentum wake zone behind the boulder. Bed roughness and permeability appears to play an important role in this flow, it affects the near-bed velocity profiles and subsurface fluid is entrained into the low pressure zone behind the boulder, specifically into the funnel vortex, moreover surface-subsurface flow exchange in particular in the vicinity of the boulders disrupts the secondary flow in the wake. The instantaneous flow features various turbulence structures which are educed by means of isosurfaces of the Q-criterion. Shear layer roll-up leads to high-frequency shedding of roller vortices, which are subsequently stretched by significant velocity gradients and deformed into a hairpin or horseshoe form. Vertical and horizontal shear layer flapping is revealed and quantified via vorticity contour plots and velocity spectra.

TET1-Mediated Oxidation of 5-Formylcytosine (5fC) to 5-Carboxycytosine (5caC) in RNA.

It was recently revealed that 5-methylcytosine (5mC) in mRNA, similar to its behavior in DNA, can be oxidized to produce 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), implying the potential regulatory roles of this post-transcriptional RNA modification. In this study, we demonstrate the in vitro oxidation of 5fC to 5-carboxycytidine (5caC) by the catalytic domain of mammalian ten-eleven translocation enzyme (TET1) in different RNA contexts. We observed that this oxidation process has very low sequence dependence and can take place in single-stranded, double-stranded, or hairpin forms of RNA sequences, although the overall conversion yields are low.

The Aggregation Free Energy Landscapes of Polyglutamine Repeats.

Aggregates of proteins containing polyglutamine (polyQ) repeats are strongly associated with several neurodegenerative diseases. The length of the repeats correlates with the severity of the disease. Previous studies have shown that pure polyQ peptides aggregate by nucleated growth polymerization and that the size of the critical nucleus (n*) decreases from tetrameric to dimeric and monomeric as length increases from Q18 to Q26. Why the critical nucleus size changes with repeat-length has been unclear. Using the associative memory, water-mediated, structure and energy model, we construct the aggregation free energy landscapes for polyQ peptides of different repeat-lengths. These studies show that the monomer of the shorter repeat-length (Q20) prefers an extended conformation and that its aggregation indeed has a trimeric nucleus (n* ∼ 3), while a longer repeat-length monomer (Q30) prefers a β-hairpin conformation which then aggregates in a downhill fashion at 0.1 mM. For an intermediate length peptide (Q26), there is an equal preference for hairpin and extended forms in the monomer which leads to a mixed inhomogeneous nucleation mechanism for fibrils. The predicted changes of monomeric structure and nucleation mechanism are confirmed by studying the aggregation free energy profile for a polyglutamine repeat with site-specific PG mutations that favor the hairpin form, giving results in harmony with experiments on this system.