Hybrid Duplex Research Articles

A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides.

Several varieties of small nucleic acid constructs are able to modulate gene expression via one of a number of different pathways and mechanisms. These constructs can be synthesized, assembled and delivered to cells where they are able to impart regulatory functions, presenting a potential avenue for the development of nucleic acid-based therapeutics. However, distinguishing aberrant cells in need of therapeutic treatment and limiting the activity of deliverable nucleic acid constructs to these specific cells remains a challenge. Here, we designed and characterized a collection of nucleic acids systems able to generate and/or release sequence-specific oligonucleotide constructs in a conditional manner based on the presence or absence of specific RNA trigger molecules. The conditional function of these systems utilizes the implementation of AND and NOT Boolean logic elements, which could ultimately be used to restrict the release of functionally relevant nucleic acid constructs to specific cellular environments defined by the high or low expression of particular RNA biomarkers. Each system is generalizable and designed with future therapeutic development in mind. Every construct assembles through nuclease-resistant RNA/DNA hybrid duplex formation, removing the need for additional 2′-modifications, while none contain any sequence restrictions on what can define the diagnostic trigger sequence or the functional oligonucleotide output.

A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication.

Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5′ end of nascent DNA. A subsequent 5′-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5′-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA–DNA junctions to remove the 5′-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA–DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA–DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5′-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.

Molecular beacon immobilized on graphene oxide for enzyme-free signal amplification in electrochemiluminescent determination of microRNA.

An electrochemiluminescence (ECL) based biosensor is described for determination of microRNAs in the A549 cell line. Firstly, graphene oxide (GO) is dripped onto a glassy carbon electrode surface to form an interface to which one end of the capture probe (with a stem-loop structure) can be anchored through π-interaction via dangling unpaired bases. The other end of the capture probe is directed away from the GO surface to make it stand upright. Target microRNAs can open the hairpin structure to form a double-stranded DNA-RNA structure. Two auxiliary probes, generating a hybridization chain reaction, are used to elongate the DNA duplex. Finally, doxorubicin-modified cadmium telluride quantum dot nanoparticles (Dox-CdTe QD) are intercalated into the base pairs of the hybrid duplexes to act as signalling molecules. The ECL signal of the Dox-CdTe QD increases proportionally with the concentration of microRNAs, specifically for microRNA-21. The assay covers a wide linear range (1 fM to 0.1nM), has a low detection limit for microRNA-21 (1 fM), and is selective, reproducible, and stable. Graphical abstract An enzyme-free amplification electrochemiluminescent assay is described to quantitative detection of microRNA in the A549 cell line. Graphene oxide was used to immobilize capture probes obviating the special modification.Doxorubicin-modified cadmium telluride quantum dot nanoparticles are intercalated into the base pairs of the hybrid duplexes to act as signalling molecules.

Investigations on structural performance of hybrid stainless steel I-beams based on slenderness

A numerical investigation on the flexural behaviour of Lean Duplex Stainless Steel (LDSS), Hybrid Stainless Steel (HSS) and Duplex Stainless Steel (DSS) I-beams using the commercial finite element software, ABAQUS are presented in this paper based on cross-section slenderness i.e. flange and web critical sections. 3-point bending (3PB) and 4-point bending (4PB) specimens were chosen for the current numerical investigations. Based on the study, it has been observed that for both Class 1 flange-critical and web-critical sections, LDSS I-beams has a higher R (rotation capacity) as compared to HSS and DSS I-beams. Significant increase in Mu (ultimate bending strength) can be seen in case of HSS I-beams, as compared to DSS and LDSS I-beams. In general, EN 1993–1-4 is found to be reliable and applicable for LDSS, HSS and DSS I-beams. New DSM formulation for HSS I-beams has been proposed based on the full-range modified Direct Strength Method (DSM) formulation for carbon steel.

A Power Series Approach for Hybrid-Duplex UAV Communication Systems Under Rician Shadowed Fading

A hybrid-duplex (HBD) UAV communication system (UCS), i.e., HBD-UCS, to improve spectrum utilization is investigated in this work. By considering the combined effect of fading and shadowing, a comprehensive outage probability analysis of the HBD-UCS under various inter-UAV interference and shadowing scenarios over Rician shadowed fading channels is conducted. It is demonstrated that the ground station (GS) in full-duplex (FD) mode operates at lower outage probability than in half-duplex (HD) mode. Furthermore, the joint detector is shown to achieve lower outage probability than the interference ignorant (II) detector and HD-UCS, even when severe shadowing is encountered. As such, utilizing joint detectors in an HBD-UCS enables multi-UAV networks to achieve high reliability when operating in urban environments.

QCM sensing of miR-21 by formation of microRNA-DNA hybrid duplexes and intercalation on surface-functionalized pyrene.

MicroRNAs (miRNAs) are small non-coding RNA molecules that serve as important biomarkers for a variety of diseases such as cancer and vascular disease. However, sensitive and accurate detection of miR-21 is very challenging in that up-regulation of miR-21 is highly associated with several types of malignant tumors. Here, quartz crystal microbalance (QCM) biosensors were developed for sensitive and specific detection of miR-21 through formation of miR-21-DNA hybrid duplexes and non-specific intercalation of surface-modified pyrene molecules. High selectivity for miR-21 over other miRNAs came from the specific hybridization between miR-21 and gold nanoparticle (AuNP)-conjugated complementary oligonucleotides of miR-21. High sensitivity was obtained through formation of intercalated complexes on the surface with subsequent gold staining signal amplification. Under optimum condition using this strategic approach, our novel QCM biosensors could detect miR-21 concentration as low as 3.6 pM in the entire linear range from 2.5 pM to 2.5 μM with a correlation coefficient of 0.989. In addition, these sensors did not work at all for other miRNAs based on their high selectivity. miR-21 in human brain total RNA and total RNA extracted from A549 cell line could also be successfully detected. Therefore, miRNA detection technology using QCM biosensors and their detection mechanisms have potential as alternatives in biological studies and clinical diagnosis.

The Mechanical Properties of RNA-DNA Hybrid Duplex Stretched by Magnetic Tweezers

RNA can anneal to its DNA template to generate an RNA-DNA hybrid (RDH) duplex and a displaced DNA strand, termed R-loop. RDH duplex occupies up to 5% of the mammalian genome and plays important roles in many biological processes. The functions of RDH duplex are affected by its mechanical properties, including the elasticity and the conformation transitions. The mechanical properties of RDH duplex, however, are still unclear. In this work, we studied the mechanical properties of RDH duplex using magnetic tweezers in comparison with those of DNA and RNA duplexes with the same sequences. We report that the contour length of RDH duplex is ∼0.30 nm/bp, and the stretching modulus of RDH duplex is ∼660 pN, neither of which is sensitive to NaCl concentration. The persistence length of RDH duplex depends on NaCl concentration, decreasing from ∼63 nm at 1 mM NaCl to ∼49 nm at 500 mM NaCl. Under high tension of ∼60 pN, the end-opened RDH duplex undergoes two distinct overstretching transitions; at high salt in which the basepairs are stable, it undergoes the nonhysteretic transition, leading to a basepaired elongated structure, whereas at low salt, it undergoes a hysteretic peeling transition, leading to the single-stranded DNA strand under force and the single-stranded RNA strand coils. The peeled RDH is difficult to reanneal back to the duplex conformation, which may be due to the secondary structures formed in the coiled single-stranded RNA strand. These results help us understand the full picture of the structures and mechanical properties of nucleic acid duplexes in solution and provide a baseline for studying the interaction of RDH with proteins at the single-molecule level.

Isolation of RNA from a mixture and its detection by utilizing a microgel-based optical device

In this investigation, we show that RNA can be separated from a solution containing DNA and RNA and the isolated RNA can be detected using poly (N-isopropylacrylamide-co-N-(3-aminopropyl) methacrylamide hydrochloride) microgel-based optical devices (etalons). The isolation of RNA was accomplished by using hairpin-functionalized magnetic beads (MMPDNA) and differential melting, based on the fact that the DNA–RNA hybrid duplex is stronger (i.e., high melting temperature) than the DNA–DNA duplex (i.e., low melting temperature). By performing concurrent etalon sensing and fluorescent studies, we found that the MMPDNA combined with differential melting was capable of selectively separating RNA from DNA. This selective separation and simple colorimetric detection of RNA from a mixture will help lead to future RNA-based disease diagnostic devices.

Classical Triplex Molecular Beacons for MicroRNA-21 and Vascular Endothelial Growth Factor Detection

Triplex molecular beacons (tMBs) possess great potential in biological sensing because of the pH responsiveness and controllability of binding strength. Here, we systematically investigate and rationally design a classical tMB for convenient detection of microRNA-21, a well-known biomarker of cardio-cerebrovascular diseases. In the tMB, we employ the complementary sequence of miR-21 as the loop and the sequences of protonated cytosine-guanine-cytosine (C-G•C+) and thymine-adenine-thymine (T-A•T) as the triplex stem, in which both the Watson-Crick and Hoogsteen base-pairing control the binding strength in cooperation. It is demonstrated for the first time that the presence of miR-21 would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure. These would hinder the fluorescence resonance energy transfer (FRET) between the fluorophore (FAM) and quencher (BHQ1) labeled at the ends of the oligonucleotide, and the fluorescence recovery degree of FAM can be used as the standard to quantitate the miR-21. More significantly, the excellent adjustability and sensitivity of our tMBs have been confirmed by constructing the corresponding duplex molecular beacon (dMB) for comparison. The fluorophore FAM in the tMB could be replaced by the fluorescent DNA/silver nanoclusters, which exhibits the universal applicability of energy donor and receptor selection for tMB. Furthermore, our proposed tMB could also be developed as an aptasensor for the detection of vascular endothelial growth factor (VEGF) by only introducing the complementary sequence of its aptamer into the tMB. This work is of great significance for the systematic study of tMBs for the detection of biomarkers such as nucleic acids and proteins.

Analysis of Coverage and Binary Rate in a Hybrid/Full Duplex Heterogeneous Cellular Network under Various Association Criteria

In recent years, Full Duplex cellular systems are under investigation. Previous works found that in full duplex systems, rate does not double because of not only self interference but also due to full duplex BSs interference. In this paper, a Hybrid duplex (H) two tier Heterogeneous Cellular Network is considered: Small pico Base Stations (BSs) use full duplex mode while macro BSs run usual Half Duplex transmission to avoid high interference. Different duplexing modes and association criteria are analyzed and their performance compared in terms of coverage, binary rate and Energy Efficiency. The simulation results show that hybrid mode rate and energy efficiency are the highest compared to full or half duplex modes while coverage does not reduce much. From association criteria point of view, binary rate and energy efficiency are better under maximum receive power in average association criteria whereas coverage is better in minimum path loss association criteria.

Abstract 4838: R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells

Abstract G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan. Citation Format: Alessio De Magis, Stefano Giustino Manzo, Marco Russo, Olivier Sordet, Rita Morigi, Giovanni Capranico. R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4838.

Energy-Efficient Hybrid Duplexing Strategy for Bidirectional Distributed Antenna Systems

We propose a bidirectional distributed antenna (DA) system where DAs are capable of working dynamically in hybrid duplex modes: full duplex (FD), half duplex, and sleep, which enables higher degree of freedom and hence much higher energy efficiency (EE) than DA sole-FD systems, with just marginal loss in spectral efficiency (SE). The proposed system also demonstrates significant EE and SE enhancement over FD colocated antenna systems. Compared to the above two benchmark systems, the proposed system also requires much simpler self-interference cancelation design for FD mode due to less cross talks between antennas. A low-complexity EE maximization scheme is proposed for the bidirectional DA system. A channel gain based DA clustering algorithm is first performed to activate/deactivate transmit/receive chains, which highlights the characteristics of DA deployment, and then a distributed hybrid duplexing algorithm is performed to optimize the downlink beamformer and the uplink transmission power. Various practical aspects are taken into account for system design, such as self-interference at DAs in FD mode, cochannel interference from uplink users to downlink users, and multiuser interference in both uplink and downlink. The effectiveness of the proposed system is verified by simulation results.

Activity and fidelity of human DNA polymerase α depend on primer structure

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

A new aptamer microarray method on the TiO2-porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO2-PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B1 (AFB1), and 0.001-10 ng/mL for fumonisin B1 (FB1) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB1, and FB1, respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Hybrid Duplex Wireless Peer Discovery With Imperfect Self-Interference Cancellation

This letter investigates the hybrid duplex wireless peer discovery (Hyb-WPD) that increases the average number of discovered peers (ADP), through arbitrating between half duplex and full duplex (FD) operations. FD allowing simultaneous tranceiving inherently introduces residual self-interference (RSI) due to non-ideal implementation; thus, this impairment yields a new design issue for transmission probability, which determines whether a peer receives or tranceives. This letter proposes the design principles for the Hyb-WPD depending on RSI through mathematically analyzing the ADP, and the numerical results demonstrate their performance.

Phosphoglucose Isomerase Deficiency in <i>Escherichia coli</i> K-12 Is Associated with Increased Spontaneous Mutation Rate

Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.

Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides.

This study assessed the effects of artificial cationic oligopeptides on a DNA/RNA hybrid duplex. An oligopeptide containing the octamer of l-2,4-diaminobutyric acid (Dab8) was found to enhance both the RNase A resistance and RNase H activity of the DNA/RNA hybrid, which are important for developing nucleic acid drugs.

Methylene-linked bis-phenylbenzimidazoles - a new scaffold to target telomeric DNA/RNA hybrid duplex.

We report a series of novel methylene-linked bis-phenylbenzimidazoles intercalators that stabilize telomeric DNA/RNA hybrid (tDRH) structures by up to 7.2 °C at a 1 μM ligand concentration while having negligible affinity for DNA/DNA duplexes, although with a low affinity for quadruplex DNA. We have used molecular modelling studies to rationalize this selectivity, concluding that the methylene spacer between the terminal benzimidazole and phenylene moieties plays a key role in facilitating the bis-intercalating process. This scaffold may be used to develop chemical tools or new therapeutics to selectively target the telomeric DNA/RNA duplex without affecting normal genomic DNA.

Correction: Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides.

Correction for 'Enhancement in RNase H activity of a DNA/RNA hybrid duplex using artificial cationic oligopeptides' by Rintaro Iwata Hara et al., Chem. Commun., 2018, 54, 8526-8529.

Fluorescence sensing platform based on ruthenium(II) complexes as high 3S (sensitivity, specificity, speed) and “on-off-on” sensors for the miR-185 detection

Inspired by the enormous importance attributed to the biological function of miRNA, we pour our attention into the design and synthesis of four ruthenium(II) complexes and evaluate their applications as miR-185 detection agents by spectroscopic measurements. It was found that all complexes can form sensing platform for the detection of the complementary target miR-185 through the introduction of carboxyfluorescein (FAM) labeled single stranded DNA (P-DNA), giving the detection limits of 0.42nM for Ru 1, 0.28nM for Ru 2, 0.32nM for Ru 3, 0.85nM for Ru 4, all with instantaneous detection time in 1min. The results of the binding constant, fluorescence anisotropy (FA) and polyacrylamide gel electrophoresis experiments (PAGE) revealed that the ruthenium(II) complexes prefer to bind P-DNA other than hybrid duplexes DNA@RNA upon recognition, resulting in the detection of miR-185. These results provide useful suggestions in the new type of metal-based miRNA detection agents.