Complementary Base Research Articles

Electrochemical determination of hantavirus using gold nanoparticle-modified graphene as an electrode material and Cu-based metal-organic framework assisted signal generation

An electrochemical biosensor was prepared for nucleic acid-based hantavirus detection using a Cu-based metal-organic framework (CuMOF) as a signal tag. The CuMOF was synthesized by the solvothermal method and then covalently bonded with signal DNA (sDNA) probes. The Au nanoparticles and reduced graphene oxide composite were deposited on the electrode surface by electroreduction as support substrate and was then functionalized with capture DNA (cDNA) probes by self-assembly. Through the complementary base pairing, the target DNA (tDNA) fragment of hantavirus hybridized with the cDNA and the sDNA in a sandwich-type format. The tDNA was detected according to the current signal of the CuMOF catalyzed reaction using o-phenylenediamine as redox substrate. The peak current of the biosensor at - 0.55V increased linearly in proportion to the logarithmic value of the tDNA concentration from 10-15 to 10-9mol/L, with a detection limit of 0.74 × 10-15mol/L. Moreover, the proposed biosensor was successfully applied to detect hantavirus and was able to distinguish hantavirus from other arboviruses.

The role of HOTAIR/miR-152-3p/LIN28B in regulating the progression of endometrial squamous carcinoma

IntroductionThere is growing evidence that long non-coding RNAs (lncRNAs) are correlated with malignancy in the modulation of tumor progression. This study aims to investigate the effect of homeobox protein (HOX) transcript antisense RNA (HOTAIR) on the migration and invasion of ESC.Material and methodsStarbase was used to identify miRNAs with complementary base pairing with HOTAIR. RNA pull-down and qRT-PCR were employed to investigate the effect of HOTAIR on miR-152-3p. In vitro cell migration and invasion assays were performed to assess the effects of HOTAIR and miR-152-3p on ESC. Computational software, TargetScan, was then used to identify the potential target of miR-152-3p, and their relationship was verified by immunoblotting analysis, qRT-PCR and luciferase reporter assay.ResultsStarbase predicted a potential miR-152-3p binding site in HOTAIR, which was validated by RNA pull-down assay. HOTAIR was negatively correlated with miR-152-3p in ESC. Moreover, HOTAIR promoted migration and invasion of ESC. The oncogenic activity of HOTAIR was partly through its negative regulation of miR-152-3p. LIN28B was identified to be a direct target of miR-152-3p. A negative correlation between LIN28B and miR-152-3p was observed in ESC. In addition, overexpression of miR-152-3p suppressed the progression of ESC by directly targeting and regulating LIN28B.ConclusionsOur results reveal that HOTAIR may be a driver of ESC through inhibiting miR-152-3p, a tumor suppressor, suggesting that miR-152-3p may be a potential target for advanced ESC therapeutic treatment.

Rapid Visualized Detection of Escherichia Coli O157:H7 by DNA Hydrogel Based on Rolling Circle Amplification

Abstract Food-borne illnesses caused by pathogenic bacteria are prominent issues in food safety. Rapid and sensitive detection of pathogenic bacteria is significantly essential for food safety. In this work, an aptamer biosensor (aptasensor) based on aldehyde magnetic beads (Mbs), rolling circle amplification (RCA) and DNA hydrogel was developed for visualized, simple and rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). Firstly, the Mbs@double-stranded DNA (dsDNA) complexes were prepared. In the presence of target, the aptamer combined with E. coli O157:H7, releasing E. coli aptamer-initiator (EA-I). The supernatant collected by magnetic separation and those released primers were hybridized with circular sequence to form padlock probe. Then, RCA reaction was initiated by adding T4 DNA ligase, phi29 DNA polymerase, and deoxynucleotides (dNTPs), while RCA cannot be triggered in the absence of target. Two circular sequences were designed to contain partially complementary bases. After RCA, two long single-stranded DNA (ssDNA) products were generated using those two circular sequences. They hybridized with each other and formed the naked-eye visible DNA hydrogel. The method showed high sensitivity and specificity with a detection limit of 4 × 103 CFU/mL for E. coli O157:H7 within 1 h, and the detection time could be finished within 30 min. The aptasensor offers many advantages such as high specificity, easy operation, efficient amplification, rapid and visualized readout, which has potential applications in food safety detection

Buildings and energy: architectural history in the climate emergency

As the current climate emergency deepens, it is no longer adequate to leave ideas of sustainability to engineers, practitioners, and studio-based educators. Ways of understanding and teaching architecture’s history must also respond. This needs to go beyond highlighting exemplars and models from the past for what they may teach us practically in terms of passive environmental conditioning. The very terms and frames of reference we use to discuss buildings in the context of history require reconsideration. This article proposes that understanding architecture from a radical material perspective has the potential to foreground the entrenched relationship between architecture and energy consumption in the history of architecture. Energy consumption is the key factor in climate change. Making historians and students more aware of how this critical relationship shaped the built environment through time places an emphasis, and thus responsibility, on the very high energy consumption of architecture. We propose two essential questions: how has humanity’s changing ability to harness useful energy interacted with the history of architecture? And how might we understand buildings through time not as objects fashioned solely by individual genius, patronage, stylistic movements and/or theoretical considerations, but as products that also result from the powerful nexus between assemblage and energy? This article attempts to demonstrate a possible approach, by sketching out three historical ‘scenarios’ that speak to different periods in time (pre-industrial, agrarian; industrial, coal- and steam-based; and late industrial, oil- and electricity-based). In these scenarios we trace regimes of energy consumption and their attendant networks of production, suggesting the centrality of such an approach to a full appreciation of building as a material process. We propose that this approach might form a new and complementary basis for research and teaching in the history of architecture.

Study on Defective T Junction-Mediated Strand Displacement Amplification and Its Application in Microchip Electrophoretic Detection of Longer Bacterial 16S rDNA

Current strand displacement amplification (SDA)-based nucleic acid sensing methods generally rely on a ssDNA template that involves complementary bases to the endonuclease recognition sequence, which has the limitation of detecting only short nucleic acids. Herein, a new SDA method in which the defective T junction structure is first used to support SDA (dT-SDA) was proposed and applied in longer DNA detection. In dT-SDA, an auxiliary probe and a primer were designed to specifically identify the target gene, following the formation of a stable defective T junction structure through proximity hybridization, and the formation of defective T junctions could further trigger cascade SDA cycling to produce numerous ssDNA products. The quantity of these ssDNA products was detected through microchip electrophoresis (MCE) and could be transformed to the concentration of the target gene. Moreover, the applicability of this developed strategy in detecting long genomic DNA was verified by detecting bacterial 16S rDNA. This proposed dT-SDA strategy consumes less time and has satisfactory sensitivity, which has great potential for effective bacterial screening and infection diagnosis.

Políticas, dinâmicas e perfis dos agrupamentos de escolas em Portugal

The rationalization of the Portuguese school system, which has taken place over the last twenty years, has led to a profound reconfiguration of the network, the typology of the schools and their management. We studied the process of building school clusters and the resulting organizational configurations, problematizing their contributions to the democratization of school education. The analysis was based on statistical data requested from the Ministry of Education and, on a complementary basis, on External Evaluation Reports from the Inspector General of Education and Science. It was possible to identify the organizational diversity of the “cluster” configuration, as well as to observe its limited impact on democratization and improvement of schooling processes.

Steganographic Model for encrypted messages based on DNA Encoding

Information has become an inseparable part of human life. Some information that is considered important, such as state or company documents, require more security to ensure its confidentiality. One way of securing information is by hiding the information in certain media using steganography techniques. Steganography is a method of hiding information into other files to make it invisible. One of the most frequently used steganographic methods is Least Significant Bit (LSB).In this study, the LSB method will be modified using DNA Encoding and Chargaff's Rule. Chargaff's Rule or complementary base pairing rule is used to construct a complementary strand. The modification of the LSB method using DNA encoding and Chargaff's Rule is expected to increase the security of the information.The MSE test results show the average value of the LSB method is 0.000236368, while the average value for the DNA Encoding-based Steganography method is 0.000770917. The average PSNR value for the LSB method was 76.82 dB while the DNA Encoding-based Steganography method had an average value of 70.88 dB. The time of inserting and extracting messages using the Steganography method based on DNA Encoding is relatively longer than the LSB method because of its higher algorithmic complexity. The message security of the DNA Encoding-based Steganography method is better because there is encryption in the algorithm compared to the LSB method which does not have encryption.

Sensitive detection of p53 DNA based on spatially confined fluorescence resonance energy transfer and multivalent assembly of branched DNA.

A key challenge for the discrete distribution-based Förster resonance energy transfer system (D-FRET) is the reduced intensity and stability of signal probes in complex biological matrices. Here, we present a spatially confined FRET (SC-FRET) probe with a stable structure and strong signal output. It consists of multivalent FRET pairs labeled with FAM or TAMRA. In this assay, p53 DNA was chosen as a model hairpin probe (HP), and two kinds of branched DNA probes (ssDNA-FAM, ssDNA-TAMRA) were involved. Under the action of p53 DNA, the unfolded HP acts as a primer to initiate polymerization extension of KFP polymerase and cleavage of Nb.BbvCI endonuclease, which produces plenty of ssDNA (primer-DNA). The branched DNA is designed to have the same binding core and different sticky ends, the core part of which can self-assemble to form X-shaped branched DNA (X-FAM or X-TAMRA), and the sticky ends of which are complementary to the primer-DNA. Therefore, the primer-DNAs released during the polymerization cleavage process will combine a large number of X-FAM and X-TAMRA in a limited space through complementary base pairing. Fluorescence was transferred from FAM to TAMRA, and a strong FRET response was generated by the locational effects. The proposed SC-FRET system based on the multivalent assembly of branched DNA exhibited a strong FRET response with an LOD of 0.01394 pM. Importantly, it also showed a high-contrast and stable FRET response in HeLa cells. Its superior biological stability is attributed to the large steric hindrance of the compact and rigid frame of the SC-FRET probe, which helps prevent intracellular degradation and provides a powerful tool for biomedical research.

Hybrid Time-Scale Optimal Scheduling Considering Multi-Energy Complementary Characteristic

Evaluating the potential utilization of hybrid energy systems and determining the multi-scale optimal operation strategy is critical to power system planning in the context of energy structure adjustment, especially for large-scale hybrid energy systems. Considering the long-term and short-term complementary characteristics, this paper puts forward a coordinated optimization framework for the integrated energy system in the world's largest multi-energy complementary base on Yellow River's upper reaches. The main procedures are as follows: 1) cross-correlation method is introduced for individually analyzing the long- and short-term complementary characteristics of wind power, photovoltaic, and hydropower in this multi-energy complementary base; 2) a double-layer model combining the long-term optimal operation model and short-term optimal operation model for determining the proportion of multiple energy and optimizing the maximum peak-shaving ability; 3) Large-Scale System Decomposition-Coordination Method is applied for solving the proposed double-layer operation model. The results show that wind power 23%, photovoltaic 35%, hydropower 42% can keep the most stable generation in the long-term complementary operation. This proportion results can improve the system peak regulation capacity with 50.8% (sunny day's morning peak) and 24.2% (rainy day's morning peak) in the optimal short-term operation.

Evaluation of expression profile of miRNAs with regulatory functions in metabolic pathways and signaling of parasitic diseases

Background: MicroRNAs (miRNAs) are a subset of small regulatory RNAs that are expressed by almost all metazoans and protozoans. They express genes either by direct cleavage or by suppressing the translation of target mRNAs by partial complementary base pairing. The active and functional unit of miRNA is a complex of Argonaute proteins known as microRNA-induced silencing complex (miRISC). They are known to regulate various growth and physiological processes. Irregular expression of miRNA in human cells is associated with a variety of disorders such as cancer, cardiovascular dysfunction, liver damage, immune dysfunction, metabolic syndromes, and pathogenic infections. A growing number of studies have shown that miRNAs are in fact a major component of host interactions and pathogens and play an important role in host immune responses to microorganisms. Emerging miRNAs are recognized as important tools for the genetic study, therapeutic development, and diagnosis of human pathogenic infections caused by various pathogenic organisms such as viruses, bacteria, parasites, and fungi. Many pathogens use the host miRNA system for their own benefits, including pathogenesis, survival within the host cell, and crossing some host immune barriers. Other pathogens express their miRNA within the host and contribute to their replication, survival, or delay. This article aims to review the role and importance of miRNA in relation to some important parasitic diseases.

Complementary Base Lowers the Barrier in SuFEx Click Chemistry for Primary Amine Nucleophiles.

The sulfur(VI) fluoride exchange (SuFEx) reaction is an emerging scheme for connecting molecular building blocks. Due to its broad functional group tolerance and rather stable resulting linkage, it is seeing rapid adoption in various fields of chemistry. Still, to date the reaction mechanism is poorly understood, which hampers further development. Here, we show that the mechanism of the SuFEx reaction for the prototypical example of methanesulfonyl fluoride reacting with methylamine can be understood as an SN2-type reaction. By analyzing the reaction path with the help of density functional theory in vacuo and under consideration of solvent and co-reactant influence, we identify the often used complementary base as a crucial ingredient to lower the reaction barrier significantly by increasing the nucleophilicity of the primary amine. With the help of energy decomposition analysis at the transition state structures, we quantify the underlying stereoelectronic effects and propose new avenues for experimental exploration of the potential of SuFEx chemistry.

Nucleic acid biohybrid nanocarriers with high-therapeutic payload and controllable extended release of daunomycin for cancer therapy.

We have developed a novel, nanosized drug carrier with high-therapeutic payload, controllable release, and the potential for active tumor targeting. It consists of a 15 nm gold nanoparticle with dense surface loading of DNA duplexes. We utilize the natural intercalating behavior of daunomycin to load the drug between DNA base pairs. We obtained a high-therapeutic payload of >1,000 drug molecules per gold nanoparticle (AuNP), one of the highest loadings reported in literature to date. We have engineered unique DNA sequences to control release of daunomycin for over 48 hr and show higher cell death compared to equivalent concentrations of free daunomycin. We have also explored cell internalization mechanisms to identify the pathways by which our gold nanoparticles enter the cell. This nanocarrier is in the ideal size range of 16-100 nm in diameter to utilize the enhanced permeability and retention effect for passive targeting to tumors. Our AuNP platform is effective as a therapeutic drug delivery device and can easily incorporate any aptamer of choice through complementary base pairing. Our work has produced an innovative nanoscale drug-delivery platform potentially leading to personalized cancer therapies through careful selection of aptamers and an adjustable drug release profile.

Advances in capillary-based immobilized enzyme microreactor based on DNA-directed immobilization

Life processes such as metabolism and energy conversion are catalyzed by biological enzymes. The changes of enzymatic activity in organisms can lead various diseases. Thus, it is imperative to develop novel methods of analyzing enzymatic activities for gaining deeper insights into metabolic processes, disease diagnosis, and drug development. Capillary electrophoresis (CE) has the advantages of high separation efficiency, fast analysis speed, and simple operation; moreover, it requires less sample and can be combined with a variety of detection methods. Therefore, CE has attracted increasing attention for enzyme analysis. Enzyme analysis based on CE mainly includes off-line mode and on-line mode. In the off-line mode, the enzyme and substrate are incubated outside the capillary, and then the product is introduced into the CE for analysis. In the on-line mode, the capillary is not only used as a separation channel, but also as an enzyme reaction site. Therefore, the on-line mode facilitates all steps of enzymatic hydrolysis, separation, and detection within a capillary. In the on-line mode, homogeneous analysis method, electrophoretically mediated microanalysis (EMMA), and heterogeneous analysis method, immobilized enzyme microreactor (IMER), were developed. The on-line enzyme analysis method of IMER combined with capillary electrophoresis (CE-IMER) was developed into a mainstream enzyme analysis method. CE-IMER combines the advantages of immobilized enzyme and CE. By immobilizing the free enzyme in capillary, it can not only significantly improve the stability and reusability of enzyme, but also enables the automatic enzyme analysis at nanoscale. This can significantly reduce the cost of enzyme analysis. Although, there are numerous methods to prepare new IMER for enzyme analysis by CE, preparing CE-IMER with good performance, reusability, large enzyme loading, and high degree of automation is the focus of research in this field. DNA-directed immobilization (DDI) makes use of the complementary base pairs (A-T, C-G) of DNA molecules to specifically immobilize biomacromolecules under mild physiological conditions. The enzyme can be immobilized on the carrier surface by DDI and the short double helix DNA molecules possess strong mechanical strength and physicochemical stability. This can form an enzyme microarray, reduce the resistance of mass transfer, improve the contact between enzyme and substrate, and promote the enzymatic analysis process. Compared with the traditional immobilization methods of adsorption, crosslinking, encapsulation, and covalent bonding, DDI can be operated under mild physiological conditions. Further, this can significantly reduce the influence of the immobilization process on the activity, conformation, and stability of the enzyme. Meanwhile, the reversible immobilization process of DDI can regenerate the surface of the carrier, thereby significantly reducing the economic and time cost of IMER preparation. Therefore, DDI is an ideal method to prepare IMER. In this article, the preliminary research and progress of our research group in the field of IMER preparation by DDI technology are presented. At present, the research on the preparation of novel IMER based on DNA nanotechnology, such as DDI, is in the initial stage and there is much scope for development and research. Based on the previous studies, we can focus on the following aspects: (1) building a more efficient catalytic IMER cascade reaction system by immobilizing target enzymes in specific regions of the capillary based on DDI; (2) aiming at the problems existing in the preparation of IMER, such as stability, enzymatic activity, and enzyme immobilization capacity, while taking advantages of DNA structure and nanomaterials to prepare novel IMERs to promote the wide application of CE-IMER in enzyme analysis.

An electrochemical biosensor based on AuNPs/Ti3C2 MXene three-dimensional nanocomposite for microRNA-155 detection by exonuclease III-aided cascade target recycling

A novel electrochemical biosensor based on AuNPs/Ti3C2 MXene three-dimensional nanocomposite for sensitive miRNA-155 detection by exonuclease III (Exo III)-aided cascade target recycling is reported. Ti3C2 MXene is synthesized and used as a support to immobilize Au nanoparticles (AuNPs) due to its special layered structure and metallic properties. The three-dimensional (3D) structure of the AuNPs/Ti3C2 MXene nanocomposite for the biosensor platform leverages the integrated advantages of a large specific surface area, excellent electrical conductivity and electrocatalytic properties. AuNPs are used to immobilize capture DNA (C-DNA) by AuS chemical bonds. The C-DNA was modified with methylene blue (MB) at the 3′ end, resulting in an initial DPV electrochemical signal (Id). In the presence of target, miRNA-155 and C-DNA formed a double-stranded structure by complementary base pairing. Subsequently, Exo III digested the 3′ end of the C-DNA in the double-stranded structure and caused the electrochemical signal to “turn off” (the final signal denoted as Ih). By Exo III-aided cascade cleavage of the target, one miRNA-155 chain causes the release of several C-DNAs under certain conditions, which significantly amplifies the peak current difference on the fabricated biosensor (ΔI = Id﹣Ih). The electrochemical sensor achieved a detection limit of 0.35 fM (S/N = 3) with a linear range from 1.0 fM to 10 nM. This sensor also shows desirable stability, reproducibility and specificity.

Long-Range Ordered Water Correlations between A–T/C–G Nucleotides

Summary The interactions between complementary base pairs are crucial to the helical duplex structure of DNA. Although base-base interactions have been widely reported, the study of long-range interactions in the base-pairing processes is still a major challenge in this field. Here, we first study the long-range interactions between the base pairs by atomic force microscopy-based single-molecule force spectroscopy (SMFS). The SMFS results of A–T/C–G imply that there are weak long-range interactions between them, which have a range of 15–25 nm. Raman spectroscopy results imply that these weak interactions can be attributed to multiplex hydrogen bond interaction of ordered water structure between A–T/C–G nucleotides. In addition, the theoretical calculations deduce that the ideal structure of these water molecules exhibits a specific helical structure. Our findings might open up a new understanding of biological assembly processes and provide helpful strategies for bio-nanotechnology.

Solvo-Driven Dimeric Nanoplasmon Coupling Under DNA Direction

Strong coupling is a prerequisite for nanoassemblies to deliver functions and applications unrealizable by noninteracting building units. DNA-Directed metamaterials, albeit highly programmable, oft...

Complementary Kumaraswamy Weibull Power Series Distribution: Some Properties and Application

In this paper, we propose Complementary Kumaraswamy Weibull Power Series (CKWPS) Distributions. The method is obtained by compounding the Kumaraswamy-G distribution and Power Series distribution on a latent complementary distance problem base. The mathematical properties of the proposed class of distribution are studied. The method of Maximum Likelihood Estimation is used for obtaining the estimates of the model parameters. A member of the family is investigated in detail. Finally an application of the proposed class is illustrated using a real data set.

Prebiotic competition and evolution in self-replicating polynucleotides can explain the properties of DNA/RNA in modern living systems

BackgroundWe hypothesize prebiotic evolution of self-replicating macro-molecules (Alberts, Molecular biology of the cell, 2015; Orgel, Crit Rev Biochem Mol Biol 39:99-123, 2004; Hud, Nat Commun 9:5171) favoured the constituent nucleotides and biophysical properties observed in the RNA and DNA of modern organisms. Assumed initial conditions are a shallow tide pool, containing a racemic mix of diverse nucleotide monomers (Barks et al., Chembiochem 11:1240-1243, 2010; Krishnamurthy, Nat Commun 9:5175, 2018; Hirao, Curr Opin Chem Biol 10:622-627), subject to day/night thermal fluctuations (Piccirilli et al., Nature 343:33-37, 1990). Self-replication, like Polymerase Chain Reactions, followed as higher daytime thermal energy “melted” inter-strand hydrogen bonds causing strand separation while solar UV radiation increased prebiotic nucleobase formation (Szathmary, Proc Biol Sci 245:91-99, 1991; Materese et al., Astrobiology 17:761-770, 2017; Bera et al., Astrobiology 17:771-785, 2017). Lower night energies allowed free monomers to form hydrogen bonds with their template counterparts leading to daughter strand synthesis (Hirao, Biotechniques 40:711, 2006).ResultsEvolutionary selection favoured increasing strand length to maximize auto-catalytic function in RNA and polymer stability in double stranded DNA (Krishnamurthy, Chemistry 24:16708-16715, 2018; Szathmary, Nat Rev Genet 4:995-1001, 2003). However, synthesis of the full daughter strand before daytime temperatures produced strand separation, longer polymer length required increased speed of self-replication. Computer simulations demonstrate optimal polynucleotide autocatalytic speed is achieved when the constituent nucleotides possess a left-right asymmetry that decreases the hydrogen bond kinetic barrier for the free nucleotide attachment to the template on one side and increases bond barrier on the other side preventing it from releasing prior to covalent bond formation. This phenomenon is similar to asymmetric kinetics observed during polymerization of the front and the back ends of linear cytoskeletal proteins such as actin and microtubules (Orgel, Nature 343:18-20, 1990; Henry, Curr Opin Chem Biol 7:727-733, 2003; Walker et al., J Cell Biol 108:931-937, 1989; Crevenna et al., J Biol Chem 288:12102-12113, 2013). Since rotation of the nucleotide would disrupt the asymmetry, the optimal nucleotides must form two or more hydrogen bonds with their counterpart on the template strand. All nucleotides in modern RNA and DNA have these predicted properties. Our models demonstrate these constraints on the properties of constituent monomers result in biophysical properties found in modern DNA and RNA including strand directionality, anti-parallel strand orientation, homochirality, quadruplet alphabet, and complementary base pairing. Furthermore, competition between RNA and DNA auto-replicators for 3 nucleotides in common permit states coexistence and possible cooperative interactions that could be incorporated into nascent living systems.ConclusionOur findings demonstrate the molecular properties of DNA/RNA could have emerged from Darwinian competition among macromolecular replicators that selected nucleotide monomers that maximized the speed of autocatalysis.

Three-cascade cycle amplification strategy for fluorescent detection of HIV-DNA in human blood with hybrid of pentaethylenehexamine-functionalized graphene quantum dot and NaGdF4:Yb,Er@NaGdF4

Poor water solubility and low efficiency of upconversion luminescence limit the applications of rare earth (RE)-doped upconversion nanoparticles in bioanalysis. The paper reports hybrid of pentaethylenehexamine functionalized graphene quantum dots and NaGdF4:Yb,Er@NaGdF4 via coordination of RE with nitrogen in the presence of polyvinylpyrrolidone. The resulting hybrid offers hexagonal nanostructure with particle size of 25 nm, good dispersibility and rich functional groups. Interestingly, the hybrid promotes the enhancement of upconversion luminescence that depends on the structure of graphene quantum dots. The hybrid was used as fluorescence probe for construction of three-cascade cycle amplification for detection of HIV-DNA. Target DNA interacts with the pre-hybridized duplex DNA to induce release of assistant strand DNA, triggering target DNA-induced recycle. The utilization of three-cascade cycle allows one target DNA to produce many tetramethylrhodamine-labeled DNA fragments. These are connected on the hybrid by complementary base pairs, promoting a significant upconversion fluorescence quenching by fluorescence resonance energy transfer, due to proximity of tetramethyl rhodamine and hybrid. The fluorescence signal decreases with increasing HIV-DNA concentration in the range of 1.0 × 10−17-1.0 × 10-12 M with the detection limit of 4.8 × 10-18 M. The proposed method provides advantage of sensitivity, specificity and stability and was successfully applied in detection of HIV-DNA in human blood.

Inference of Bacterial Small RNA Regulatory Networks and Integration with Transcription Factor-Driven Regulatory Networks.

ABSTRACTSmall noncoding RNAs (sRNAs) are key regulators of bacterial gene expression. Through complementary base pairing, sRNAs affect mRNA stability and translation efficiency. Here, we describe a network inference approach designed to identify sRNA-mediated regulation of transcript levels. We use existing transcriptional data sets and prior knowledge to infer sRNA regulons using our network inference tool, the Inferelator. This approach produces genome-wide gene regulatory networks that include contributions by both transcription factors and sRNAs. We show the benefits of estimating and incorporating sRNA activities into network inference pipelines using available experimental data. We also demonstrate how these estimated sRNA regulatory activities can be mined to identify the experimental conditions where sRNAs are most active. We uncover 45 novel experimentally supported sRNA-mRNA interactions in Escherichia coli, outperforming previous network-based efforts. Additionally, our pipeline complements sequence-based sRNA-mRNA interaction prediction methods by adding a data-driven filtering step. Finally, we show the general applicability of our approach by identifying 24 novel, experimentally supported, sRNA-mRNA interactions in Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. Overall, our strategy generates novel insights into the functional context of sRNA regulation in multiple bacterial species.IMPORTANCE Individual bacterial genomes can have dozens of small noncoding RNAs with largely unexplored regulatory functions. Although bacterial sRNAs influence a wide range of biological processes, including antibiotic resistance and pathogenicity, our current understanding of sRNA-mediated regulation is far from complete. Most of the available information is restricted to a few well-studied bacterial species; and even in those species, only partial sets of sRNA targets have been characterized in detail. To close this information gap, we developed a computational strategy that takes advantage of available transcriptional data and knowledge about validated and putative sRNA-mRNA interactions for inferring expanded sRNA regulons. Our approach facilitates the identification of experimentally supported novel interactions while filtering out false-positive results. Due to its data-driven nature, our method prioritizes biologically relevant interactions among lists of candidate sRNA-target pairs predicted in silico from sequence analysis or derived from sRNA-mRNA binding experiments.