Single-stranded Polynucleotides Research Articles

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

SPECTROPHOTOMETRIC STUDY OF COMPLEXES OF SOME INTERCALATORS WITH DOUBLE-STRANDED AND SINGLE-STRANDED NUCLEIC ACIDS

Interaction of ethidium bromide (EtBr), acridine orange (AO) and non-intercalator methylene blue (MB) with DNA as well as interaction of EtBr and MB with single-stranded synthetic polynucleotides poly(rA) and poly(rU) have been studied. It was revealed that in absorption spectra of EtBr and MB with DNA or single-stranded polynucleotides a real- or pseudo-isosbestic point appears. Particularly, in absorption spectra of the complexes of classical intercalator EtBr with DNA the real isosbestic point is formed, but in the case of non-classical intercalator MB a pseudo-isosbestic point is formed, while in the absorption spectra of their complexes with single-stranded polynucleotides a pseudo-isosbestic point is formed. It was found out that for another classical intercalator AO there is no a real or pseudo-isosbestic point.

Взаимодействие метиленового синего с одноцепочечными полинуклеотидами poly(rA) и poly(rU) и с двухцепочечным полинуклеотидом poly(rA)-poly(rU)

Interaction of dye-intercalator methylene blue (MB) with single-stranded (ss-) polynucleotides poly(rA), poly(rU) and double-stranded poly(rA)-poly(rU) has been studied by the method of absorption spectroscopy at various concentration ratios ligand/phosphate (concentration of ss-polynucleotides by phosphate residue). It was revealed that in the absorption layer of methylene blue, in the wavelength change interval 500≤λ≤750 nm, the absorption spectra of the complexes MB-ss-poly(rA) decrease more sharply, as compared to MB spectrum, along with polynucleotide concentration enhancement in the solution. It was also revealed that the absorption spectra of the complexes MB-ss-poly(rU) decrease moderately at the increase of this polynucleotide concentration in the solution. In the spectra of the complexes MB-ss-poly(rU) a pseudo-isosbestic point is formed, while in the spectra of the complexes MB-ss-poly(rA) there is no such point. The pseudo-isosbestic point is formed also in the spectra of the complexes MB-poly(rA)-poly(rU). It was also revealed that the absorption spectra of the complexes of MB with ss-poly(rA) and poly(rA)-poly(rU) are shifted to the longer wavelengths by ~5-7 nm, while the shift in the absorption spectra of the complexes MB-ss-poly(rU) composes almost ~2 nm. The absorption spectra changes of the complexes of MB with the mentioned polynucleotides indicate that MB shows higher specificity to poly(rA), as compared to poly(rU) and poly(rA)-poly(rU). The obtained data also indicate that MB binds to poly(rA) and poly(rA)-poly(rU) by intercalation and electrostatic modes, and with ss-poly(rU) – mainly by electrostatic mechanism.

A novel ratiometric electrochemical aptasensor for highly sensitive detection of carcinoembryonic antigen

A novel ratiometric electrochemical aptasensor was proposed for carcinoembryonic antigen (CEA) detection based on exonuclease III (Exo III)-assisted recycling and rolling circle amplification (RCA) strategies. A thiolated ferrocene-labeled hairpin probe 2 (Fc-HP2) was fixed on the gold nanoparticles (AuNPs)-modified gold electrode (AuE) surface through Au–S bonds. The presence of CEA led to the release of trigger, which hybridized with the 3′-protruding of hairpin probe 1 (HP1) and triggered the Exo III cleavage reaction, accompanied by the releasing of trigger and generation of new DNA fragment which was used for the successive hybridization with Fc-HP2. After the Exo III cleavage process, the remaining Fc-HP2 fragments hybridized as primers with the RCA template to initiate the RCA process, and long single-stranded polynucleotides were produced for methylene blue (MB) binding. Such changes resulted in the signal of Fc (IFc) decreased and that of MB (IMB) increased, achieving a linear relationship between IMB/IFc and logarithm of CEA concentrations ranging from 1.0 pg mL−1 to 100.0 ng mL−1 with a detection limit of 0.59 pg mL−1. Additionally, the developed aptasensor had been successfully applied to detect CEA in human serum samples. Therefore, the proposed strategy might provide a new platform for clinical detections of CEA.

A New DNA Repair-Related Platform for Pharmaceutical Outlook in Cancer Therapies: Ultrashort Single-Stranded Polynucleotides

Thio- and cyano- modified single-stranded poly(dNTP) sequences of different molecular sizes (20–200 n) and the same lengths routine poly(dNTP) and poly(NTP) species were tested for their impact on catalytic activities of β-like DNA polymerases from chromatin of HL-60, WERI-1A and Y-79 cells as well as for the affinity patterns in DNApolβ-poly(dNTP)/(NTP) pairs, respectively. An essential link between the lengths of ultrashort (50–100 n) single-stranded poly(dNTP) sequences of different structures and their inhibitory effects towards the cancer-specific DNA polymerases β was found. A possible significance of this phenomenon for both DNA repair suppression in tumors and a consequent anti-cancer activity of the DNA repair related short poly(dNTP) fragments is under discussion.

DNA/RNA recognition controlled by the glycine linker and the guanidine moiety of phenanthridine peptides

The binding of four phenanthridine-guanidine peptides to DNA/RNA was evaluated via spectrophotometric/microcalorimetric methods and computations. The minor structural modifications-the type of the guanidine group (pyrrole guanidine (GCP) and arginine) and the linker length (presence or absence of glycine)-greatly affected the conformation of compounds and consequently the binding to double- (ds-) and single-stranded (ss-) polynucleotides. GCP peptide with shorter linker was able to distinguish between RNA (A-helix) and DNA (B-helix) by different circular dichroism response at 295 nm and thus can be used as a chiral probe. Opposed to the dominant stretched conformation of GCP peptide with shorter linker, the more flexible and longer linker of its analogue enabled the molecule to adopt the intramolecularly stacked form which resulted in weaker yet selective binding to DNA. Beside efficient organization of ss-polynucleotide structures, GCP peptide with shorter linker bound stronger to ss-DNA/RNA compared to arginine peptides which emphasize the importance of GCP unit.

LPI-KTASLP: Prediction of LncRNA-Protein Interaction by Semi-Supervised Link Learning With Multivariate Information

Long non-coding RNA, also known as lncRNA, is a series of single-stranded polynucleotides (no less than 200 nucleotides each), consisting of non-protein coding transcripts. LncRNA plays a crucial role in regulating gene expression, during the transcriptional, post-transcriptional, and epigenetic processes. This is achieved by lncRNA interacts with the corresponding RNA-binding proteins. It has been drawn to a lot of attention that the reduction of the excessive laboratory cost and the increase in speed and accuracy gains benefits from the employment of computational intelligence in lncRNA-protein interaction (LPI) identification. Although numerous pertinent in silico studies of LPI prediction have been proposed, there is still room for enhancing the accuracy of the existing LPI prediction methods. In this paper, we have proposed a novel method for identifying LPI with kernel target alignment based on semi-supervised link prediction (LPI-KTASLP), which adopts multivariate information to predict lncRNAs-proteins interactions. To integrate the heterogeneous kernels, kernel target alignment has been applied to deal with kernel fusion. We have calculated the low-rank approximation matrices of lncRNA and protein, where eigendecomposition is used to reduce computing pressure. The prediction model has been obtained by producing the ultimate LPI prediction matrix. Experimental results show that the prediction ability of the LPI-KTASLP algorithm has surpassed many other LPI prediction schemes. Our method of lncRNA-protein interaction prediction has been evaluated on a standard benchmark dataset of LPIs. We have observed that the highest AUPR of 0.6148 is obtained by our proposed model (LPI-KTASLP). This is superior to the integrated LPLNP (AUPR: 0.4584), the RWR (AUPR: 0.2827), the CF (AUPR: 0.2357), the LPIHN (AUPR: 0.2299), and the LPBNI (AUPR: 0.3302). It is very encouraging that most of the LPI predictions have been confirmed to be close to relevant concentrations.

Adventures with RNA graphs.

The structure of RNA has been a natural subject for mathematical modeling, inviting many innovative computational frameworks. This single-stranded polynucleotide chain can fold upon itself in numerous ways to form hydrogen-bonded segments, imperfect with single-stranded loops. Illustrating these paired and non-paired interaction networks, known as RNA's secondary (2D) structure, using mathematical graph objects has been illuminating for RNA structure analysis. Building upon such seminal work from the 1970s and 1980s, graph models are now used to study not only RNA structure but also describe RNA's recurring modular units, sample the conformational space accessible to RNAs, predict RNA's three-dimensional folds, and apply the combined aspects to novel RNA design. In this article, we outline the development of the RNA-As-Graphs (or RAG) approach and highlight current applications to RNA structure prediction and design.

Synergistic and non-specific nucleic acid production by T7 RNA polymerase and Bsu DNA polymerase catalyzed by single-stranded polynucleotides

Point-of-care molecular diagnostic tests show great promise for providing accurate, timely results in low-infrastructure healthcare settings and at home. The design space for these tests is limited by a variety of possible background reactions, which often originate from relatively weak promiscuous activities of the enzymes used for nucleic acid amplification. When this background signal is amplified alongside the signal of the intended biomarker, the dynamic range of the test can be severely compromised. Therefore, a detailed knowledge of potential side reactions arising from enzyme promiscuity can improve rational design of point-of-care molecular diagnostic tests. Towards this end, we report a previously unknown synergistic reaction between T7 RNA polymerase and Bsu DNA polymerase that produces nucleic acid in the presence of single-stranded DNA or RNA. This reaction occurs in the absence of any previously reported substrates for either polymerases and compromises a theoretical microRNA amplification scheme utilizing these polymerases.

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates.

The Apolipoprotein B mRNA editing complex (APOBEC) family of enzymes contains single-stranded polynucleotide cytidine deaminases. These enzymes catalyze the deamination of cytidine in RNA or single-stranded DNA, which forms uracil. From this 11 member enzyme family in humans, the deamination of single-stranded DNA by the seven APOBEC3 family members is considered here. The APOBEC3 family has many roles, such as restricting endogenous and exogenous retrovirus replication and retrotransposon insertion events and reducing DNA-induced inflammation. Similar to other APOBEC family members, the APOBEC3 enzymes are a double-edged sword that can catalyze deamination of cytosine in genomic DNA, which results in potential genomic instability due to the many mutagenic fates of uracil in DNA. Here, we discuss how these enzymes find their single-stranded DNA substrate in different biological contexts such as during human immunodeficiency virus (HIV) proviral DNA synthesis, retrotransposition of the LINE-1 element, and the "off-target" genomic DNA substrate. The enzymes must be able to efficiently deaminate transiently available single-stranded DNA during reverse transcription, replication, or transcription. Specific biochemical characteristics promote deamination in each situation to increase enzyme efficiency through processivity, rapid enzyme cycling between substrates, or oligomerization state. The use of biochemical data to clarify biological functions and alignment with cellular data is discussed. Models to bridge knowledge from biochemical, structural, and single molecule experiments are presented.

Binding Mode and Selectivity of a Scorpiand-Like Polyamine Ligand to Single- and Double-Stranded DNA and RNA: Metal- and pH-Driven Modulation.

The interaction of a polyazacyclophane ligand having an ethylamine pendant arm functionalized with an anthryl group (L), with the single-stranded polynucleotides polyA, polyG, polyU, and polyC as well as with the double-stranded polynucleotides polyA-polyU, poly(dAT)2 , and poly(dGC)2 has been followed by UV/Vis titration, steady state fluorescence spectroscopy, and thermal denaturation measurements. In the case of the single-stranded polynucleotides, the UV/Vis and fluorescence titrations permit to distinguish between sequences containing purine and pyrimidine bases. For the double-stranded polynucleotides the UV/Vis measurements show for all of them hypochromicity and bathochromic shifts. However, the fluorescence studies reveal that both polyA-polyU and poly(dAT)2 induce a twofold increase in the fluorescence, whereas interaction of poly(dGC)2 with the ligand L induces a quenching of the fluorescence. Cu2+ modulates the interaction with the double-stranded polynucleotides due to the conformation changes that its coordination induces in compound L. In general, the spectroscopic studies show that intercalation seems to be blocked by the formation of the metal complex. All these features suggest the possibility of using compound L as a sequence-selective fluorescence probe.

Molecular dynamics study on the mechanism of polynucleotide encapsulation by chitosan

The safe and effective delivery of therapeutic genes into target cell interiors is of great importance in gene therapy. Chitosan has been extensively studied as a gene delivery carrier due to its good biocompatibility and biodegradability. Understanding the atomic interaction mechanism between chitosan and DNA is important in the design and application of chitosan-based drug and gene delivery systems. In this work, the interactions between single-stranded polynucleotides and different types of chitosan were systematically investigated by using molecular dynamics (MD) simulation. Our results demonstrate that the functional groups of chitosan, the types of base and length of polynucleotides regulate the interaction behavior between chitosan and polynucleotides. The encapsulation capacity of polynucleotide by chitosan is mainly balanced by two factors: the strength of polynucleotide binding to chitosan and the tendency of self-aggregation of polynucleotide in the solution. For –NH3+ chitosan, due to the strong electrostatic interaction, especially the H-bond between –NH3+ groups in chitosan and phosphate groups in polynucleotide, the aggregation effect could be partially eliminated. The good dispersal capacity of polynucleotides may improve the encapsulation of polynucleotides by chitosan, and hence increase the delivery and transfection efficiency of chitosan-based gene carrier.

Complex Thermodynamic Behavior of Single-Stranded Nucleic Acid Adsorption to Graphene Surfaces.

In just over a decade since its discovery, research on graphene has exploded due to a number of potential applications in electronics, materials, and medicine. In its water-soluble form of graphene oxide, the material has shown promise as a biosensor due to its preferential absorption of single-stranded polynucleotides and fluorescence quenching properties. The rational design of these biosensors, however, requires an improved understanding of the binding thermodynamics and ultimately a predictive model of sequence-specific binding. Toward these goals, here we directly measured the binding of nucleosides and oligonucleotides to graphene oxide nanoparticles using isothermal titration calorimetry and used the results to develop molecular models of graphene-nucleic acid interactions. We found individual nucleosides binding KD values lie in the submillimolar range with binding order of rG < rA < rC < dT < rU, while 5mer and 15mer oligonucleotides had markedly higher binding affinities in the range of micromolar and submicromolar KD values, respectively. The molecular models developed here are calibrated to quantitatively reproduce the above-mentioned experimental results. For oligonucleotides, our model predicts complex binding features such as double-stacked bases and a decrease in the fraction of graphene stacked bases with increasing oligonucleotide length until plateauing beyond ∼10-15 nucleotides. These experimental and computational results set the platform for informed design of graphene-based biosensors, further increasing their potential and application.

Interaction of Zn(2+) ions with single-stranded polyU and polyC in neutral solutions.

Effect of Zn(2+) ions on the conformation of single-stranded polynucleotides polyU and polyC in a wide temperature range at pH 7 was studied by differential UV spectroscopy and by thermal denaturation. The atoms coordinating Zn(2+) ions were determined (O4 and N3 in polyU and N3 in polyC). A three-dimensional phase diagram and its two-dimensional components were constructed for a polyC-Zn(2+) system. The phase diagram revealed a region in which ordered single-stranded structures, stabilized by Zn(2+)-mediated cross-links involving N3 atom of cytosine, are formed. The phase diagram also demonstrated that the behavior of the polyC-Zn(2+) system is similar to the effect of retrograde condensation observed in some binary solutions of simple liquids. A dependence of Zn(2+)-polyC binding constant on the metal ion concentration was obtained. The reason why zinc-induced transition of the sequences with adenine-uracil (AU) base pairs from A-form geometry to a metallized m-form requires higher pH compared to the sequences comprised of guanine-cytosine (GC) base pairs is explained. This information can be useful for the development of possible technological applications based on m-DNA.

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.

What history tells us XXXIII. Molecular hybridization: A problematic tool for the study of differentiation and development (1960–1980)

Molecular hybridization consists of the formation of doublestranded DNA-DNA or DNA-RNA (less frequently RNARNA) duplexes between single-stranded polynucleotides with complementary sequences. It is one of the techniques that has been most extensively used in molecular biology. DNA sequencing and amplification of DNA by PCR are based on it. The same is true for the Northern and Southern blot experiments, extensively used by molecular biologists during the early days of genetic engineering, before PCR and the advances of the sequencing programmes. In situ hybridization has been and still is a highly used technique to map genes on chromosomes, and to study gene expression – both its level and cellular localization – during development. But there was another dimension to molecular hybridization that occupied a major place in the 1960s and 1970s, and completely disappeared at the beginning of the 1980s: the use of this technology to access the structural organization of the genome and to investigate the way the genetic information contained in the genome is expressed during differentiation and development. It was a global approach, ignoring the precise nature of the DNA and RNA sequences. This contribution is devoted to this use of molecular hybridization and to the difficulties encountered.

Band structure of the spectra of Hamiltonians of regular polynucleotide duplexes

We calculate the band structure of the spectra of Hamiltonians of regular DNA duplexes and show that in single-stranded periodic polynucleotides whose period is determined by the number m of nucleotides in an elementary cell, the spectrum consists of m nonintersecting energy bands. In DNA duplexes, the number of energy bands is equal to 2m, and the bands can intersect. Discrete energy levels can be present in forbidden bands in the case of (semi)bounded chains or duplexes.

Antiviral Drug Development – Success and Failure: A Personal Perspective with a Japanese Connection

At the 25th International Conference on Antiviral Research, I received a special recognition for my contribution to the International Society of Antiviral Research over a period of 25 years (from 1987 until 2012). This review follows the theme of my presentation at that event, which comprised 10 reminiscences, all with a Japanese connection concerning the success, or otherwise, in the clinical development of: double- and single-stranded polynucleotides; suramin, a polysulfonate; dextran sulfate, a polysulfate; brivudin; BVaraU; 2',3'-dideoxynucleoside analogues; HEPT; adefovir and tenofovir; CXCR4 antagonists; and elvitegravir.

Binding of a Phenanthridine-Biguanide Derivative with DNA/RNA Polynucleotides Studied by Surface-Enhanced Raman Spectroscopy (SERS)

Surface-enhanced Raman spectroscopy (SERS) in the near-infrared region has been applied to study interactions between a phenanthridine-biguanide derivative (PB) and polynucleotides. The PB molecules scatter radiation in the silver citrate colloid most intensively at concentration of 2.5 6 10−5 mol dm−3, at which they are optimally oriented towards the nanoparticles, with the phenanthridine plane placed perpendicularly to the silver surface. A band at 228 cm−1, attributed to the Ag–N stretching mode, implies that chemical mechanism, along with the electromagnetic mechanism, contributes to the total enhancement of the scattered radiation. A decrease in intensity and shifts of the biguanide-related bands in the SERS spectra of the mixtures of PB with DNA indicate binding of the small molecules with the nucleic acid. Studies with double-stranded DNA analogues reveal that the molecules of PB intercalate into the G–C base pairs by the phenanthridine system and bind within the minor groove of the A–T sequences through the biguanide substituent. Intensity decrease in the SERS spectrum of the PB/RNA mixture confirms insertion of the molecules between A–U base pairs, whereas an upward shift of the amino groups bending mode (1078 cm−1) as well as diminution of the CN stretching band at 1421 cm−1 imply hydrogen bonding of the biguanide moiety with the nucleobases. SERS spectra of the mixtures containing the single-stranded polynucleotides show that the polynucleotide secondary structure does not affect binding of PB with the polynucleotides but that affinity towards the polynucleotides depends solely on the molecular structure of the nucleic bases.

Vibronic Model for the Quantum Dynamical Study of the Competition between Bright and Charge-Transfer Excited States in Single-Strand Polynucleotides: The Adenine Dimer Case

A simple vibronic model aimed at investigating the interplay between bright excitonic states and dark charge-transfer (CT) states in stacked adenine (Ade) nucleobases is presented. Two orbitals (the HOMO and the LUMO) for each Ade site have been included in the electronic Hamiltonian, whose parameters have been fitted to reproduce the main features of the absorption spectra of two stacked 9-methyladenine (9Me-A) molecules, computed in aqueous solution at the PCM/TD-PBE0 level. Three modes for each adenine unit have been included in the Hamiltonian, to describe the main structural changes among the different excited state minima of the adenine stacked dimer, as described at the TD-DFT level. The developed vibronic Hamiltonian (four electronic states and six nuclear coordinates) has been adopted to perform quantum dynamical calculations of a photoexcited Ade stacked dimer, utilizing the multiconfigurational time-dependent Hartree method. The obtained results indicate that the transfer between the bright excitonic state and the CT state is fast and effective.