Poly rA Research Articles

Polycationic Monomeric and Homodimeric Asymmetric Monomethine Cyanine Dyes with Hydroxypropyl Functionality-Strong Affinity Nucleic Acids Binders.

New analogs of the commercial asymmetric monomethine cyanine dyes thiazole orange (TO) and thiazole orange homodimer (TOTO) with hydroxypropyl functionality were synthesized and their properties in the presence of different nucleic acids were studied. The novel compounds showed strong, micromolar and submicromolar affinities to all examined DNA ds-polynucleotides and poly rA–poly rU. The compounds studied showed selectivity towards GC-DNA base pairs over AT-DNA, which included both binding affinity and a strong fluorescence response. CD titrations showed aggregation along the polynucleotide with well-defined supramolecular chirality. The single dipyridinium-bridged dimer showed intercalation at low dye-DNA/RNA ratios. All new cyanine dyes showed potent micromolar antiproliferative activity against cancer cell lines, making them promising theranostic agents.

Study of complexes of Hoechst 33258 with poly(rA)-poly(rU) depending on various ionic strengths in the water-saline solution

Comparative study of the complexes of groove-binding ligand Hoechst 33258 (H33258) with synthetic homopolynucleotides poly(rA)-poly(rU) and poly(dA)-poly(dT) has been carried out at various concentration ratios of r = ligand/nucleic acids (NA) and different ionic strengths of the water-saline solution 0.02, 0.04 and 0.1 M, using the method of UV-melting. It was revealed that the melting curves of the complexes of poly(dA)-poly(dT) with H33258 at the low concentrations of ligand are biphasic, which actually does not depend on the solution ionic strength. In the case of the complexes of poly(rA)-poly(rU)-H33258, the melting curves become quasi-biphasic only at the ionic strength 0.02 M and relatively high concentrations of the ligand. Differential melting curves (DMC) of the mentioned polynucleotides and their complexes with H33258 were obtained as well. DMC of poly(rA)-poly(rU) were found to be significantly wide at the ionic strengths of the solution 0.02 and 0.04 M and to show an intrinsic heterogeneity of double-stranded structure of this polynucleotide. Communicated by Ramaswamy H. Sarma

Adsorption of Polyadenylic acid on graphene oxide: experiments and computer modeling

In this work, we study the adsorption of poly(rA) on graphene oxide (GO) using AFM and UV absorption spectroscopies. A transformation of the homopolynucleotide structure on the GO surface is observed. It is found that an energetically favorable conformation of poly(rA) on GO is achieved after a considerable amount of time (days). It is revealed that GO can induce formation of self-structures of single-stranded poly(rA) including a duplex at pH 7. The phenomenon is analyzed by polymer melting measurements and observed by AFM. Details of the noncovalent interaction of poly(rA) with graphene are also investigated using molecular dynamics simulations. The adsorption of (rA)10 oligonucleotide on graphene is compared with the graphene adsorption of (rC)10. DFT calculations are used to determine equilibrium structures and the corresponding interaction energies of the adenine-GO complexes with different numbers of the oxygen-containing groups. The IR intensities and vibrational frequencies of free and adsorbed adenines on the GO surface are calculated. The obtained spectral transformations are caused by the interaction of adenine with GO.

STUDY OF METHYLENE BLUE INTERACTION WITH SYNTHETIC POLYNUCLEOTIDE POLY(rA)-POLY(rU)

A study of the interaction of methylene blue (MB) with poly(rA)-poly(rU) by the method of fluorescence spectroscopy has been carried out. The data obtained revealed that MB, being a DNA-specific ligand, can bind to double-stranded regions of RNA. In this regard, as in the case of DNA, semi-intercalation was the most preferable mechanism for the binding of this ligand to poly(rA)-poly(rU). On the other hand, non-linear curves of dependence of F0/F on concentration of polynucleotide might result from two binding modes, the second of which was probably of an electrostatic nature. Proceeding from the data obtained, the value of KSV was revealed to be almost an order of magnitude less than for DNA, which may indicate that RNA is a less preferable target for MB.

Synthesis and nucleic acid binding evaluation of a thyminyl l-diaminobutanoic acid-based nucleopeptide.

Herein we present the synthesis of a l-diaminobutanoic acid (DABA)-based nucleopeptide (3), with an oligocationic backbone, realized by solid phase peptide synthesis using thymine-bearing DABA moieties alternating in the sequence with free ones. CD studies evidenced the ability of this oligothymine nucleopeptide, well soluble in aqueous solution, to alter the secondary structure particularly of complementary RNA (poly rA vs poly rU) and inosine-rich RNAs, like poly rI and poly rIC, and showed its preference in binding double vs single-stranded DNAs. Furthermore, ESI mass spectrometry revealed that 3 bound also G-quadruplex (G4) DNAs, with either parallel or antiparallel topologies (adopted in our experimental conditions by c-myc and tel22, respectively). However, it caused detectable changes only in the CD of c-myc (whose parallel G4 structure was also thermally stabilized by ~3°C), while leaving unaltered the antiparallel structure of tel22. Interestingly, CD and UV analyses suggested that 3 induced a hybrid mixed parallel/antiparallel G4 DNA structure in a random-coil tel22 DNA obtained under salt-free buffer conditions. Titration of the random-coil telomeric DNA with 3 gave quantitative information on the stoichiometry of the obtained complex. Overall, the findings of this work suggest that DABA-based nucleopeptides are synthetic nucleic acid analogues potentially useful in antigene and antisense strategies. Nevertheless, the hexathymine DABA-nucleopeptide shows an interesting behaviour as molecular tool per se thanks to its efficacy in provoking G4 induction in random coil G-rich DNA, as well as for the possibility to bind and stabilize c-myc oncogene in a G4 structure.

STUDY OF BINDING PECULIARITIES OF ETHIDIUM BROMIDE AND METHYLENE BLUE WITH DOUBLE-STRANDED RNA

The paper presents the results of studies on the interaction of intercalators ethidium bromide (EtBr) and methylene blue (MB) with synthetic double-stranded (ds) ribonucleotide ds-poly(rA)-poly(rU) at a solution ionic strengths of 0.02, 0.04 and 0.1 M. These results revealed that poly(rA)-poly(rU) has a relatively unstable structure at the ionic strengths μ$\leq$0.02 M, which leads to the interaction of the mentioned ligands with this polynucleotide. EtBr affinity for ds-poly(rA)-poly(rU) was revealed not to depend on the ionic strength of the solution, while the affinity of MB for this polynucleotide depends on this factor, as well as on the structural state of the polynucleotide.

Spectroscopic study on binding of TMPyP4+ porphyrin to single-stranded poly(rA)

Binding of tetracationic porphyrin (TMPyP4+) to poly(rA) has been studied in neutral buffered solution of low ionic strength in a wide range of molar phosphate-to-dye ratios (P/D) using absorption spectroscopy, polarized fluorescence and fluorimetric titration. Two competitive binding modes were identified: partial intercalation of porphyrin chromophores between adenine bases prevailing at P/D > 20 and its outside binding to poly(rA) backbone dominating at P/D < 6. Both of them were accompanied by enhancement of the porphyrin emission. Absence of the emission quenching near stoichiometric P/D ratios allowed us to assume that external binding occurs without the self-stacking of the chromophores.

Thermodynamics of the Interaction of Synthetic Polynucleotide Poly(rA)-Poly(rU) with Intercalators

The melting of poly(rA)-poly(rU) and its complexes with the intercalators — methylene blue (MB) and ethidium bromide (EtBr) has been carried out at the ionic strength of the solution 0.02, 0.04, and 0.1 M. It was revealed that this polynucleotide has the non-stable double-stranded structure at the ionic strength of 0.02 M and it becomes more stable at the complex-formation with the mentioned ligands. The increase of the solution ionic strength also results in the stabilization of the double-stranded structure of the polynucleotide, in consequence of which the interaction of the MB and EtBr with the poly(rA)-poly(rU) becomes more preferable and more beneficial thermodynamically. It was shown that at the ionic strength of 0.1 M the poly(rA)-poly(rU) takes a structure that is more available to the intercalation of both ligands. In the case of the MB the main binding mode becomes a semi-intercalation, while in the case of the EtBr all the intrinsic binding modes are displayed. It was also shown that in the case of the EtBr the values of ΔH and ΔS increase, while in the case of the MB, ΔH and ΔS those values increase, then decrease at the enhancement of the ionic strength of the solution.

Spectroscopic Study of the Binding of Netropsin and Hoechst 33258 to Nucleic Acids

The interaction of groove binding compounds — peptide antibiotic (polyamide) netropsin and fluorescent dye (bisbenzimidazole) Hoechst 33258 — with the double-stranded DNA and synthetic double-stranded polynucleotide poly(rA)-poly(rU) has been studied by spectrophotometry. Absorption spectra of these ligand complexes with nucleic acids have been obtained. Spectral changes at the complexation of individual ligands with the mentioned nucleic acids reveal the similarity of binding of each of these ligands with both DNA and RNA. Based on the spectroscopic measurements, the binding parameters of netropsin and Hoechst 33258 binding to DNA and poly(rA)-poly(rU) – K and n, as well as the thermodynamic parameters ΔS, ΔG, and ΔH have been determined. It was found that the binding of Hoechst 33258 to both nucleic acids is accompanied by a positive change in enthalpy, while in the case of netropsin the change in enthalpy is negative. Moreover, the contribution of entropy to the formation of the complexes is more pronounced in the case of Hoechst 33258.

Impact of linker between triazolyluracil and phenanthridine on recognition of DNA and RNA. Recognition of uracil-containing RNA

A phenanthridine-triazolyluracilyl multifunctional ligand, linked by a lysine–glycine peptide, binds to poly rA–poly rU with micromolar affinity and selective fluorescence response.

Small molecule probes finely differentiate between various ds- and ss-DNA and RNA by fluorescence, CD and NMR response

Two small molecules showed intriguing properties of analytical multipurpose probes, whereby one chromophore gives different signal for many different DNA/RNA by application of several highly sensitive spectroscopic methods. Dyes revealed pronounced fluorescence ratiomeric differentiation between ds-AU-RNA, AT-DNA and GC-DNA in approximate order 10:8:1. Particularly interesting, dyes showed specific fluorimetric response for poly rA even at 10-fold excess of any other ss-RNA, and moreover such emission selectivity is preserved in multicomponent ss-RNA mixtures. The dyes also showed specific chiral recognition of poly rU in respect to the other ss-RNA by induced CD (ICD) pattern in visible range (400–500 nm), which was attributed to the dye-side-chain contribution to binding (confirmed by absence of any ICD band for reference compound lacking side-chain). Most intriguingly, minor difference in the side-chain attached to dye chromophore resulted in opposite sign of dye-ICD pattern, whereby differences in NMR NOESY contacts and proton chemical shifts between two dye/oligo rU complexes combined with MD simulations and CD calculations attributed observed bisignate ICD to the dimeric dye aggregate within oligo rU.

Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells.

Inhibition of the DNA polymerase and RNase H activities of HIV-1 reverse transcriptase and HIV-1 replication by Brasenia schreberi (Junsai) and Petasites japonicus (Fuki) components

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses two distinct enzymatic activities: those of RNA- and DNA-dependent DNA polymerases and RNase H. In the current HIV-1 therapy, all HIV-1 RT inhibitors inhibit the activity of DNA polymerase, but not that of RNase H. We previously reported that ethanol and water extracts of Brasenia schreberi (Junsai) inhibited the DNA polymerase activity of HIV-1 RT [Hisayoshi et al. (2014) J Biol Macromol 14:59-65]. In this study, we screened 43 edible plants and found that ethanol and water extracts of Brasenia schreberi and water extract of Petasites japonicus strongly inhibit not only the activity of DNA polymerase to incorporate dTTP into poly(rA)-p(dT)15 but also the activity of RNase H to hydrolyze the RNA strand of an RNA/DNA hybrid. In addition, these three extracts inhibit HIV-1 replication in human cells, with EC50 values of 1-2µg/ml. These results suggest that Brasenia schreberi and Petasites japonicus contain substances that block HIV-1 replication by inhibiting the DNA polymerase activity and/or RNase H activity of HIV-1 RT.

Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome

The archaeal exosome is a phosphorolytic 3′–5′ exoribonuclease complex. In a reverse reaction it synthesizes A-rich RNA tails. Its RNA-binding cap comprises the eukaryotic orthologs Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. In Sulfolobus solfataricus DnaG and Rrp4 but not Csl4 show preference for poly(rA). Archaeal DnaG contains N- and C-terminal domains (NTD and CTD) of unknown function flanking a TOPRIM domain. We found that the NT and TOPRIM domains have comparable, high conservation in all archaea, while the CTD conservation correlates with the presence of exosome. We show that the NTD is a novel RNA-binding domain with poly(rA)-preference cooperating with the TOPRIM domain in binding of RNA. Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. We also found that DnaG strongly binds native and in vitro transcribed rRNA and enables its polynucleotidylation by the exosome. Furthermore, rRNA-derived transcripts with heteropolymeric tails were degraded faster by the exosome than their non-tailed variants. Based on our data, we propose that archaeal DnaG is an RNA-binding protein, which, in the context of the exosome, is involved in targeting of stable RNA for degradation.

Binding ability of a thymine-functionalized oligolysine towards nucleic acids

In this Letter, we investigated the binding properties towards nucleic acids of a thymine-functionalized oligolysine, composed of nucleobase-bearing amino acid moieties and underivatized l-lysine residues alternate in the backbone. The basic nucleopeptide proved to be well soluble in water and able to interact with both DNA and RNA, as suggested by circular dichroism, UV and surface plasmon resonance studies performed on the thymine-containing oligomer with both adenine-containing DNA (dA12) and RNA (rA12 and poly rA) molecules. In both cases the thymine-functionalized oligolysine was proven to form complexes characterized by a 1:1 T/A stoichiometric ratio, as evidenced by CD titration. UV melting experiments revealed that the complex formed between the homothymine oligolysine and rA12 RNA was more stable than the complex with dA12 DNA probably due to the additional H-bonding of the 2′-OH groups in RNA, that reinforces the overall interaction with the nucleopeptide. Finally, human serum stability assays were conducted on the thymine-bearing nucleopeptide which showed a half-life of 45min.

Synthesis of a novel benzodifuran derivative and its molecular recognition of poly rA RNA

In this manuscript, we describe a synthetic approach to a novel benzodifuran derivative as well as CD studies regarding its ability to interact with DNA and RNA. After the chemical synthesis and ESI-MS and NMR characterization, this heteroaromatic molecule was investigated by CD spectroscopy in order to evaluate it as a potential nucleic acid binder. Interestingly, the benzodifuran compound was found to be able to induce conformational changes in both DNA and RNA homoadenine molecules forming in the latter case a complex with a 6:1 benzodifuran/nucleobase stoichiometric ratio, as evidenced by CD titration experiments.

Nanohybrid Structures Formed by Carbon Nanotubes with Long Polynucleotide

Adsorption of poly(rA) on the single-walled carbon nanotubes (SWNTs) surface have been studied by AFM and computer modeling. It follows from AFM images that relatively long fragments of polymers attached to two individual nanotubes in aqueous suspension form branched structures. The size of these structures increases after the addition of a complementary poly(rU) to the aqueous suspension of nanotubes with adsorbed poly(rA), which is induced by hybridization of the adsorbed and free polymer. Molecular dynamic simulation demonstrates different possible structures of these nanohybrids.

Application of equilibrium binding model for analysis of conformational transitions in poly(rA)poly(rU) complexes with metal ions

Application of equilibrium binding model for analysis of conformational transitions in poly(rA)poly(rU) complexes with metal ions

Synthetic polycation: Polynucleotide interactions determined using liquid chromatography with short monolithic columns

LC on short monolithic columns (Convective Interaction Medium Disks) was applied to investigate several specially synthesized water soluble polycations of different charge type (primary, tertiary, quaternary amine), as well as a copolymer of neutral saccharide and cationic monomers, regarding their ability to form reversible complexes with DNA. For this purpose, two separation modes were used, namely, pseudo-affinity and cation-exchange chromatography. Synthetic polynucleotides, namely, polyriboadenylic acid (poly(rA)) and polyribocytidylic acid (poly(rC)), were used as approximate structural analogues of DNA. In first case, the hypothetical specific binding between dissolved polymers and polynucleotide (poly(rA) or poly(rC)), covalently attached to epoxy-bearing monolithic sorbent, has been studied and compared to the results obtained using cation exchange chromatography. Quantitative parameters of interactions between macromolecules were established using frontal elution method.

Determination of the solution structure of nucleic acid double helices using NOE difference NMR spectroscopy, chemical shift calculations, and computer modeling

A procedure involving the combined use of NOE measurements, chemical shift shielding calculations, and stereochemical model building is discussed to determine the solution conformation of nucleic acids. Using this procedure it is shown that the DNA, poly(dA)-poly(dT), and the RNA-DNA hybrid, poly(rA)-poly(dT), in solution exist as right-handed B structures and that the heteronomous model is not true for them in solution. Even though both the DNA and the hybrid have the gross morphology of the B form, there are significant differences in the ultimate details such as base displacement, tilt, and twist.