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International Journal of Biological... Jun 2022Two Ru(II) complexes containing different substituents, [Ru(phen)(7-CH-dppz)] (Ru1) and [Ru(phen)(7-Br-dppz)] (Ru2), have been synthesized in this study. The binding...
Two Ru(II) complexes containing different substituents, [Ru(phen)(7-CH-dppz)] (Ru1) and [Ru(phen)(7-Br-dppz)] (Ru2), have been synthesized in this study. The binding properties of Ru1 and Ru2 with the duplex RNA poly(U)•poly(A) (where "•" denotes the Watson - Crick base pairing) have been researched by biophysical techniques and viscosity measurements. Analysis of spectral titrations and viscosity measurements indicate that Ru1 and Ru2 bind to the duplex via intercalative, and the binding affinity of Ru1 with the duplex is remarkably higher than that of Ru2. Furthermore, fluorescence emission spectra demonstrates that although complexes Ru1 and Ru2 can act as molecular "light switches" for the duplex RNA, alters in fluorescence emission of Ru1 and Ru2 are prominent differences, and the effectiveness of Ru1 is more remarkable compared with that of Ru2. The melting experiments suggest that the duplex RNA stabilizing effects of Ru1 and Ru2 differ from each other, among them, complex Ru1 can obviously enhance the stability of the duplex RNA, while Ru2 has only a slightly stabilizing effect for the duplex RNA, indicating that Ru1 preferentially binds to RNA duplex over Ru2. The obtained results indicate that subtle modifications of the intercalative ligand of Ru(II) polypyridyl complex with either methyl or bromide group have a significant effect on the duplex-binding discrimination.
Topics: Bromine; Coordination Complexes; Poly A; RNA; RNA, Messenger; Ruthenium
PubMed: 35489619
DOI: 10.1016/j.ijbiomac.2022.04.091 -
Chembiochem : a European Journal of... Apr 2018Fluorescence-based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as...
Fluorescence-based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment-sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8-naphthalimide) at the 5-position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells-poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.
Topics: Fluorescent Dyes; Peptide Nucleic Acids; Poly A; RNA
PubMed: 29396904
DOI: 10.1002/cbic.201700661 -
Biochimica Et Biophysica Acta Jan 1981poly(A)+ RNA was isolated from maize by affinity chromatography on columns of oligo(dT)-cellulose. A modified nucleotide ('X') was detected in ribonuclease T2 digests of...
poly(A)+ RNA was isolated from maize by affinity chromatography on columns of oligo(dT)-cellulose. A modified nucleotide ('X') was detected in ribonuclease T2 digests of the RNA as part of a resistant dinucleotide. The dinucleotide was detected by means of the polynucleotide kinase-mediated transfer of a radioactive phosphate atom from adenosine triphosphate to the 5'-OH position of the dinucleotide. Intact poly(A) tracts were released from poly(A)+ RNA by digestion with ribonuclease T1 and A in a high salt buffer and were isolated by oligo(dT)-cellulose chromatography. The poly(A) preparation was found to consist of a series of polyadenylate fragments which varied in chain length from approximately 17 to greater than 70. The modified nucleotide was shown to occupy an internal position in these poly(A) tracts.
Topics: Chromatography, Affinity; Chromatography, Thin Layer; Endonucleases; Endoribonucleases; Plants; Poly A; RNA; RNA, Messenger; Ribonucleases
PubMed: 6163465
DOI: 10.1016/0005-2787(81)90213-6 -
Cell Systems Jun 2024Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small...
Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates but is decoupled from translation. Interestingly, an optimal tail length of ∼100 nt displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability-a feature of endogenous mRNAs-impacts translation efficiency but not mRNA degradation rates. Stochastic modeling combined with single-cell tracking reveals that poly(A) tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.
Topics: Humans; RNA, Messenger; Poly A; Protein Biosynthesis; RNA Stability; HEK293 Cells; Gene Expression Regulation
PubMed: 38901403
DOI: 10.1016/j.cels.2024.05.004 -
Nucleic Acids Research Sep 1983The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively...
The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect.
Topics: Animals; Dictyostelium; Kinetics; Poly A; Protein Biosynthesis; RNA, Messenger; Rabbits; Reticulocytes
PubMed: 6137807
DOI: 10.1093/nar/11.18.6353 -
Methods in Molecular Biology (Clifton,... 2014The binding of small molecules to non-canonical nucleic acid structures has been a major focus of rational drug design. Among the non-canonical nucleic acid structures,...
The binding of small molecules to non-canonical nucleic acid structures has been a major focus of rational drug design. Among the non-canonical nucleic acid structures, targeting poly(A) using small molecules has attracted a special interest due to the cellular functions of poly(A) tails. Here, the methods for determining the binding of a small molecule to poly(A) using UV-visible(UV-Vis) and Circular Dichroism (CD) Spectroscopy are described. Experiments used in determining the melting temperature, binding stoichiometry and dissociation constant of poly(A)-small molecule systems are depicted.
Topics: Circular Dichroism; Nucleic Acid Conformation; Poly A
PubMed: 24590782
DOI: 10.1007/978-1-62703-971-0_8 -
Computational Biology and Chemistry Feb 2015Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since...
Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since the anti-tumour agent cisplatin preferentially forms DNA adducts at runs of consecutive guanine nucleotides (poly(G)), the position and frequency of occurrence of poly(G) sequences in the updated human genome was investigated. There are more runs of consecutive guanines than would be expected by random chance. This especially true for poly(G) sequences longer than approximately n=9. A plot of poly(G) length against log(observed/expected) frequency produced a straight line for n>9. A similar observation was also found for poly(A) DNA sequence repeats. This data implied that the increase in observed/expected frequency is directly related to length of DNA repeat. It was proposed that long runs of consecutive guanine nucleotides could be a sensitive sensor of cellular DNA damage since a number of DNA damaging agents cause lesions at poly(G) sequences.
Topics: Base Sequence; DNA; DNA Damage; Genome, Human; Humans; Microsatellite Repeats; Molecular Probes; Molecular Sequence Data; Poly A; Poly G
PubMed: 25479163
DOI: 10.1016/j.compbiolchem.2014.11.006 -
Methods in Molecular Biology (Clifton,... 2014The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient "hook" to isolate mRNA. However the length of this tail and its position within the...
The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient "hook" to isolate mRNA. However the length of this tail and its position within the primary RNA transcript can also hold diagnostic value for RNA metabolism. In general, mRNA with a long poly(A)-tail is well translated, whereas a short poly(A)-tail can indicate translational silencing. A short poly(A)-tail is also appended to RNA-decay intermediates via the TRAMP complex. A number of approaches have been developed to measure the length and position of the poly(A)-tail. Here, we describe a simple method to "tag" adenylated RNA using the native function of DNA polymerase I to extend an RNA primer on a DNA template in second-strand DNA synthesis. This function can be harnessed as a means to purify, visualize, and quantitate poly(A)-dynamics of individual RNA and the transcriptome en masse.
Topics: Genetic Techniques; Poly A; RNA, Messenger; Transcriptome
PubMed: 24590777
DOI: 10.1007/978-1-62703-971-0_3 -
Journal of the American Chemical Society Mar 2013We developed a model for hole migration along relatively short DNA hairpins with fewer that seven adenine (A):thymine (T) base pairs. The model was used to simulate hole...
We developed a model for hole migration along relatively short DNA hairpins with fewer that seven adenine (A):thymine (T) base pairs. The model was used to simulate hole migration along poly(A)-poly(T) sequences with a particular emphasis on the impact of partial hole localization on the different rate processes. The simulations, performed within the framework of the stochastic surrogate Hamiltonian approach, give values for the arrival rate in good agreement with experimental data. Theoretical results obtained for hairpins with fewer than three A:T base pairs suggest that hole transfer along short hairpins occurs via superexchange. This mechanism is characterized by the exponential distance dependence of the arrival rate on the donor/acceptor distance, k(a) ≃ e(-βR), with β = 0.9 Å(-1). For longer systems, up to six A:T pairs, the distance dependence follows a power law k(a) ≃ R(-η) with η = 2. Despite this seemingly clear signature of unbiased hopping, our simulations show the complete delocalization of the hole density along the entire hairpin. According to our analysis, the hole transfer along relatively long sequences may proceed through a mechanism which is distinct from both coherent single-step superexchange and incoherent multistep hopping. The criterion for the validity of this mechanism intermediate between superexchange and hopping is proposed. The impact of partial localization on the rate of hole transfer between neighboring A bases was also investigated.
Topics: DNA; Models, Molecular; Poly A; Poly T; Quantum Theory; Time Factors
PubMed: 23402652
DOI: 10.1021/ja3113998 -
Nucleic Acids Research Oct 2016PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported...
PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported recently to participate in piRNA biogenesis in silkworm and C. elegans, respectively. To get insights on the role of mammalian PNLDC1, we characterized the human and mouse enzymes. PNLDC1 shows limited conservation compared to PARN and represents an evolutionary related but distinct group of enzymes. It is expressed specifically in mouse embryonic stem cells, human and mouse testes and during early mouse embryo development, while it fades during differentiation. Its expression in differentiated cells, is suppressed through methylation of its promoter by the de novo methyltransferase DNMT3B. Both enzymes are localized mainly in the ER and exhibit in vitro specificity restricted solely to 3' RNA or DNA polyadenylates. Knockdown of Pnldc1 in mESCs and subsequent NGS analysis showed that although the expression of the remaining deadenylases remains unaffected, it affects genes involved mainly in reprogramming, cell cycle and translational regulation. Mammalian PNLDC1 is a novel deadenylase expressed specifically in cell types which share regulatory mechanisms required for multipotency maintenance. Moreover, it could be involved both in posttranscriptional regulation through deadenylation and genome surveillance during early development.
Topics: Animals; Cell Differentiation; Cell Line; Embryonic Development; Embryonic Stem Cells; Endoplasmic Reticulum; Exoribonucleases; Gene Expression; Gene Expression Regulation, Developmental; Humans; Methylation; Mice; Models, Molecular; Molecular Conformation; Poly A; Polyadenylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Transport
PubMed: 27515512
DOI: 10.1093/nar/gkw709