RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50) : 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50 : 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide: RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.Peer reviewe

Natural Occurrence of 2′,5′-Linked Heteronucleotides in Marine Sponges

2′,5′-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2′,5′-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2′-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2′,5′-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2′,5′-linked heteronucleotides which accompanied high levels of 2′,5′-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2′p5′G, A2′ p5′U, A2′p5′C, G2′p5′A and G2′ p5′U. The natural occurrence of 2′-adenylated NAD+ was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2′,5′-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family

Pyrimidinoceptor potentiation by ATP in NG108-15 cells

AbstractRegulation of inositol phospholipid hydrolysis by UTP and UDP in neuroblastoma×glioma hybrid cell line NG108-15 was potentiated in the presence of ATP. The effect of ATP was dose dependent and shifted the EC50 value for these uracil nucleotides up to three powers of magnitude, having no influence on the maximal value of the response. Adenine nucleotides (ADP, AMP, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), β,γ-methyleneadenosine 5′-triphosphate (βγMeATP), 3′-O-(4-benzoyl)benzoyl ATP (BzATP) and 3′-deoxyadenosine 5′-O-(1-thio)triphosphate (dATPαS)) as well as adenosine, had no influence on the pyrimidinoceptor response. The potentiation effect was abolished by excess of EDTA. The results were in agreement with the hypothesis of pyrimidinoceptor affinity regulation via extracellular phosphorylation of the receptor protein, initiated by ATP. This mechanism may have physiological implication for functioning of uracil nucleotides as endogenous signaling molecules

Comparison of ITS1 profiles of specimens of the same strain and of different strains.

<p>Comparison of ITS1 profiles of specimens of the same strain and of different strains.</p

Examples of results from direct sequencing of ITS2.

<p>Aligned fragments of sequence electropherograms (sequenced using the reverse primer). Samples (from top to bottom): Pe2, R, K2, and V2. Dashed arrows indicate the position of an indel.</p

Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater Sponge <i>Ephydatia fluviatilis</i>

<div><p>Multicopy genes, like ribosomal RNA genes (rDNA), are widely used to describe and distinguish individuals. Despite concerted evolution that homogenizes a large number of rDNA gene copies, the presence of different gene variants within a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge <i>Ephydatia fluviatilis</i> as an example. In addition to using conventional substitution analysis, we have developed a mathematical method, the proportion model method, to quantify the relative amounts of allelic variants of different length using data from direct sequencing of the heterogeneous amplicon. This method is based on determining the expected signal intensity values (corresponding to peak heights from the sequencing electropherogram) by sequencing clones from the same or highly similar amplicon and comparing hypothesized combinations against the values obtained by direct sequencing of the heterogeneous amplicon. This method allowed to differentiate between all specimens analysed.</p></div

Comparison of ITS2 profiles of specimens of the same strain and of different strains.

<p>An asterisk (*) marks the substitution results calculated from sequencing data of mixed sequences (the region following an indel).</p><p>Two asterisks (**) mark the results calculated using other results of length analysis obtained using the proportion model.</p

ITS2 heterogeneities in different individuals: percentages of ITS2 sequences of different length and those of nucleotides in the analysed substitution positions.

<p>The substitution results that are calculated from sequencing data of mixed sequences (the area following indels) are marked with an asterisk (*).</p

Sites of samples taken from a single individual (specimen V3).

<p>The yellow labels indicate the sites of the sponge where the samples were taken. The excised sponge areas were always smaller than the label area.</p

Schematic representation of heterogeneities found in ITS1 and ITS2 revealed by cloning from 5 specimens.

<p>Red and blue rectangles mark the locations of indels and substitutions, respectively. The heterogenous positions (accompanied by a few surrounding nucleotides) found in those locations are given in the boxes below.</p