The influenza A virus is a human pathogen that poses a serious public health threat due to rapid antigen changes and emergence of new, highly pathogenic strains with the potential to become easily transmitted in the human population. The viral genome is encoded by eight RNA segments, and all stages of the replication cycle are dependent on RNA. In this study, we designed small interfering RNA (siRNA) targeting influenza segment 5 nucleoprotein (NP) mRNA structural motifs that encode important functions. The new criterion for choosing the siRNA target was the prediction of accessible regions based on the secondary structure of segment 5 (+)RNA. This design led to siRNAs that significantly inhibit influenza virus type A replication in Madin-Darby canine kidney (MDCK) cells. Additionally, chemical modifications with the potential to improve siRNA properties were introduced and systematically validated in MDCK cells against the virus. A substantial and maximum inhibitory effect was achieved at concentrations as low as 8 nM. The inhibition of viral replication reached approximately 90% for the best siRNA variants. Additionally, selected siRNAs were compared with antisense oligonucleotides targeting the same regions; this revealed that effectiveness depends on both the target accessibility and oligonucleotide antiviral strategy. Our new approach of target-site preselection based on segment 5 (+)RNA secondary structure led to effective viral inhibition and a better understanding of the impact of RNA structural motifs on the influenza replication cycle.

Influenza A virus (IAV) affects 5%–10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2′- O -methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11–15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5′ and 3′ ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches.

Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (−)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics.

Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by in vitro chemical structure mapping, thermodynamics, bioinformatics, and binding to isoenergetic microarrays of an entire natural sequence of the 875 nt segment 8 vRNA and of a smaller fragment. Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle.

The aim of this study was to identify the group of highly polymorphic microsatellite markers for the identification of six pear cultivars (P. communis) and two individuals of wild pear (P. pyraster). From among 40 tested SSR markers, 19 were selected to profile genetic diversity in pear genotypes due to high polymorphisms. These markers showed high heterozygosity levels (0.5–1) and, on average, 6.4 alleles per marker were found. The set of microsatellite markers employed in this study demonstrated usefulness of microsatellite markers for the identification of pear genotypes. The examined wild forms were represented in this study by only two individuals of P. pyraster. It can be assumed that these forms were distinctly different from the cultivated pear cultivars.

Alternative splicing at the MAPT gene exon 10 yields similar levels of the 3R and 4R tau protein isoforms. ¹ Lee CJ , Irizarry K . Alternative splicing in the nervous system: an emerging source of diversity and regulation. Biol Psychiatry 2003; 54:771–6; PMID:14550676; http://dx.doi.org/10.1016/S0006-3223(03)00375-5 [Crossref], [PubMed], [Web of Science ®] [Google Scholar] The presence of mutations, particularly in exon 10 and intron 10–11, changes the quantity of tau isoforms. Domination each of the isoform yields tau protein aggregation and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we report for the first time the secondary structure of the 194/195 nucleotide region for the wild type (WT) and 10 mutants of the MAPT gene pre-mRNA determined using both chemical and microarray mapping. Thermodynamic analyses indicate that single nucleotide mutations in the splicing regulatory element (SRE) that form a hairpin affect its stability by up to 4 and 7 kcal/mol. Moreover, binding the regulatory hairpin of small molecule ligands (neomycin, kanamycin, tobramycin and mitoxantrone) enhance its stability depending on the nature of the ligands and the RNA mutations. Experiments using the cos-7 cell line indicate that the presence of ligands and modified antisense oligonucleotides affect the quantity of 3R and 4R isoforms. This finding correlates with the thermodynamic stability of the regulatory hairpin. An alternative splicing regulation mechanism for exon 10 is postulated based on our experimental data and on published data.