Dofinansowanie ze środków budżetu państwa
Deklaracja dostępności
Prof. Elżbieta Kierzek Head of Department elzbieta.kierzek@ibch.poznan.pl ext. 1113, 1112
Dr. Marta Soszyńska-Jóźwiak adiunkt
Dr. Marta Szabat adiunkt
Ulanowska Izabela MSc PhD student
Maria Nalewaj MSc PhD student
Agnieszka Baliga-Gil MSc PhD student
Kinga Maziec MSc, BEng PhD student
Mariia Sokulska MSc PhD student
Upper panel: isoenergetic microarrays with hybridized hairpins H4 and H6 along with bar graph of relative binding intensity to the probes and secondary structures of these model RNAs with marked results. Microarrays are built with chimera 2'-O-methyl-LNA oligonucleotide probes complementary step-by-step to RNA models. Probes binding sites are marked with middle nucleotide of region binding to pentamer probe (filled square - strong binding; unfilled square - medium binding). Lower panel: microarrays, bar graph and structures of Pk1 and Pk-H1. Each probe was spotted six times and the average intensity is plotted. Conditions: 1 M NaCl, 20 mM sodium cacodylate, 0.5 mM Na2EDTA, pH 7.0, 4°C.
A/ Secondary structure of R2 5'RNA from Bombyx mori based on microarray mapping, chemical mapping and structures/sequences comparison of R2 5'RNA. B/ Secondary structure of Saturnia pyri pseudoknot annotated with mutations from the other four R2 pseudoknots (from Bombyx mori, Callosamia promethea, Coscinocera hercules and Samia cynthia). In frame, a cartoon of the pseudoknot that combines the annotation of conserved features.
A/ Secondary structure of segment 8 vRNA predicted by RNAstructure 5.3 using as constraints: strong reactivity of DMS; consensus base pairs from sequence and structure analysis (orange bars); SHAPE reactivities converted to pseudo- free energies. B/ Isoenergetic microarrays with hybridized segment 8 vRNA of strain A/Vietnam/1203/2004 (H5N1) in different temperatures. C/ Secondary structure of mini-vRNA8 predicted by RNAstructure 5.3 using as constraints: strong reactivity of DMS and SHAPE reactivities converted to pseudo-energy. Efficient packaging of a segment 8 encoding only GFP protein required nucleotides 1-177 and 198-376 (mini-vRNA8 nomenclature). D/ Example of antisense oligonucleotide transfection. MDCK cells transfected with FAM6 labeled oligonucleotide (left picture - green). Nucleus were stained with DAPI (right picture - blue). E/ Antiviral activity of 230 nM antisense oligonucleotides in MDCK cells. Logarithm of average titer of influenza in cell culture supernatant was analyzed by immunofocus assay: red bars - for influenza A/California/04/2009 (IAV) after 36 h postinfection; blue bars - for influenza B/Brisbane/60/2008 (IBV) after 48 h postinfection. V- control infected cells without treatment; LPF – control, infected cells treated only with lipofectamine 2000; 68-11L and 404-14L antisense oligonucleotides. Statistic were calculated using T-test (*** P<0.001). No statistically differences in virus titer were observed for IBVDAPI (prawe zdjęcie - niebieskie). E/ Aktywność antywirusowa 230 nM oligonukleotydów antysensowych w linii komórkowej MDCK. Uśredniony logarytm z miana wirusa został zanalizowany za pomocą testu immunofluorescencji pośredniej: czerwone słupki - dla wirusa grypy A/California/04/2009 (IAV) po 36 h po infekcji; niebieskie słupki - dla wirusa grypy B/Brisbane/60/2008 (IBV) po 48 h po infekcji. V – kontrola, infekowane komórki; LPF – kontrola, infekowane komórki traktowane lipofektaminy 2000; 68-11L oraz 404-14L oligonukleotydy antysensowe. Statystyka została obliczona z zastosowaniem testu T- studenta (*** P<0.001). Nie wykazano statystycznej różnicy w mianie wirusa dla wirusa IBV.DAPI (prawe zdjęcie - niebieskie). E/ Aktywność antywirusowa 230 nM oligonukleotydów antysensowych w linii komórkowej MDCK. Uśredniony logarytm z miana wirusa został zanalizowany za pomocą testu immunofluorescencji pośredniej: czerwone słupki - dla wirusa grypy A/California/04/2009 (IAV) po 36 h po infekcji; niebieskie słupki - dla wirusa grypy B/Brisbane/60/2008 (IBV) po 48 h po infekcji. V – kontrola, infekowane komórki; LPF – kontrola, infekowane komórki traktowane lipofektaminy 2000; 68-11L oraz 404-14L oligonukleotydy antysensowe. Statystyka została obliczona z zastosowaniem testu T- studenta (*** P&amp;lt;0.001). Nie wykazano statystycznej różnicy w mianie wirusa dla wirusa IBV.
A/ Antiviral activity of the two most effective unmodified siRNAs in the IFA assay. The results obtained for siRNAs were compared with the negative control K1 (100%). Cells treated with lipofectamine, without siRNA, are labeled as LF, remaining tags are siRNAs names. Error bars represent the standard deviation of independent experiments. Statistical analysis was performed using the student's t-test (*** p <0.001, ** p <0.01, * p <0.05). B/ Exemplary fluorescence microscopy images of the influenza virus-infected MDCK cell culture in the IFA assay. Green fluorescence (FITC) comes from NP-targeting anti-influenza antibodies, blue is DAPI nucleus staining. The signal from antibodies against the influenza NP protein is reduced in the sample treated with siRNA 613 demonstrating viral inhibition.
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