PROJECT SUMMARY Seasonal influenza A viruses (IAV) cause millions of infections and thousands of deaths each year in the US. Low vaccine efficacy and rapid antiviral resistance requires development of alternative therapeutic strategies to limit the burden of influenza on the population. The genome of IAV consists of eight negative- sense RNA segments that upon infection are first copied into positive-sense complementary RNA (cRNA), which in turn are replicated into genomic negative-sense viral RNA (vRNA) segments that will be packaged into progeny viruses. As the viral life cycle critically depends on template-mediated vRNA replication, an appealing approach to halt virus multiplication would be to place complementary antisense oligonucleotides (ASOs) as roadblocks on vRNA segments to stall viral polymerase progression. Our objective in this proposal is to examine the suitability of gamma-peptide nucleic acids (PNAs) as an antiviral against influenza virus replication. Gamma-PNAs are second-generation analogues of PNAs that show enhanced affinity, water solubility and biological activity when targeted to cellular RNA or genomic DNA. As this modified backbone enhances the hybridization affinity with target RNAs by significantly increasing the melting temperature of the resulting duplex, gamma-PNAs, unlike conventional DNA or RNA oligomers, could indeed function as stable roadblocks for viral polymerase. We have assembled an interdisciplinary team with expertise in the relevant areas, i.e. RNA biology, influenza virology, and PNA chemistry, to successfully complete the proposed experiments. Taken together, our study will lay the groundwork for future in vivo pharmacokinetic studies in animals.