Incorporating G-C pair-recognizing Guanidinium into PNAs for sequence and structure specific recognition of dsRNAs over dsDNAs and ssRNAs

Recognition of RNAs under physiological conditions is important for the development of chemical probes and therapeutic ligands. Nucleobase-modified dsRNA-binding PNAs (dbPNAs) are promising for the recognition of dsRNAs in a sequence and structure specific manner under near-physiological conditions. Guanidinium is often present in proteins and small molecules for the recognition of G bases in nucleic acids, in cell-penetrating carriers, and in bioactive drug molecules, which might be due to the fact that guanidinium is amphiphilic and has unique hydrogen bonding and stacking properties. We hypothesized that a simple guanidinium moiety can be directly incorporated into PNAs to facilitate enhanced molecular recognition of G-C pairs in dsRNAs and improved bioactivity. We grafted a guanidinium moiety directly into a PNA monomer (designated as R) using a two-carbon linker as guided by computational modeling studies. The synthetic scheme of the PNA R monomer is relatively simple compared to that of the previously reported L monomer. We incorporated the R residue into various dbPNAs for binding studies. dbPNAs incorporated with R residues are excellent in sequence specifically recognizing G-C pairs in dsRNAs over dsDNA and ssRNAs. We demonstrated that the R residue is compatible with unmodified T and C and previously developed modified L and Q residues in dbPNAs for targeting model dsRNAs, the influenza A viral panhandle duplex structure, and the HIV-1 frameshift site RNA hairpin. Furthermore, R residues enhance the cellular uptake of PNAs.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Nanyang Technological UniversityAccepted versionThis work was supported by a NTU-A*STAR Seed Funding Research Award (2018), a Singapore Ministry of Education (MOE) Tier 1 grant (RG152/17), and a MOE Tier 2 grant (MOE2015-T2-1-028) to G.C. The work was also supported by a MOE Tier 1 grant (RG146/17) to Y.M

General recognition of U‑G, U‑A, and C‑G pairs by double-stranded RNA-binding PNAs incorporated with an artificial nucleobase

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA–RNA triplex formation. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dsRNA-binding PNAs (dbPNAs) can recognize internal Watson–Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases.Ministry of Education (MOE)Nanyang Technological UniversityWe thank Prof. Tom Brown for providing us a detailed protocol for the synthesis of the S base. This work was supported by NTU start-up grant, Singapore Ministry of Education (MOE) Tier 1 grants (RGT3/13, RG42/15, and RG152/17), and MOE Tier 2 grants (MOE2013-T2-2-024 and MOE2015-T2-1-028) to G.C. The work was also supported by MOE Tier 1 (RG126/16 and RG31/18) and MOE Tier 2 (MOE2018-T2-1-033) to K.X

A short chemically modified dsRNA-Binding PNA (dbPNA) inhibits influenza viral replication by targeting viral RNA panhandle structure

RNAs play critical roles in diverse catalytic and regulatory biological processes and are emerging as important disease biomarkers and therapeutic targets. Thus, developing chemical compounds for targeting any desired RNA structures has great potential in biomedical applications. The viral and cellular RNA sequence and structure databases lay the groundwork for developing RNA-binding chemical ligands through the recognition of both RNA sequence and RNA structure. Influenza A virion consists of eight segments of negative-strand viral RNA (vRNA), all of which contain a highly conserved panhandle duplex structure formed between the first 13 nucleotides at the 5' end and the last 12 nucleotides at the 3' end. Here, we report our binding and cell culture anti-influenza assays of a short 10-mer chemically modified double-stranded RNA (dsRNA)-binding peptide nucleic acid (PNA) designed to bind to the panhandle duplex structure through novel major-groove PNA·RNA2 triplex formation. We demonstrated that incorporation of chemically modified PNA residues thio-pseudoisocytosine (L) and guanidine-modified 5-methyl cytosine (Q) previously developed by us facilitates the sequence-specific recognition of Watson-Crick G-C and C-G pairs, respectively, at physiologically relevant conditions. Significantly, the chemically modified dsRNA-binding PNA (dbPNA) shows selective binding to the dsRNA region in panhandle structure over a single-stranded RNA (ssRNA) and a dsDNA containing the same sequence. The panhandle structure is not accessible to traditional antisense DNA or RNA with a similar length. Conjugation of the dbPNA with an aminosugar neamine enhances the cellular uptake. We observed that 2-5 μM dbPNA-neamine conjugate results in a significant reduction of viral replication. In addition, the 10-mer dbPNA inhibits innate immune receptor RIG-I binding to panhandle structure and thus RIG-I ATPase activity. These findings would provide the foundation for developing novel dbPNAs for the detection of influenza viral RNAs and therapeutics with optimal antiviral and immunomodulatory activities.Ministry of Education (MOE)Ministry of Health (MOH)Nanyang Technological UniversityNational Medical Research Council (NMRC)This work was supported by National Science Centre Grant UMO-2015/19/B/NZ1/02803 to E.K. and Grant UMO-2016/21/N/NZ1/00565 to J.K., the Polish Ministry of Science and Higher Education under the KNOW program, Singapore Ministry of Education (MOE) Tier 1 Grants RGT3/13 and RG42/15 to G.C., MOE Tier 2 Grants MOE2013-T2-2-024 and MOE2015-T2-1-028 to G.C., NTU start-up grant and MOH NMRC Grant OFIRG17nov084 to D.L., Temasek Life Sciences Laboratory, Singapore (to M.P.), and Fondation pour la Recherche Med́icale and Agence Nationale de Recherche Programme Labex (ARCANE, ANR-11-LABX-003 to J.-L.D.)

Targeting RNA Editing of Antizyme Inhibitor 1: a Potential Oligonucleotide-Based Antisense Therapy for Cancer.

10.1016/j.ymthe.2021.05.008Mol The

Selective Binding to mRNA Duplex Regions by Chemically Modified Peptide Nucleic Acids Stimulates Ribosomal Frameshifting

Minus-one programmed ribosomal frameshifting (−1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2–8 nucleotides downstream from the slippery site. Upon −1 RF, the ribosomal reading frame is shifted from the normal zero frame to the −1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically modified peptide nucleic acid (PNA) L and Q monomers to recognize G-C and C-G Watson–Crick base pairs, respectively, through major-groove parallel PNA·RNA–RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free <i>in vitro</i> translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically modified PNAs targeting model mRNA hairpins stimulate −1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs