The diverse biological functions of RNA are determined by the complex structures of RNA stabilized by both secondary and tertiary interactions. An RNA triplex is an important tertiary structure motif that is found in many pseudoknots and other structured RNAs. A triplex structure usually forms through tertiary interactions in the major or minor groove of a Watson-Crick base-paired stem. A major-groove RNA triplex structure is stable in isolation by forming consecutive major-groove base triples such as U·A-U and C(+) ·G-C. Minor-groove RNA triplexes, e.g., A-minor motif triplexes, are found in almost all large structured RNAs. As double-stranded RNA stem regions are often involved in biologically important tertiary triplex structure formation and protein binding, the ability to sequence specifically target any desired RNA duplexes by triplex formation would have great potential for biomedical applications. Programmable chemically modified triplex-forming oligonucleotides (TFOs) and triplex-forming peptide nucleic acids (PNAs) have been developed to form TFO·RNA2 and PNA·RNA2 triplexes, respectively, with enhanced binding affinity and sequence specificity at physiological conditions. Here, we (1) provide an overview of naturally occurring RNA triplexes, (2) summarize the experimental methods for studying triplexes, and (3) review the development of TFOs and triplex-forming PNAs for targeting an HIV-1 ribosomal frameshift-inducing RNA, a bacterial ribosomal A-site RNA, and a human microRNA hairpin precursor, and for inhibiting the RNA-protein interactions involving human RNA-dependent protein kinase and HIV-1 viral protein Rev.
RNA duplex regions are often involved in tertiary interactions and protein binding and thus there is great potential in developing ligands that sequence-specifically bind to RNA duplexes. We have developed a convenient synthesis method for a modified peptide nucleic acid (PNA) monomer with a guanidine-modified 5-methyl cytosine base. We demonstrated by gel electrophoresis, fluorescence and thermal melting experiments that short PNAs incorporating the modified residue show high binding affinity and sequence specificity in the recognition of an RNA duplex containing an internal inverted Watson-Crick C-G base pair. Remarkably, the relatively short PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. The attached guanidine group stabilizes the base triple through hydrogen bonding with the G base in a C-G pair. Selective binding towards an RNA duplex over a single-stranded RNA can be rationalized by the fact that alkylation of the amine of a 5-methyl C base blocks the Watson–Crick edge. PNAs incorporating multiple guanidine-modified cytosine residues are able to enter HeLa cells without any transfection agent.
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.
Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for diverse biological functions. Hence, targeting dsRNAs through major-groove triplex formation is a promising strategy for the development of chemical probes and potential therapeutics. Short (e.g., 6–10 mer) chemically-modified Peptide Nucleic Acids (PNAs) have been developed that bind to dsRNAs sequence specifically at physiological conditions. For example, a PNA incorporating a modified base thio-pseudoisocytosine (L) has an enhanced recognition of a G–C pair in an RNA duplex through major-groove L·G–C base triple formation at physiological pH, with reduced pH dependence as observed for C+·G–C base triple formation. Currently, an unmodified T base is often incorporated into PNAs to recognize a Watson–Crick A–U pair through major-groove T·A–U base triple formation. A substitution of the 5-methyl group in T by hydrogen and halogen atoms (F, Cl, Br, and I) causes a decrease of the pKa of N3 nitrogen atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions. Here, we synthesized a series of PNAs incorporating uracil and halouracils, followed by binding studies by non-denaturing polyacrylamide gel electrophoresis, circular dichroism, and thermal melting. Our results suggest that replacing T with uracil and halouracils may enhance the recognition of an A–U pair by PNA·RNA2 triplex formation in a sequence-dependent manner, underscoring the importance of local stacking interactions. Incorporating bromouracils and chlorouracils into a PNA results in a significantly reduced pH dependence of triplex formation even for PNAs containing C bases, likely due to an upshift of the apparent pKa of N3 atoms of C bases. Thus, halogenation and other chemical modifications may be utilized to enhance hydrogen bonding of the adjacent base triples and thus triplex formation. Furthermore, our experimental and computational modelling data suggest that PNA·RNA2 triplexes may be stabilized by incorporating a BrUL step but not an LBrU step, in dsRNA-binding PNAs.
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 in vitro 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.
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