Incorporating a guanidine-modified cytosine base into triplex-forming PNAs for the recognition of a C-G pyrimidine–purine inversion site of an RNA duplex

Abstract

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.

Incorporating uracil and 5-halouracils into short peptide nucleic acids for enhanced recognition of A–U pairs in dsRNAs

“…Extensive studies have been done for facilitating the cellular uptake bioactive PNAs and other peptides and nucleic acids (32,34,(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98)(99)(100). We focused on testing the effect of bromination of PNA on cellular uptake as previous studies suggest that fluorination of PNA T base does not significantly enhance cellular uptake of PNAs (101,102).…”

“…However, PNAs have been shown to form Watson-Crick duplexes with complementary DNA, RNA, or PNA in an antiparallel (with the C-terminus of a PNA aligned with 5 end of DNA/RNA) or parallel (with the N-terminus of a PNA aligned with 5 end of DNA/RNA) orientation (20)(21)(22). In addition, PNAs can be involved in the formation of parallel major-groove triplexes with various compositions including PNA·DNA-PNA, PNA·RNA-PNA, PNA·DNA-DNA, and PNA·RNA-RNA (13,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). It is interesting to note that short PNAs show selective binding to dsRNAs over dsDNAs, suggesting PNAs' great potential as dsRNA-specific binders (13,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37).…”

“…To recognize an inverted Watson-Crick C-G pair in an RNA duplex, a guanidine-modified PNA monomer (Q monomer) (34) or other monomers may be incorporated into PNAs through PNA·RNA-RNA triplex formation. Similar to L, Q is a cytosine derivative favoring Q·C-G triple formation, while destabilizing Watson-Cricklike Q-G pair formation (34)(35)(36)(37).…”

“…To recognize an inverted Watson-Crick C-G pair in an RNA duplex, a guanidine-modified PNA monomer (Q monomer) (34) or other monomers may be incorporated into PNAs through PNA·RNA-RNA triplex formation. Similar to L, Q is a cytosine derivative favoring Q·C-G triple formation, while destabilizing Watson-Cricklike Q-G pair formation (34)(35)(36)(37). To recognize an inverted Watson-Crick U-A pair in an RNA duplex, a PNA strand incorporating an E (3-oxo-2,3-dihydropyridazine) base has been used through E·U-A base triple formation (39)(40)(41).…”

Peptide nucleic acid mediated inhibition of the bacterial signal recognition particle

“…Electrophoretic mobility shift assay (EMSA) a) EMSA experiment for RNA-PNA interaction: The RNA-PNA interaction analysis using EMSA was performed as per previously published protocols. [6][7][8][9] Briefly, the lyophilized samples of 26-mer RNA or PNA were dissolved in appropriate volume of RNA binding buffer (20mM HEPES, 150mM NaCl, 0.5mM EDTA, pH 7.0) to get the desired stock concentration. The RNA samples were heated to 95°C for 5 min for complete denaturation and then snap cooled on ice for 10 min.…”