Sequence Selective Recognition of Double-Stranded RNA Using Triple Helix-Forming Peptide Nucleic Acids

Abstract

Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

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

“…We note that the RNA duplex-binding triplex-forming PNAs (9,10) are complementary to the pyrrol-imidazole polyamides, which selectively and sequence specifically bind to the minor groove of DNA duplexes (41). …”

“…Alternatively, through the formation of a highly sequence specific triplex structure, an oligonucleotide may bind to an RNA duplex without disrupting the pre-existing structure of the RNA target (8–10). For example, even though the major groove is relatively deep and narrow, an RNA double helix can accommodate a third strand in the major groove to form a modestly stable triplex (11–13).…”

“…For example, even though the major groove is relatively deep and narrow, an RNA double helix can accommodate a third strand in the major groove to form a modestly stable triplex (11–13). In a major-groove triplex, the pyrimidine bases in the third strand recognize the sequence of the purine strand of the double helical RNA by Hoogsteen hydrogen bonding, forming U·A-U or T·A-U and C + ·G-C base triples (Figure 1A and B) (8–10). Chemically modified triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) are promising in enhancing the recognition of the structured RNAs through TFO·RNA 2 and PNA·RNA 2 triplex formation, respectively (9,10).…”

“…In a major-groove triplex, the pyrimidine bases in the third strand recognize the sequence of the purine strand of the double helical RNA by Hoogsteen hydrogen bonding, forming U·A-U or T·A-U and C + ·G-C base triples (Figure 1A and B) (8–10). Chemically modified triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs) are promising in enhancing the recognition of the structured RNAs through TFO·RNA 2 and PNA·RNA 2 triplex formation, respectively (9,10). Compared to TFOs, which typically have a negatively charged phosphate backbone, PNAs have a neutral and chemically stable backbone (14), which facilitates enhanced sequence specific recognition of RNA duplexes.…”

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

“…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).…”