RNA triplexes: from structural principles to biological and biotech applications

Abstract

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.

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

“…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.…”

“…Through the incorporation of modified bases, it is possible to develop PNAs that selectively bind to the duplex over the single-stranded regions of RNA (9,15–20). For example, we have shown previously (15) that PNAs incorporating a modified neutral base thio-pseudoisocytosine (L) (Figure 1C) can recognize a Watson–Crick G-C pair in an RNA duplex by forming a Hoogsteen L·G pair with improved stability and minimized pH dependence compared to a Hoogsteen C + ·G pair (Figure 1B).…”

“…For example, we have shown previously (15) that PNAs incorporating a modified neutral base thio-pseudoisocytosine (L) (Figure 1C) can recognize a Watson–Crick G-C pair in an RNA duplex by forming a Hoogsteen L·G pair with improved stability and minimized pH dependence compared to a Hoogsteen C + ·G pair (Figure 1B). The enhanced Hoogsteen L·G pair formation is presumably due to improved van der Waals interaction between the thio group in L and H8 in G, as well as base stacking and hydrogen bonding interactions (9,13,15). PNAs incorporating L-modified residues show selective binding to RNA duplexes over single-stranded RNAs, because a Watson–Crick-like G-L pair (Figure 1E) is destabilized compared to a Watson–Crick G-C pair due to the steric clash between the relatively bulky thio group in L and the amino group in G. Furthermore, PNAs show enhanced binding to RNA duplexes over DNA duplexes (9,15–20).…”

“…The enhanced Hoogsteen L·G pair formation is presumably due to improved van der Waals interaction between the thio group in L and H8 in G, as well as base stacking and hydrogen bonding interactions (9,13,15). PNAs incorporating L-modified residues show selective binding to RNA duplexes over single-stranded RNAs, because a Watson–Crick-like G-L pair (Figure 1E) is destabilized compared to a Watson–Crick G-C pair due to the steric clash between the relatively bulky thio group in L and the amino group in G. Furthermore, PNAs show enhanced binding to RNA duplexes over DNA duplexes (9,15–20). Thus, triplex-forming PNAs may become useful therapeutic agents that not only allow the stabilization of desired RNA duplexes, but also inhibit undesired tertiary interactions and protein binding (8,9,21,22).…”

“…PNAs incorporating L-modified residues show selective binding to RNA duplexes over single-stranded RNAs, because a Watson–Crick-like G-L pair (Figure 1E) is destabilized compared to a Watson–Crick G-C pair due to the steric clash between the relatively bulky thio group in L and the amino group in G. Furthermore, PNAs show enhanced binding to RNA duplexes over DNA duplexes (9,15–20). Thus, triplex-forming PNAs may become useful therapeutic agents that not only allow the stabilization of desired RNA duplexes, but also inhibit undesired tertiary interactions and protein binding (8,9,21,22). Compared to small molecule binders, triplex-forming PNAs are programmable and can, in principle, recognize any sequence of RNA duplex structures.…”