Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

Abstract

Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA2–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA2 triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA2–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA2–PNA triplex is significantly more stable than a DNA2–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications.

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

“…PNA monomer L was synthesized following the reported method (15). PNA oligomers were synthesized manually using Boc chemistry via a Solid-Phase Peptide Synthesis protocol.…”

“…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).…”

“…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).…”

“…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).…”

“…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).…”