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

Abstract

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.

Unnatural bases for recognition of noncoding nucleic acid interfaces

“…[ 102 ] Chen and coworkers studied the use of 5‐halouracils ( X U) in aminoethylglycine PNAs for targeting AU pairs in dsRNAs, and similarly found increased thermal stability for all halouracil‐thymine substitutions in PNA‐RNA triplexes, with the most pronounced stabilization observed for X = Br. [ 103 ] The 2‐thiouridine (s 2 U) base is a subtle O‐ > S modification of uridine, but has been shown to dramatically increase RNA duplex stability. The s 2 U sugar preferentially adopts a 3′‐endo pucker conformation due to increased steric interactions between the 2′‐hydroxyl and sulfur, which in turn favors A‐form RNA duplex formation via decreased conformational entropy.…”

Extended Peptide Nucleic Acid Nucleobases Based on Isoorotic Acid for the Recognition of A–U Base Pairs in Double‐Stranded RNA

“…Nielsen and co‐workers showed that substituting the Hoogsteen‐binding T residues for substituted 1,8‐naphthyridin‐2(1 H )‐ones (Figure 2 a) in a PNA clamp led to increased binding of the PNA*DNA–PNA triplex [11] . Chen and co‐workers modified U bases [12] and showed that replacement of the thymine methyl with hydrogen or halogens might lower the p K a of the Hoogsteen‐binding pyrimidine, increasing binding affinity for A–U compared to T (Figure 2 b, X=H, F, Cl, Br, I; Y=O) [12a] . More recently, they reported that 2‐thiouracil (Figure 2 b, X=H; Y=S) enhances A–U binding in a position independent manner [12b] .…”

“…Expanding on our strategy of using extended PNA nucleobases for RNA recognition, [13] we envisioned a scaffold containing benzamide attached through an amide linkage to an isoorotic acid derived PNA ( Io , Figure 2 e). This design relied on presumed advantages including (1) attaching the pyrimidine base to an electron‐withdrawing group to increase the H‐bond donating ability of the uracil, [12a] (2) imparting enough rigidity across the extended π‐system to afford π‐stacking opportunities and entropic advantages for binding and, (3) planning a modular synthesis allowing modification of the extended nucleobase to achieve optimum planarity and binding.…”

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

“…Our data provide important insights into developing ligands targeting the tau pre-mRNA hairpin structure. For example, double-stranded RNAs (dsRNAs) may be targeted by chemically modified dsRNA-binding PNAs that show significantly reduced binding to single-stranded RNAs (ssRNAs) [72,74,75,76,77,78] and dsDNAs [72,74,75,76,77,78,79,80,81,82,83]. However, the application of dsRNA-binding PNAs for regulating tau pre-mRNA exon 10 splicing may not be ideal, because the splice site hairpin contains a relatively short dsRNA region (seven base pairs), and residues +5 to +7 (critical for U1 snRNP recognition) are not involved in stable base pairing interactions [21,29,84].…”