Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains

Abstract

We report herein the synthesis and DNA/RNA binding properties of bPNA+, a new variant of bifacial peptide nucleic acid (bPNA) that binds oligo T/U nucleic acids to form triplex hybrids. By virtue of a new bivalent side chain on bPNA+, similar DNA affinity and hybrid thermostability can be obtained with half the molecular footprint of previously reported bPNA. Lysine derivatives bearing two melamine bases (K 2M) can be prepared on multigram scale by double reductive alkylation with melamine acetaldehyde, resulting in a tertiary amine side chain that affords both peptide solubility and selective base-triple formation with 4 T/U bases; the Fmoc-K 2M derivative can be used directly in solid phase peptide synthesis, rendering bPNA+ conveniently accessible. A compact bPNA+binding site of two U6 domains can be genetically encoded to replace existing 6 bp stem elements at virtually any location within an RNA transcript. We thus replaced internal 6 bp RNA stems that supported loop regions with 6 base-triple hybrid stems using fluorophorelabeled bPNA+. As the loop regions engaged in RNA tertiary interactions, the labeled hybrid stems provided a fluorescent readout; bPNA+ enabled this readout without covalent chemical modification or introduction of new structural elements. This strategy was demonstrated to be effective for reporting on widely observed RNA tertiary interactions such as intermolecular RNA-RNA kissing loop dimerization, RNA-protein binding, and intramolecular RNA tetralooptetraloop receptor binding, illustrating the potential general utility of this method. The modest 6 bp stem binding footprint of bPNA+ makes the hybrid stem replacement method practical for noncovalent installation of synthetic probes of RNA interactions. We anticipate that bPNA+ structural probes will be useful for the study of tertiary interactions in long noncoding RNAs.

Unnatural bases for recognition of noncoding nucleic acid interfaces

“…Synthetic targeting motifs for UU sites developed by Baranger and Zimmerman (Acr, bisamidine) [ 165,171 ] and Bong (bPNAs, t4M) [ 180,182 ] …”

“…[172,173] Interestingly, a construct equipped with tris(2-aminoethyl)amine [170] was reported to target and trigger hydrolytic nucleic acid cleavage via a general base mechanism. [174] Bong and coworkers have also used melamine as a synthetic base to bind to oligo T/U internal bulge sites, by macromolecular display of the triazine on sidechains of α-peptides [175][176][177][178][179][180] and other scaffolds, [181][182][183][184][185] yielding a family of compounds collectively called bifacial peptide nucleic acids (bPNAs) (Figure 8). Multivalent display of melamine in polymers and lipids [186][187][188] was found to encapsulate 5-fluorouracil and direct assembly and vesicle fusion with complementary hydrogen-bonding lipids and peptides directed by complementary hydrogen-bonding; [164] likewise, oligo-A DNA was found to assemble around free cyanuric acid [189] and oligo-T DNA can assemble around free melamine.…”

“…[191] Functional compatibility with native structures allows pre-existing duplex stem elements to be replaced by triplex hybrid stems, thus transforming oligo T/U domains into compact, genetically encodable sites for RNA modification with bPNA probes. It was recently demonstrated that native RNA tertiary interactions such as kissing loop dimerization, tetraloop-tetraloop receptor binding and RNA-protein binding could be reported using bPNA fluorescent probes; [180] native T-rich DNA loops such as those found in the G-quadruplex fold of the human telomere can also be targeted by bPNAs. [182] 5 | CONCLUSIONS…”

“…Multivalent display of melamine in polymers and lipids [186][187][188] was found to encapsulate 5-fluorouracil and direct assembly and vesicle fusion with complementary hydrogen-bonding lipids and peptides directed by complementary hydrogen-bonding; [164] likewise, oligo-A DNA was found to assemble around free cyanuric acid [189] and oligo-T DNA can assemble around free melamine. [190] Discrete macromolecules displaying melamine exhibit high affinity 1:1 binding to oligo T/U internal bulges in DNA [165,171] and Bong (bPNAs, t4M) [180,182] duplexes equipped with terminal oligo-U domains. [191] Functional compatibility with native structures allows pre-existing duplex stem elements to be replaced by triplex hybrid stems, thus transforming oligo T/U domains into compact, genetically encodable sites for RNA modification with bPNA probes.…”

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

“…Bong and co-workers used melamine as a Janus-wedge nucleobase (K M , Figure 9) to organize two identical strands of oligothymidine DNA tracts (or oligouridine RNA tracts) on a peptide template to form peptide−DNA(RNA) triplex structures [137]. This approach was applied to induce RNA-RNA kissing loop dimerization and RNA-protein binding [138].…”