Incorporating modified bases and base-backbone linkers into PNAs for targeting RNA secondary structures

• Main Author: Ong, Alan Ann Lerk
• Other Authors: Chen Gang
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/136984
• Institution: Nanyang Technological University

The structure diversity of RNA allows RNA to be involved in various biological processes. RNA secondary structures include the single-stranded (ss) loops and double-stranded (ds) stems and are involved in protein binding and gene regulation. Peptide nucleic acid (PNA) is a synthetic analog of DNA and RNA with a chemically stable and neutral peptide-like backbone. Targeting RNA secondary structures shows great promise in RNA-targeting therapeutics. Traditional antisense PNAs (asPNAs) can be used to target ssRNA and weakly formed dsRNA regions. Short chemically-modified PNAs have been developed to recognize dsRNAs at near physiological conditions by major-groove PNA·RNA-RNA triplex formation. However, the development of dsRNA-binding PNAs (dbPNAs) as a class of dsRNA-targeting drug is hindered by complicated RNA secondary structures and canonical or non-canonical base pairings. Here, we utilize PNAs incorporating modified bases and base-backbone linkers to improve the recognition of model RNA secondary structures and disease-related RNA structures. Project 1. Chemically-modified short PNAs recognize RNA duplexes at near physiological conditions by major-groove PNA·RNA-RNA triplex formation and show a great promise in developing RNA-targeting probes and therapeutics. Thymine (T) and uracil (U) are often incorporated into PNAs to recognize A-U pairs through major-groove T·A-U and U·A-U triplex formation. Incorporation of a modified nucleobase, 2-thiouracil (s2U), into triplex-forming oligonucleotides (TFOs) stabilizes both DNA and RNA triplexes. Thiolation of uracil causes a decrease of the pKa of N3 nitrogen atom which may result in improved hydrogen bonding in addition to enhanced base stacking interactions, similar to the previously reported thiolation effect of pseudoisocytosine (J to L substitution). Here, we incorporated s2U into short PNAs, followed by binding studies of a series of s2U-modified PNAs. We demonstrated by nondenaturing polyacrylamide gel electrophoresis (PAGE) and thermal melting experiments that s2U and L incorporated into dbPNAs enhance the recognition of A-U and G-C pairs, respectively, in RNA duplexes in a position-independent manner, with no appreciable binding to DNA duplex. Combining s2U and L modifications in dbPNAs facilitates enhanced and selective recognition of dsRNAs over ssRNAs. We further demonstrated through a cell-free assay the application of the s2U- and L-modified dbPNAs in the inhibition of the pre-microRNA-198 maturation in a substrate specific manner. Thus, s2U-modified dbPNAs may be generally useful for the enhanced and selective recognition of RNA duplexes and for the regulation of RNA functions. Project 2. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dbPNAs can recognize internal Watson−Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases. Project 3. Developing a general platform for the molecular recognition of both dsRNAs and ssRNAs is important for probing, detecting, and targeting RNA structures. Traditional asPNAs can be used to target ssRNAs and weakly formed dsRNA regions. Relatively stable dsRNA regions may be targeted by nucleobase modified dbPNAs. Since natural dsRNAs are often interrupted by ssRNA loop regions, it is of general interest to biochemists and biologists to be able to target dsRNA-ssRNA junctions in a sequence- and structure-specific manner. Here, we describe our design of combining dbPNAs and asPNAs (designated as daPNAs) for the targeting of a model dsRNA-ssRNA junction, as well as dsRNA-ssRNA regions present in a miRNA hairpin precursor and tau pre-mRNA splice site hairpin. Our data suggest that it is promising to combine traditional asPNA (with a 4-letter code: T, C, A, and G) and dbPNA (with a 4-letter code: T, L, Q, and E or S) scaffolds to achieve RNA secondary structure-specific targeting of RNA with micromolar to nanomolar affinities. Thus, daPNAs may be a useful 7-letter platform for developing novel chemical probes and therapeutic ligands targeting RNA structures.