Incorporation of modified bases into triplex-forming peptide nucleic acids for the recognition of a C-G pyrimidine-purine inversion site of an RNA duplex

Peptide nucleic acid (PNA), an analogue of DNA, can enhance the recognition of specific sequences in RNA duplexes and may be useful therapeutic reagents. In this study, a PNA monomer (Q) containing a N4-(2-guanidoethyl)-5-methylcytosine base was synthesized and incorporated into 8-mer PNAs. Together with previously reported thio-pseudoisocytosine (L) monomer, the non-denaturing polyacrylamide gel electrophoresis results suggest that the PNA is able to selectively target RNA hairpins containing a pyrimidine-purine (C-G) inversion with high binding affinity. The PNA does not bind to DNA hairpins. Thermal melting experiments indicate that PNA containing Q monomer is not able to bind to single-stranded RNA.​Master of Scienc

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.MOE (Min. of Education, S’pore)Published versio

RNA triplexes : from structural principles to biological and biotech applications

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. WIREs RNA 2015, 6:111–128. doi: 10.1002/wrna.1261MOE (Min. of Education, S’pore)Accepted versio

Intracellular delivery of antisense peptide nucleic acid by fluorescent mesoporous silica nanoparticles

In order to overcome poor cell permeability of antisense peptide nucleic acid (PNA), a fluorescent mesoporous silica nanoparticle (MSNP) carrier was developed to successfully deliver antisense PNA into cancer cells for effective silence of B-cell lymphoma 2 (Bcl-2) protein expression in vitro. First, fluorescent MSNP functionalized with disulfide bond bridged groups was fabricated and characterized. Antisense and negative control PNAs were synthesized and further conjugated with fluorescent dye cyanine 5. Then, the PNAs were covalently connected with fluorescent MSNP via amidation between amino group of PNAs and carboxylic acid group on the MSNP surface. High intracellular concentration of glutathione serves as a natural reducing agent, which could cleave the disulfide bond to trigger the PNA release in vitro. Confocal laser scanning microscopy studies prove that PNA conjugated MSNP was endocytosed by HeLa cancer cells, and redox-controlled intracellular release of antisense PNA from fluorescent MSNP was successfully achieved. Finally, effective silencing of the Bcl-2 protein expression induced by the delivered antisense PNA into HeLa cells was confirmed by Western blot assay.NRF (Natl Research Foundation, S’pore)ASTAR (Agency for Sci., Tech. and Research, S’pore)MOE (Min. of Education, S’pore)Accepted versio

Tertiary base triple formation in the SRV-1 frameshifting pseudoknot stabilizes secondary structure components

Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot are essential in stimulating -1 ribosomal frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus ribosomal frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA-RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1-loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1-loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.Ministry of Education (MOE)This work was supported by grants from Singapore Ministry of Education (MOE) Tier 2 (MOE2015-T2-1-028 and MOE2019-T2-1-069 to G.C.) and The Chinese University of Hong Kong, Shenzhen (CUHK-Shenzhen) University Development Fund (to G.C.). This work was also supported by the National Natural Science Foundation of China (Grant 32000914 to L.Y.)

Intracellular Delivery of Antisense Peptide Nucleic Acid by Fluorescent Mesoporous Silica Nanoparticles

In order to overcome poor cell permeability of antisense peptide nucleic acid (PNA), a fluorescent mesoporous silica nanoparticle (MSNP) carrier was developed to successfully deliver antisense PNA into cancer cells for effective silence of B-cell lymphoma 2 (Bcl-2) protein expression <i>in vitro</i>. First, fluorescent MSNP functionalized with disulfide bond bridged groups was fabricated and characterized. Antisense and negative control PNAs were synthesized and further conjugated with fluorescent dye cyanine 5. Then, the PNAs were covalently connected with fluorescent MSNP via amidation between amino group of PNAs and carboxylic acid group on the MSNP surface. High intracellular concentration of glutathione serves as a natural reducing agent, which could cleave the disulfide bond to trigger the PNA release <i>in vitro</i>. Confocal laser scanning microscopy studies prove that PNA conjugated MSNP was endocytosed by HeLa cancer cells, and redox-controlled intracellular release of antisense PNA from fluorescent MSNP was successfully achieved. Finally, effective silencing of the Bcl-2 protein expression induced by the delivered antisense PNA into HeLa cells was confirmed by Western blot assay

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

RNA duplex regions are often involved in tertiary interactions and protein binding and thus there is great potential in developing ligands that sequence-specifically bind to RNA duplexes. We have developed a convenient synthesis method for a modified peptide nucleic acid (PNA) monomer with a guanidine-modified 5-methyl cytosine base. We demonstrated by gel electrophoresis, fluorescence and thermal melting experiments that short PNAs incorporating the modified residue show high binding affinity and sequence specificity in the recognition of an RNA duplex containing an internal inverted Watson-Crick C-G base pair. Remarkably, the relatively short PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. The attached guanidine group stabilizes the base triple through hydrogen bonding with the G base in a C-G pair. Selective binding towards an RNA duplex over a single-stranded RNA can be rationalized by the fact that alkylation of the amine of a 5-methyl C base blocks the Watson–Crick edge. PNAs incorporating multiple guanidine-modified cytosine residues are able to enter HeLa cells without any transfection agent.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Sequence- and structure-specific probing of RNAs by short nucleobase-modified dsRNA-binding PNAs incorporating a fluorescent light-up uracil analog

RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil (btU). The incorporation of btU into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of btU-modified dbPNAs is sequence- and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1–1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating btU-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Nanyang Technological UniversityThis work was supported by NTU-A*STAR Seed Funding Research Award (2018), Singapore Ministry of Education (MOE) Tier 1 grants (RGT3/13, RG42/15, and RG152/17), and MOE Tier 2 grants (MOE2013-T2-2-024 and MOE2015-T2-1-028) to G.C. G.C. thanks Prof. Dongping Zhong for the helpful discussions on TICT

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

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.MOE (Min. of Education, S’pore)Published versio

General recognition of U‑G, U‑A, and C‑G pairs by double-stranded RNA-binding PNAs incorporated with an artificial nucleobase

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA–RNA triplex formation. 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 dsRNA-binding PNAs (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.Ministry of Education (MOE)Nanyang Technological UniversityWe thank Prof. Tom Brown for providing us a detailed protocol for the synthesis of the S base. This work was supported by NTU start-up grant, Singapore Ministry of Education (MOE) Tier 1 grants (RGT3/13, RG42/15, and RG152/17), and MOE Tier 2 grants (MOE2013-T2-2-024 and MOE2015-T2-1-028) to G.C. The work was also supported by MOE Tier 1 (RG126/16 and RG31/18) and MOE Tier 2 (MOE2018-T2-1-033) to K.X