Articles by Desiree-Faye Kaixin Toh in JoVE
Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids Desiree-Faye Kaixin Toh*1, Kiran M. Patil*1, Gang Chen1 1Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.
Other articles by Desiree-Faye Kaixin Toh on PubMed
Intracellular Delivery of Antisense Peptide Nucleic Acid by Fluorescent Mesoporous Silica Nanoparticles Bioconjugate Chemistry. Aug, 2014 | Pubmed ID: 25055196 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.
RNA Triplexes: from Structural Principles to Biological and Biotech Applications Wiley Interdisciplinary Reviews. RNA. Jan-Feb, 2015 | Pubmed ID: 25146348 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.
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 Nucleic Acids Research. Nov, 2016 | Pubmed ID: 27596599 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.