γ-C-Substituted Multifunctional Peptide Nucleic Acids: Design, synthesis and bioevaluation

Abstract

Peptide nucleic acids (PNAs) are a promising class of oligonucleotide analogs with great potential for gene therapeutic applications. PNAs are DNA analogs in which the normal phosphodiester backbone is replaced by acyclic, achiral and neutral amino-ethyl-glycyl backbone. PNAs can bind to complementary DNA/RNA sequences with high affinity and sequence specificity. PNAs and their analogs are resistant to proteases and nucleases. Besides these advantages, PNA suffers from some limitations like low aqueous solubility, poor cell permeability and ambiguity in binding orientation. To overcome these limitations, in the present work, we have designed and synthesized PNA analogs which have substitutions at γ-C on the backbone. The substituted chains carry terminal cationic amino/guanidino groups to improve cell permeability or have an azide group to link fluorophore via click chemistry. The stability of PNA:DNA duplex was determined by temperature dependent UV spectroscopy. The γ-C modified PNAs are shown to stabilize duplexes with cDNA better than control unmodified PNA. Among all modifications guanidino modified PNA showed highest thermal stability. The specificity of PNA binding to the target DNA has been studied by mismatch binding studies. The CD studies showed that the modification does not alter the conformation of PNA:DNA duplexes. The live cell imaging was carried out to check the cell permeability of modified PNA oligomers in NIH 3T3 and MCF-7 cell lines by confocal microscopy. The quantification of cellular uptake was carried out using FACS studies which showed that cationic functional groups enhance the efficiency of cell penetration.