Synthesis and Evaluation of Modified Peptide Nucleic Acid (PNA) for Improved DNA/RNA Binding Selectivity

Abstract

Peptide Nucleic Acid (PNA) is an achiral synthetic analogue of DNA / RNA oligonucleotide. Its backbone is made of repeating units of N-(2-aminoethyl) glycine (aeg) to which purine and pyrimidine nucleobases are attached. This pseudo-peptide backbone of PNA is uncharged which facilitates binding to cDNA / cRNA duplex or triplex better than the corresponding DNA/RNA due to the decreased electrostatic repulsion. A noticeable property of PNA is that they are resistant to hydrolytic cleavage by nucleases or proteases. Although PNAs have the above mentioned advantages, they still suffer from some limitations like less aqueous solubility, ambiguity in binding orientation and poor cell permeability. The rationale of my thesis is to synthesize an ether containing PNA backbone which includes synthesis, characterization and biophysical studies. The PNA monomer contains three modifications: the backbone N-3 is replaced with oxygen, the nucleobase is attached at C-γ position and this renders the PNA monomer chiral. A homo-octamer was synthesized with the modified PNA monomer using solid phase synthesis method. UV-Tm studies were performed to check the thermal stability of duplexes or triplexes formed by modified PNA with cDNA / cRNA and compared with aeg PNA. Circular dichroism was used to study the conformation of the duplexes or triplexes formed. The conclusion drawn from the present work is that in spite of these drastic changes to the PNA backbone, the modified PNA forms more stable duplexes and triplexes than control aeg PNA. The binding is stronger in case of triplex structure as compared to that in duplex structure. Moreover, the modified PNA was found to differentiate between RNA and DNA, by binding specifically only to RNA.