Synthesizing Cell-Permeable Peptide Nucleic Acid Molecules which Release Guanidinium Groups upon Entering the Cell

In order to fully understand the several ten thousand genes in the human genome and to better diagnose and treat diseases, such as cancer, it is crucial to develop a cell-permeable molecule that modulates gene expression. Peptide Nucleic Acid, a nucleic acid analog, can hybridize with DNA and RNA strands in order to modulate gene expression in cells. It is an ideal molecule, because of its hybridizing properties and unnatural backbone, to synthesize and use in gene expression and medicinal applications. Most of my work within the lab will concern the synthesis of the PNA molecule that is able to traverse the cell effectively and efficiently; therefore, I will be analyzing the cellular uptake of the PNA molecules. The new class of PNA derivatives developed is the g-PNA molecule, which is guanidine-based PNA. It has been ascertained that the guanidinium groups are essential for the cellular uptake of the PNA molecule, but once in the cell the charged molecule hinders the ability of the PNA to hybridize with the intended RNA molecule. Therefore, the current work in the lab will deal with the synthesis of a second generation g-PNA molecule that is able to release the guanidinium groups after cellular uptake. This work will involve installing cysteine side-chains to the PNA molecule that will covalently bond, using a disulfide bond, the PNA molecule to the guanidinium group. The importance of the disulfide bond is that once inside the cell the disulfide bond between the PNA and the guanidinium groups will be reduced, resulting in a nearly charge-neutral molecule. My current work is synthesizing these various “linkers,” and analyzing the cellular uptake properties of them, once they have detached from their PNA molecules within the cell. This research can be divided into different steps in order to fully analyze the effectiveness and efficiency of the PNA molecule binding to the intended RNA target without electrostatic interference: the synthesis of disulfide-containing monomers, the synthesis of the corresponding oligomers, the characterization of cellular uptake, and the assessment of antisense effects.