Design and synthesis of peptide nucleic acid (PNA) agents for imaging gene expression

The ability to non-invasively image gene expression in vitro and in vivo would greatly impact the study of biological systems and the diagnosis and treatment of human disease. The challenge is that the number of mRNAs expressed is usually very low, ranging from 10s to 1000 copies per cell. Imaging agents must therefore be highly specific, be stable inside cells, and generate a low background signal. Peptide nucleic acids (PNAs) are an ideal nucleic acid analog for imaging gene expression in vivo since they can recognize mRNAs with great stability and specificity, are stable inside cells, and do not cause RNaseH mediated degradation of the target mRNA.; In one approach to the design of such agents we investigated the use of hydroxamic acid-based catalysts for mRNA triggered fluorogenic ester activation by the nucleic acid-triggered fluorescent probe activation (NATPA) strategy. In this strategy a disease-specific mRNA is used as a template to bring together a catalytic component and a fluorogenic component which then react to produce a fluorescent compound. We found that the hydroxamic acid catalysts were 30 to 50 better at hydrolyzing activated esters than the previously utilized imidazole catalyst. We also developed a synthetic method for incorporating the hydroxamic acid catalyst into PNA.; We also investigated an approach to detect mRNA by using the mRNA as a template to bring together a fluorescent donor and acceptor probe and produce a signal by FRET (fluorescence energy transfer). To this end we developed a novel PNA building block, Fmoc-PNA-U'(Dde)-OH, for attaching the required fluorescent molecules to terminal and internal positions of the PNAs. The building block enables the post-synthetic attachment of reporter groups to an amino group attached to the 5-position of uracil (U) following selective deprotection of a Dde group that is orthogonal to the standard protecting groups for amino acids and PNAs. We then used this building block to synthesize various PNA FRET pairs and demonstrated their use for detecting mRNA in vitro and in living cells by confocal microscopy.; In a third approach, we synthesized PNA and 2'-O-methyl-mRNA molecular beacons for imaging 28s rRNA and a transgene mRNA in living cells. In these systems, a quencher was attached to the carboxy terminus and a fluorophore to the amino terminus of a hairpin antisense agent. When the agent bound to the target mRNA, the quencher could no longer quench the fluorophore effectively which then became fluorescent.; Finally, we evaluated the ability of a hypertonic shock cell loading system to deliver various types of anti-sense PNAs into cells using a luciferase splicing correction assay. Using this system, we were able to demonstrate effective intracellular delivery of PNAs and 2'-OMe-phosphorothioate RNAs into cells.