Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis

The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improve-ment of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

Automated summary

In this study, a capillary electrophoresis-based gel free hybridization assay using fluorescently labeled peptide nucleic acids (PNAs) is proposed to evaluate the identity of nucleic acid species in gene therapy products. Various proof-of-concept studies demonstrate the potential of PNA probes for advanced analytical characterization of therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. The method is highly specific for single-stranded nucleic acids up to 1000 nucleotides, with a limit of quantification in the picomolar range when multiple probes are used.