Peptide nucleic acid–dependent artifact can lead to false-positive triplex gene editing signals

Abstract

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

Shorter Peptide Nucleic Acid Probes Improve Affibody-Mediated Peptide Nucleic Acid-Based Pretargeting

“…PNA aggregates have been shown to interact nonspecifically with proteins and oligonucleotides, 44,45 which can impair their usefulness for in vivo applications such as gene editing. 46 The solubility of our secondary probes of different lengths, including HP18 (15-mer), HP17 (12-mer), HP16 (9-mer), and HP20 (8-mer), was assessed in a buffer used for lutetium-177 labeling (0.2 M NH 4 Ac; pH 5.5) at 25 °C (see Figure S19). Among these probes, the longest variant, HP18 (15mer), exhibited the lowest solubility, measuring at just 3 μM.…”

Perspectives on conformationally constrained peptide nucleic acid (PNA): insights into the structural design, properties and applications

“…However, despite the seemingly solid evidence of gene-editing by gPNA including in vivo 320 and in utero 321 studies in mouse models, a question has been raised whether the observed effects are real or just artifacts resulting from the possible aggregation of the (miniPEG) PNA onto the donor DNA. 322 The pyrrolidinyl acpcPNA has not yet been extensively studied for biological applications. In one report, the acpcPNA designed to target the NF-kB binding site in the promoter region of the Il6 gene was introduced to macrophage cells with the help of oxidized carbon nanoparticles and was shown to inhibit the stimulated production of Il6 mRNA as well as IL-6 protein levels in vitro.…”

“…However, despite the seemingly solid evidence of gene-editing by γPNA including in vivo 320 and in utero 321 studies in mouse models, a question has been raised whether the observed effects are real or just artifacts resulting from the possible aggregation of the (miniPEG) PNA onto the donor DNA. 322 …”

Lowering Entropic Barriers in Triplex DNA Switches Facilitating Biomedical Applications at Physiological pH

“…To enhance the formation of triplex structures, various strategies have been developed, including modifying the third oligo (e.g., base modification, [3,14] artificial base substitution, [15] sugar modification, [16,17] pseudopeptide backbone replacement [18,19] ) to improve its enthalpy contribution and promote binding to homopurine‐homopyrimidine duplexes. However, these chemical modifications are often costly and may lead to unpredictable thermodynamic consequences and potential biosafety concerns [20–23] . Another commonly used strategy involves the noncovalent intercalation of DNA binders to induce triplex formation by providing additional stacking forces [8,24] .…”