DNA recognition and induced genome modification by a hydroxymethyl-γ tail-clamp peptide nucleic acid

Abstract

Peptide nucleic acids (PNAs) can target and stimulate recombination reactions in genomic DNA. We have reported that γPNA oligomers possessing the diethylene glycol γ-substituent show improved efficacy over unmodified PNAs in stimulating recombination-induced gene modification. However, this structural modification poses a challenge because of the inherent racemization risk in O-alkylation of the precursory serine side chain. To circumvent this risk and improve γPNA accessibility, we explore the utility of γPNA oligomers possessing the hydroxymethyl-γ moiety for gene-editing applications. We demonstrate that a γPNA oligomer possessing the hydroxymethyl modification, despite weaker preorganization, retains the ability to form a hybrid with the double-stranded DNA target of comparable stability and with higher affinity than that of the diethylene glycol-γPNA. When formulated into poly(lactic-co-glycolic acid) nanoparticles, the hydroxymethyl-γPNA stimulates higher frequencies (≥ 1.5-fold) of gene modification than the diethylene glycol γPNA in mouse bone marrow cells.

Figure 1.. CD spectra for PNA, ^mpγPNA, and ^serγPNA

All samples contained 20 μM of the respective oligomer in 10 mM NaPi buffer. Spectra were recorded at 37°C. Regular PNA (black), ^mpγPNA (blue), and ^serγPNA (red).

Figure 2.. CD spectra for hybrid triplexes containing ssDNA and PNA, ^mpγPNA, or ^serγPNA

All samples contained 1 μM ssDNA and 2 μM PNA/γPNA in 10 mM NaPi buffer. Spectra were recorded at 37°C. ssDNA and PNA (black), ^mpγPNA (blue), ^serγPNA (red).

Figure 3.. UV-thermal denaturation analyses on a 90-mer dsDNA alone or in combination with PNA, ^mpγPNA, or ^serγPNA

All samples contained 2.5 μM dsDNA and 5 μM PNA/γPNA in 10 mM NaPi (A) or 100 mM NaPi (B). All samples were annealed as described in the experimental procedures, and melting temperature (Tm) values are presented in parentheses in the legend. 90-mer dsDNA alone (broken black) or in combination with PNA (solid black), ^mpγPNA (blue), and seryPNA (solid red).

Figure 4.. Titration of 100-mer dsDNA target with increasing equivalents of PNA, ^mpγPNA, ^serγPNA, or ^serγPNA-scr

(A) Each sample contained 50 nM DNA in 100 mM NaPi buffer, was annealed as in the experimental procedures, and was run on an 8% PAGE gel. (B) The band intensities for the PNA-DNA hybrids were measured on ImageJ and normalized to the 0 equiv condition.

Figure 5.

Langmuir binding isotherms for PNA/γPNA complexes with dsDNA in 100 mM NaPi

Figure 6.. NP characterization

(A) NP diameter as measured by dynamic light scattering (DLS). (B) NP surface charge as measured by zeta potential. (C) Total nucleic acid loading (PNA + donor DNA) in PLGA NPs. (D) Release of nucleic acids from PLGA NPs. Error bars: mean with SD.

Figure 7.. Modification of primary bone marrow cells with the β-thalassemia-causing mutation at IVS2-654

Bone marrow cells (BMCs) were left untreated (black) or were treated with NPs containing ^mpγPNA (blue) or ^serγPNA (red). Error bars: mean with SD. *p < 0.05.

Figure 8.. Correction of the β-thalassemia-causing mutation in primary BMCs

BMCs were left untreated (black) or were treated with NPs containing ^mpγPNA (blue) or ^serγPNA (red). Error bars: mean with SD. **p < 0.005.

Scheme 1.

Chemical structures of PNA, ^mpγPNA, and ^serγPNA monomer units