Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA

Abstract

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10−30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3−10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable −80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion−ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.

Impact of charges on the hybridization kinetics and thermal stability of PNA duplexes

“…However, when comparing the effect of charges at the γ-position on the hybridization kinetics, the charge on the PNA impacts the association kinetics more (6-fold difference between P3 vs. G3) than the dissociation (2-fold difference between P3 vs. G3). The faster k on of cationic PNA is in-line with the findings of Windsor and co-workers 29 but a direct comparison should be treated with caution because the charges were incorporated on the nucleobase (achiral backbone) in this prior study rather than at the γ-position of the backbone as in the present study.…”

“…19–21 Extensive SAR studies have been performed to further optimise PNA properties. 22 Notably, alternative nucleobases 23–30 have been advanced for improved affinity and cellular uptake; conformationally restricted backbones 31–35 have been shown to enhance duplex stability significantly; and substitution of the achiral 2-aminoethyl glycine has been used to tune and improve the properties of PNAs. In particular, substitution at the γ-position of the 2-aminoethyl glycine backbone has been found to enhance hybridization to DNA by chirality-induced preorganization (Fig.…”