Parameterization of the miniPEG-Modified γPNA Backbone: Toward Induced γPNA Duplex Dissociation

Abstract

γ-Modified peptide nucleic acids (γPNAs) serve as potential therapeutic agents against genetic diseases. Miniature poly(ethylene glycol) (miniPEG) has been reported to increase solubility and binding affinity toward genetic targets, yet details of γPNA structure and dynamics are not understood. Within our work, we parameterized missing torsional and electrostatic terms for the miniPEG substituent on the γ-carbon atom of the γPNA backbone in the CHARMM force field. Microsecond timescale molecular dynamics simulations were carried out on six miniPEG-modified γPNA duplexes from NMR structures (PDB ID: 2KVJ). Three NMR models for the γPNA duplex (PDB ID: 2KVJ) were simulated as a reference for structural and dynamic changes captured for the miniPEG-modified γPNA duplex. Principal component analysis performed on the γPNA backbone atoms identified a single isotropic conformational substate (CS) for the NMR simulations, whereas four anisotropic CSs were identified for the ensemble of miniPEG-modified γPNA simulations. The NMR structures were found to have a 23° helical bend toward the major groove, consistent with our simulated CS structure of 19.0°. However, a significant difference between simulated methyl- and miniPEG-modified γPNAs involved the opportunistic invasion of miniPEG through the minor and major groves. Specifically, hydrogen bond fractional analysis showed that the invasion was particularly prone to affect the second G-C base pair, reducing the Watson-Crick base pair hydrogen bond by 60% over the six simulations, whereas the A-T base pairs decreased by only 20%. Ultimately, the invasion led to base stack reshuffling, where the well-ordered base stacking was reduced to segmented nucleobase stacking interactions. Our 6 μs timescale simulations indicate that duplex dissociation suggests the onset toward γPNA single strands, consistent with the experimental observation of decreased aggregation. To complement the insight of miniPEG-modified γPNA structure and dynamics, the new miniPEG force field parameters allow for further exploration of such modified γPNA single strands as potential therapeutic agents against genetic diseases.

Figure 1

Chemical structure of a γPNA monomer. The Greek lettering identifies the carbon atom position labeling with R designating modifications at the γ-position and B designating nucleobases attached to the γPNA monomer.

Figure 2

Chemical structure of an (R-)miniPEG-modified γPNA monomer with B representing the nucleobase.

Figure 3

Chemical structure of the model compound used for parameter development. The Greek lettering represents the torsions for the backbone.

Figure 4

Model compound used for torsional parameterization represented in licorice. The carbon atoms are colored according to residue number; the first residue carbon atoms are colored in green, and the second are colored in gray. The remaining atoms are colored as follows: red for oxygen, blue for nitrogen, and white for hydrogen. The Greek lettering identifies the carbon atom position labeling. The solid blue line indicates the noncovalent interaction between C···O.

Figure 5

PES for the optimization of the miniPEG linkage to the PNA backbone. The chemical structure of the model compound is shown with the blue atoms detailing the dihedral optimized.

Figure 6

RMSD for the miniPEG-modified γPNA duplexes for the backbone atoms on the central six residues in each simulation.

Figure 7

RMSF computed for the methyl-modified γPNA duplex (left) and the miniPEG-modified γPNA duplex (right) for the backbone atoms on the central six residues.

Figure 8

Hydrogen bonding fractions for the central six WC base pairing nucleobases on the miniPEG-modified γPNA duplex. The red bar lines indicate standard deviation for the hydrogen bond fraction analysis across all six 1 μs simulations.

Figure 9

Probability distribution for the backbone torsion values of the miniPEG-modified γPNA. The red dashed line indicates the average torsional value from NMR observations. See Figure 3 for the chemical structure and torsional definitions.

Figure 10

(A) Scree plot of the PCs captured for the six 1 μs MD simulations of miniPEG-modified γPNA duplex backbone atom coordinates. (B–D) 2D projections for the reduced dimensionality of the backbone coordinates (top right and bottom row).

Figure 11

MiniPEG-modified CS1 for the 6 μs simulation of the miniPEG-modified γPNA. The γPNA duplex is represented in CPK, with the carbon atoms for the backbone atoms and nucleobases in gray and the carbon atoms associated with the miniPEG modification in green. All other atoms are colored as follows: blue for nitrogen and red for oxygen. Encircled in red are the areas of destabilization found in duplex. The nucleobases encircled represent destabilization of WC base pairing. The orange dashed line represents base stacking interactions. The blue solid line represents a hydrogen bond.

Figure 12

MiniPEG-modified CS2 for the 6 μs simulation of the miniPEG-modified γPNA. The γPNA duplex is represented in CPK, with the carbon atoms for the backbone atoms and nucleobases in gray and the carbon atoms associated with the miniPEG modification in green. All other atoms are colored as follows: blue for nitrogen and red for oxygen. Encircled in red are the areas of destabilization found in duplex. The orange dashed line represents base stacking interactions.

Figure 13

MiniPEG-modified CS3 for the 6 μs simulation of the miniPEG-modified γPNA. The γPNA duplex is represented in CPK, with the carbon atoms for the backbone atoms and nucleobases in gray and the carbon atoms associated with the miniPEG modification in green. All other atoms are colored as follows: blue for nitrogen and red for oxygen. Encircled in red are the areas of destabilization found in duplex. The orange dashed line represents base stacking interactions.

Figure 14

CS4 for the 6 μs simulation of the miniPEG-modified γPNA. The γPNA duplex is represented in CPK, with the carbon atoms for the backbone atoms and nucleobases in gray and the carbon atoms associated with the miniPEG modification in green. All other atoms are colored as follows: blue for nitrogen and red for oxygen. Encircled in red are the areas of destabilization found in duplex. The orange dashed line represents base stacking interactions. The blue line represents a hydrogen bond.