Hydrophilic and Cell-Penetrable Pyrrolidinyl Peptide Nucleic Acid via Post-synthetic Modification with Hydrophilic Side Chains

Abstract

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose-phosphate was replaced by a peptide-like backbone. The absence of negative charge in the PNA backbone leads to several unique behaviors including a stronger binding and salt independency of the PNA-DNA duplex stability. However, PNA possesses poor aqueous solubility and cannot directly penetrate cell membranes. These are major obstacles that limit in vivo applications of PNA. In previous strategies, the PNA can be conjugated to macromolecular carriers or modified with positively charged side chains such as guanidinium groups to improve the aqueous solubility and cell permeability. In general, a preformed modified PNA monomer was required. In this study, a new approach for post-synthetic modification of PNA backbone with one or more hydrophilic groups was proposed. The PNA used in this study was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic acid dipeptide backbone (acpcPNA) that shows several advantages over the conventional PNA. The aldehyde modifiers carrying different linkers (alkylene and oligo(ethylene glycol)) and end groups (-OH, -NH, and guanidinium) were synthesized and attached to the backbone of modified acpcPNA by reductive alkylation. The hybrids between the modified acpcPNAs and DNA exhibited comparable or superior thermal stability with base-pairing specificity similar to those of unmodified acpcPNA. Moreover, the modified apcPNAs also showed the improvement of aqueous solubility (10-20 folds compared to unmodified PNA) and readily penetrate cell membranes without requiring any special delivery agents. This study not only demonstrates the practicality of the proposed post-synthetic modification approach for PNA modification, which could be readily applied to other systems, but also opens up opportunities for using pyrrolidinyl PNA in various applications such as intracellular RNA sensing, specific gene detection, and antisense and antigene therapy.

<p>Preparation and SPECT/CT Imaging of ¹⁷⁷Lu-Labeled Peptide Nucleic Acid (PNA) Targeting CITED1: Therapeutic Evaluation in Tumor-Bearing Nude Mice</p>

“…17,30,31 We then used 177 Lu to label PNA with a chelating agent 1,4,7,10-tetraazacyclododecane-N, N', N, N'-tetraacetic acid (DOTA), the PNA sequence is combined with DOTA and can be incorporated into any position of the peptide-PNA coupling and incorporated into the peptide that mediates cell penetration and PNA internalization. 32,33 In cell internalization experiments, the uptake rate of the 177 Lu-nonsense-PNA group also increased slightly in the cellular uptake and retention experiments. It may be that 177 Lu-nonsense-PNA has a certain non-specific binding in K1 cells, which is why the retention rate of 177 Lu-nonsense-PNA has decreased so quickly.…”

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

“…The nitrogen atom can be used as a convenient handle for subsequent modification to create functional PNA probes 133,134 or cell-permeable PNA. 135 The effect of ring size of the cyclic b-amino acid in the (1S,2S)-configuration was explored in a series of pyrrolidinyl PNAs with (2R,4R)-proline stereochemistry (Fig. 27).…”

“…323 Enhanced cell membrane permeation of the acpcPNA was also realized by backbone modification with positively charged groups. 135…”

Peptide nucleic acids and their role in gene regulation and editing

“…Another common delivery strategy is by modification of the PNA backbone to include positively charged groups such as guanidinium groups. [ 90–92 ] However, the aforementioned delivery techniques are not easy to implement for large‐scale in vivo applications. In addition, during gene editing applications the need for co‐delivery of donor DNA would not be feasible using these delivery methods.…”