Peptide Nucleic Acids

Peptide nucleic acids (PNAs) are synthetic nucleic acid analogs, which were first synthesized in 1991 by Peter E. Nielsen and colleagues. As shown in figure 1, unlike DNA and RNA, the phosphate sugar backbone of PNA is replaced by repeating N-(2-aminoethyl)-glycine units. Remarkably, PNAs are electrically neutral so that they generate no charge repulsion to DNA (or RNA) strands. Because of structural differences, PNAs show several attractive features including high thermal stability, resistance to enzymatic degradation, and strong binding to DNA or RNA and so on. In this article, we review the general features of PNAs and their applications.

 

Fig.1 Structure of peptide nucleic acid composed to DNA.(A) The PNA polypeptide backbone. (B)The sugar and phosphate backbone of normal DNA. B = nucleic acid base. The brackets with “n” indicate a polymer with multiple repeats (David P.Clark, et al.)

Significant Properties of PNAs

As mentioned above, PNAs display strong binding affinity towards DNA and RNA, besides, the unique properties of PNAs also including: 

1. Thermal stability (Tm) of PNAs

The thermal stability is measured by melting temperature. On average, the Tm of a PNA/DNA duplex is 1 higher per base pair compared to that of the corresponding DNA/DNA or DNA/RNA duplex. Given the higher thermal stability and specificity of a PNA/DNA, the PNA can able to block PCR in a sequence specific manner if targeted against one of the PCR primer sites.

2. Stronger binding independent of salt concentration

Because of unnatural backbone of PNAs, the thermal stability of a PNA/DNA duplex is bind independent of the salt concentration, whereas low ionic strength reduces the stability of DNA/DNA duplexes. The Tm of a 15-mer PNAs decrease by only 5 as the NaCl concentration is raised from 10 mM to 1M. Furthermore, at acidic pH (pH 4.5-6.5), PNAs show higher stability than DNA.

3. Resistance to enzymatic degradation

An additional consequence of unnatural backbone of PNA is resistance to nucleases and proteases-mediated degradation, extending the life time both in vitro and in vivo.

4. Solubility of PNAs

PNAs show low aqueous solubility, generally, the solubility of PNAs decreases as the polymer length. To improve the solubility, some strategies have been developed including terminal modification with charged amino acids such as L-lysine as well as modification of aminoehlyglycyl backbone.

Synthesis and modifications of PNAs

The procedures of PNAs synthesis are similar to peptides, which usually synthesized via solid phase techniques, either by manual or automated. However, with the growth of PNAs chain, PNA has the tendency of aggregation, particularly of purine-rich sequences. Additionally, low coupling efficiency is also common problems in solid phase synthesis. With more than 15 years experiences in peptide services, GenScript has develop a strategy to overcome these problems, for example, a PNA Lys-AGCTAGCT-Gly is successfully delivered with high coupling efficiency and without aggregate.

Fig.2. HPLC (A) and MS (B) result in GenScript. PNAs sequence: Lys-AGCTAGCT-Gly.

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As stated above, despite the attractive features of PNAs make it as a potential candidate to develop therapeutic and diagnostic agents, there are basic challenges still remaining for such applications such as poor uptake by cells, low aqueous solubility. To solve these problems, many researcher are focusing on PNAs modifications (α-, β- and γ- position). For instance, hybridization properties are enhanced with γ- substituents. To enhance the cellular uptake of PNAs, current main methods are conjugate with cell penetrating peptides (CPPs), use of complementary DNA to mediate PNAs uptake or incorporate into liposomes, as shown in Fig.3. 

Fig.3. Two methods for cellular delivery. (A) Transfection of PNAs. (B) CPP-PNA conjugate. (Saarbach et al.)

To improve solubility of PNAs, introduction of cationic functional groups is recommended because of good solubility of charged PNAs. Moreover, introduction of a G-clamp modified nucleobase, via both base pairing and base stacking enhanced the duplex stability while increasing the solubility because of the presence of positively charged side chains. Gupta with colleagues summarized a list of PNAs modifications in backbone and nucleobase modifications, find more in Table 1 and Table 2.


Table 1. Modifications in PNAs backbone along with the impact of modifications in properties. (A. Gupta et al.)