Abstract
The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.
Highlights
Peptide nucleic acid (PNA) [1] is an artificial nucleic acid having unique physicochemical properties, which can largely be attributed to the fact that PNA has an achiral, peptide-like N-(2aminoethyl)glycine backbone in place of the sugar-phosphate backbone found in DNA and RNA (Figure 1)
[12] The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone
In the case of csubstituted PNA, positively charged or neutral side chains increase binding affinity with DNA, [16,17,18,19,20] but this increase is primarily attributed to steric or hydrogen-bonding effects leading to conformational preorganization of the PNA backbone. [21,22] There is evidence that negatively charged side chains are tolerated at the c-position, [17,23] but their effect on binding affinity with DNA at varying ionic strength has not been thoroughly studied
Summary
Peptide nucleic acid (PNA) [1] is an artificial nucleic acid having unique physicochemical properties, which can largely be attributed to the fact that PNA has an achiral, peptide-like N-(2aminoethyl)glycine backbone in place of the sugar-phosphate backbone found in DNA and RNA (Figure 1). [5] there are no reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. In the case of csubstituted PNA, positively charged or neutral side chains increase binding affinity with DNA, [16,17,18,19,20] but this increase is primarily attributed to steric or hydrogen-bonding effects leading to conformational preorganization of the PNA backbone. [21,22] There is evidence that negatively charged side chains are tolerated at the c-position, [17,23] but their effect on binding affinity with DNA at varying ionic strength has not been thoroughly studied. [26] The results of these studies could be interpreted to conclude that increasing negative charge decreases PNA duplex stability via electrostatic repulsion. The decreased duplex stability of pPNA may result predominantly from structural, rather than electrostatic effects
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