Search tips
Search criteria 


Logo of adnaLink to Publisher's site
Artif DNA PNA XNA. 2014 Sep-Dec; 5(3): e1131801.
Published online 2016 January 8. doi:  10.1080/1949095X.2015.1131801
PMCID: PMC5329894

Effect of chirality in gamma-PNA: PNA interaction, another piece in the picture


Modification of the PNA backbone can be used to broaden their utility by introducing new functional groups. In particular, gamma-modified PNA have been found to be quite effective in a number of applications, and exhibit particularly high DNA binding affinity. The introduction of one side chain imply that the achiral backbone of PNA becomes chiral, and binding properties depend on the stereochemistry. A new paper on gamma-modified PNA by Ly and co-workers complete the existing knowledge by displaying that in binding to complementary PNA stereochemical orthogonality can be demonstrated. This opens the way to the exploitation of stereochemical features in diagnostic assays and in nanofabrication.

Keywords: gamma-PNA, nanofabrication, orthogonality, stereochemistry, self-assembly

Among nucleic acid analogs, peptide nucleic acids (Fig. 1) have several peculiar properties. One of these is that they have an achiral structure and thus they can form double-strands with both right or left handedness.1

Figure 1.
Structure of achiral (A), chiral α-PNA (B), and chiral γ-PNA (C). Frequently, “R” are amino acid side chains, but can also be more complex and unnatural structures. Preferred stereochemistry is shown and is that derived ...

Moreover, their structure can be changed using synthetic methods starting from synthons from the chiral pool, most notably D- and L-amino acids.2 Therefore, it is possible to obtain derivatives with a definite stereochemistry and analyze the effect of chirality on the binding properties. In a recent work, Danith Ly and coworkers3 made an important step forward in the understanding of these properties.

The first moves in this field were done by Nielsen and co-workers, who first produced a series of derivatives using substitution at C2 (α) position (Fig. 1B),4 which were produced from L- or D-amino acids, and studied their interactions with DNA. Binding of these α-PNA depends both on the side-chain and stereochemistry.5 The side chain can be used to change the PNA properties, most notably solubility. Later on it was found that a general rule for these α-PNA was that DNA binding is favored with D-amino acid derivatives, mainly due to intrastrand interactions.6 Analysis of crystal structure of a PNA:DNA duplex,7 and later on molecular modeling studies8 confirmed this hypothesis. Furthermore, substitution at α position was found to increase the sequence selectivity in DNA binding, especially if a stretch of consecutive chiral monomers were used.9 Ly and coworkers showed that using these derivatives with guanidinium-bearing side chains (G-PNA) increased the cellular delivery.10 The α-PNA did not show a definite pre-organization, as circular dichroism spectra of single-strands gave rise to very weak signal not showing the exciton coupling pattern; however, when they were bound to complementary achiral PNA they were shown to be prone to form either right- or left-handed helical PNA:PNA double helices. The circular dichroism signature of these complexes can be linked to the propensity of the PNA for either of the two handednesses,11 and aggregation of cyanine dyes on these PNA:PNA duplexes supported these conclusions.12

On the other hand, a substituent can be introduced also in the aminoethyl part of the PNA backbone (Fig. 1C). Liang and coworkers first synthesized C5-substituted (γ) PNA backbone in 1994,13 and Falkiewicz's group devised a general method, based on Mitsunobu reactions, for the synthesis of C2 and C5-modified PNA;14 γ-modified PNA became a successful story after several groups proposed this type of modification as a simple and very effective strategy for the introduction of functional groups, spacers or fluorophores.15,16,17 It was demonstrated by our group that γ-PNA synthesized from L-amino acids were strong inducer of right-handedness of PNA:PNA duplexes;18 L-lysine- or L-arginine based γ-PNA showed increased DNA binding affinity.18,19 Thus stereochemistry also in this case can be used as a tool for strengthening DNA binding. Absolute stereochemistry can change according to the nature of atoms in the side chain (Fig. 1); we therefore prefer to use relative D, and L-notations which indicate unequivocally the position of the R group with respect of the PNA chain for the following discussion.

Danith Ly and coworkers also went deeper into the study of the properties of γ-PNA in the single strands. Their findings, based on NMR and CD studies, were consistent with the general concept of pre-organization.20 In fact, it was found that even the modification with small groups such as those of Ala and Ser can induce an intense circular dichroism signal in the nucleobase region, pointing out that this modification was able to induce a definite conformation with helical arrangement of the bases. While in the lysine and arginine based derivatives the electrostatic interactions can favor the binding with negatively charged DNA, in the case of γ-PNA derived from alanine or serine, a stabilizing effect of the PNA:DNA and PNA:RNA duplexes was also observed in the absence of such interactions, finding that a favorable contribution to the overall binding energy was coming from the entropic term, which was less negative in these PNA systems than in (achiral) unmodified PNA.20 Both these experimental observations (CD and entropy data) pointed out to less flexible, constrained single-strand conformation of γ-PNA, which, with appropriate L-stereochemistry, can already be organized in order to better bind right-handed natural nucleic acids. This view is corroborated by the fact that the crystal structure of γ-PNA:DNA showed conformation of the PNA similar to those previously found in other type of duplexes,21 thus presumably with an entropy not different from that of achiral PNA duplexes. The observed favorable entropy should come from change in entropy of the single-stranded initial state. The good DNA binding properties of γ-modified PNA allowed to favor strand invasion of duplex PNA, which is very important for applications on genomic DNA.22-24 C5-substitution is also very useful for attaching reporter groups, but can also be used to tether PNA probes to a solid surface, which is especially useful when the captured DNA should be brought as close as possible to the surface to maximize the signal, such as in electrochemical sensors.25 All these properties demonstrated that this class of PNAs has very good performances; not surprisingly they have also become commercially available products with improved binding properties and better solubility.

In the paper recently published by Ly and coworkers on JACS,3 another important feature, yet not addressed, was studied: the effect of γ-substitution on PNA:PNA duplex stability when both strands are chiral, i.e. the compatibility of stereochemically different γ-PNA backbones.

The authors used as model system a minimal sequence with five nucleobases on each strand, and two L-lysine residues at both termini. Single-stranded PNA were found to have CD signals independent of the terminal lysine chirality, but essentially affected by the chirality of the modified monomers and by the sequence, being less intense for thymine containing PNA. Also in this case, binding to complementary oligonucleotide was found to be driven by a less negative entropy change.

Furthermore, the effect of stereochemistry on PNA:PNA interactions was studied by melting temperature experiments, and two major results can be inferred: a very high sequence selectivity, with ΔTm of at least −20°C, and a complete orthogonality of the two enantiomeric PNA, i.e., L-PNA and D-PNA can both bind to achiral PNA, but they do not bind each other (Fig. 2). Even more importantly, since only one of the two enantiomeric PNA stretch is able to bind to nucleic acid targets, but both of them can bind to achiral PNA, inhibition of achiral PNA action in diagnostic tests and in biological systems can be efficiently accomplished with the binding of complementary and stereochemically orthogonal to DNA and RNA left handed γ-PNA. Ly and coworkers therefore proposed that this orthogonality could be used in applications where two PNA stands can be programmed to bind only if the appropriate stereochemistry is used, most notably in hybridization chain reaction (HCR) methodologies26 and in micro- and nanotechnological fabrication.

Figure 2.
Stereochemical orthogonality of D- and L-γ-PNA. Only L-PNA binds to natural nucleic acids. D-PNA and L-PNA do not bind to each other, but they both bind to achiral PNA and to PNA with the same stereochemistry.

The PNA structure has been proposed to be an excellent candidate for nanofabrication by self-assembly, having the characteristics of high fidelity of sequence recognition, high chemical and biological stability, and higher compatibility with organic solvents compared to DNA and modified oligonucleotides.27 For PNA:PNA duplexes in which only one strand is chiral, different strategies have been described enabling to modulate their helicity.28,29 Switching elements can be introduced to produce sensing upon hybridization of the target DNA/RNA, and some PNA-containing nanomaterials can act as sensing elements in cells.30 In addition, as shown in the paper by Ly and co-workers, generation of a fluorescent signal from two pyrene modified PNA, and aggregation of microbeads can be regulated by the stereochemistry of the PNA duplex; in particular, for microbeads, positive aggregation of fluorescently labeled particles with γ-PNA of different stereochemistry linked on the surface could be obtained; in this case, the PNA was modified in order to contain a mini-PEG residue on the γ-side chain and at N-terminus, in order to prevent collapse on microparticles; association of small (2 microns) particles to larger ones (10 microns) through complementary PNA:PNA sequences only occurred when these were loaded with PNA of compatible stereochemistry. Thus, a new application of chirality or γ-PNA, namely stereo-orthogonality has been introduced, and this tool seems to be apt to simplify the design of micro- and nanoscale architectures. Aggregation induced by hybridization of sequences as short as 5 nucleobases was thus possible. Hence once again chirality, if properly used, can be exploited to improve the molecular toolbox of artificial DNA design.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.


1. Rasmussen H., Sandholm J.. Crystal structure of a peptide nucleic acid (PNA) duplex at 1.7 angstrom resolution. Nat Struct Biol 1997; 4:98–101; PMID:9033585; [PubMed] [Cross Ref]
2. Sugiyama T., Kittaka A. Chiral Peptide Nucleic Acids with a Substituent in the N-(2-Aminoethy)glycine Backbone. Molecules 2013; 18:287–310; [PubMed] [Cross Ref]
3. Sacui I., Hsieh WC., Manna A., Sahu B., Ly DH.. Gamma Peptide Nucleic Acids: As Orthogonal Nucleic Acid Recognition Codes for Organizing Molecular Self-Assembly. J Am Chem Soc 2015; 137:8603–10; PMID:26079820; [PubMed] [Cross Ref]
4. Haaima G., Lohse A., Buchardt O., Nielsen PE. Peptide nucleic acids (PNAs) containing thymine monomers derived from chiral amino acids: hybridization and solubility properties of D-lysine PNA. Angew Chem Int Ed Engl 1996; 35:1939–42
5. Püschl A., Sforza S., Haaima G., Dahl O., Nielsen PE. Peptide nucleic acids (PNAs) with a functional backbone. Tetrahedron Lett 1998; 39:4707–10; [Cross Ref]
6. Sforza S., Galaverna G., Dossena A., Corradini R., Marchelli R. Role of Chirality and Optical Purity in Nucleic Acid Recognition by PNA and PNA analogs. Chirality 2002; 14:591–8 [PubMed]
7. Menchise V., De Simone G., Tedeschi T., Corradini R., Sforza S., Marchelli R., Capasso D., Saviano M., Pedone C.. Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine-based chiral PNA-DNA duplex. Proc Natl Acad Sci USA 2003; 100:12021–6; PMID:14512516; [PubMed] [Cross Ref]
8. Topham CM., Smithyz JC.. Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: Quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form. Biophys J 2007; 92:769–86; PMID:17071666; [PubMed] [Cross Ref]
9. Corradini R., Feriotto G., Sforza S., Marchelli R., Gambari R.. Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acids probes by a surface plasmon resonance biosensor. J Mol Recognit 2004; 17:76–84; PMID:14872540; [PubMed] [Cross Ref]
10. Dragulescu-Andrasi A., Zhou P., He G., Ly DH. Cell-permeable GPNA with appropriate backbone stereochemistry and spacing binds sequence-specifically to RNA. Chem Commun 2005; 14(2):244–6; 10.1039/b412522c [PubMed] [Cross Ref]
11. Corradini R., Tedeschi T., Sforza S., Marchelli R. Electronic Circular Dichroism of Peptide Nucleic acids and their analogues. In: Berova N, Polavarapu P, Nakanishi K, Woody RW editors. Advances in Chiroptical Methods Volume 2. New York: John Wiley and Sons Inc; 2012. Chapter 2.IV.5 p 587–614
12. Dilek I., Madrid M., Singh R., Urrea CP., Armitage BA.. Effect of PNA backbone modifications on cyanine dye binding to PNA-DNA duplexes investigated by optical spectroscopy and molecular dynamics simulations. J Am Chem Soc 2005; 127:3339–45; PMID:15755150; [PubMed] [Cross Ref]
13. Kosynkina L., Wang W., Liang TC. A convenient synthesis of chiral peptide nucleic acid (PNA) monomer. Tetrahedron Lett 1994; 35:5173–6; [Cross Ref]
14. Falkiewicz B., Kolodziejczyk AS., Liberek B., Wisniewski K. Synthesis of achiral and chiral peptide nucleic acid (PNA) monomers using Mitsunobu reaction. Tetrahedron 2001; 57:7909–17; [Cross Ref]
15. Englund EA., Appella DH. g-substituted peptide nucleic acids constructed from L-lysine are a versatile scaffold for multifunctional display. Angew Chem Int Ed Engl 2007; 46:1414–8 [PubMed]
16. Ficht S., Dose C., Seitz O.. As fast and selective as enzymatic ligations: unpaired nucleobases increase the selectivity of DNAcontrolled native chemical PNA ligation. ChemBioChem 2005; 6:2098–103; PMID:16208732; [PubMed] [Cross Ref]
17. Totsingan F., Tedeschi T., Sforza S., Corradini R., Marchelli R.. Highly Selective Single Nucleotide Polymorphism (SNP) Recogniton by a Chiral (5S) PNA Beacon. Chirality 2009; 21:245–53; PMID:18853465; [PubMed] [Cross Ref]
18. Sforza S., Tedeschi T., Corradini R., Marchelli R. Induction of helical handedness and DNA binding properties of peptide nucleic acids (PNAs) with two stereogenic centres. Eur J Org Chem 2007:5879–85; [Cross Ref]
19. Manicardi A., Fabbri E., Tedeschi T., Sforza S., Bianchi N., Brognara E., Gambari R., Marchelli R., Corradini R.. Cellular uptake, biostability and anti-miR-210 activity of chiral arginine-PNA in leukemic K562 cells. ChemBiochem 2012; 13:1327–37; PMID:22639449; [PMC free article] [PubMed] [Cross Ref]
20. Dragulescu-Andrasi A., Rapireddy S., Frezza BM., Gayathri C., Gil RR., Ly DH.. A simple g-backbone modification preorganizes peptide nucleic acid into a helical structure. J Am Chem Soc 2006; 128:10258–67; PMID:16881656; [PubMed] [Cross Ref]
21. Yeh JI., Shivachev B., Rapireddy S., Crawford MJ., Gil RR., Du S., Madrid M., Ly DH.. Crystal structure of chiral γPNA with complementary DNA strand: Insights into the stability and specificity of recognition and conformational preorganization. J Am Chem Soc 2010; 132:10717–27; PMID:20681704; [PMC free article] [PubMed] [Cross Ref]
22. Rapireddy S., He G., Roy S., Armitage BA., Ly DH.. Strand invasion of mixed-sequence B-DNA by acridine-linked, g-peptide nucleic acid (gPNA). J Am Chem Soc 2007; 129:15596–600; PMID:18027941; [PubMed] [Cross Ref]
23. Rapireddy S., Bahal R., Ly DH.. Strand invasion of mixed-sequence, double helical B-DNA by γPNAs containing G-Clamp nucleobases under physiological conditions. Biochemistry 2011; 50:3913–8; PMID:21476606; [PMC free article] [PubMed] [Cross Ref]
24. He G., Rapireddy S., Bahal R., Sahu B., Ly DH.. Strand invasion of extended, mixed-sequence B-DNA by gPNAs. J Am Chem Soc 2009; 131:12088–90; PMID:19663424; [PMC free article] [PubMed] [Cross Ref]
25. De A., Souchelnytskyi S., van den Berg A., Carlen ET.. Peptide Nucleic Acid (PNA)−DNA duplexes: comparison of hybridization affinity between vertically and horizontally tethered PNA probes. ACS Appl Mater Interfaces 2013; 5:4607–12; PMID:23668364; [PubMed] [Cross Ref]
26. Bonifazi D., Carloni LE., Corvaglia V., Delforge A. Peptide nucleic acids in materials science. Artificial DNA, PNA & XNA 2012; 3:112–22; [PMC free article] [PubMed] [Cross Ref]
27. Dirks RM., Pierce NA.. Triggered amplification by hybridization chain reaction. Proc Nat Acad Sci USA 2004; 101:15275–8; PMID:15492210; [PubMed] [Cross Ref]
28. Wittung P., Eriksson M., Lyng R., Nielsen PE., Nordén BJ. Induced Chirality in PNA-PNA Duplexes. J Am Chem Soc 1995; 117:10167–73 ; [Cross Ref]
29. Corradini R., Tedeschi T., Sforza S., Green MM., Marchelli R. Control of Helical Handedness in DNA and PNA Nanostructures, in, Zuccheri G, Samorì B editors. DNA Nanotechnology: Methods and Protocols. Methods in Molecular Biology, vol. 749, New York: Springer Science+Business Media; 2011. p. 79–92
30. Ryoo SR., Lee J., Yeo J., Na HK., Kim YK., Jang H., Lee JH., Han SW., Lee Y., 0 Kim VN., Min DH.. Quantitative and multiplexed microRNA sensing in living cells based on peptide nucleic acid and nano graphene oxide (PANGO). ACS Nano 2013; 7:5882–5891; PMID:23767402; [PubMed] [Cross Ref]

Articles from Artificial DNA, PNA & XNA are provided here courtesy of Taylor & Francis