γ Sulphate PNA (PNA S): highly selective DNA binding molecule showing promising antigene activity

Abstract

Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.

Figure 1. PNA monomers.

Schematic representation of standard and modified PNA monomers.

Figure 2. NMR characterization of the monomers.

Superimposition of the 2D-TOCSY, expansion of the backbone region, for the g^S and g^OH monomers.

Figure 3. Monomers rotamers.

Representation of the two rotamers found for the sulphate monomer g^S.

Figure 4. Sulphate monomers conformation.

The two hypothesized conformations for the g^S monomers.

Figure 5. CD of single strands.

CD spectra of PNA S (–) and PNA OH (–) in 10 mM Phosphate buffer 100 mM NaCl, 5 mM MgCl₂, pH 7 at 10 µM strand concentration.

Figure 6. CD of triplexes.

CD spectra of (PNA S)₂DNAc (–), (PNA)₂DNAc (–) in 10 mM Na Phosphate buffer, 100 mM NaCl, 5 mM MgCl₂, pH 7 at 3 µM triplex concentration.

Figure 7. Uptake analysis of FITC-oligomers in SKBR3 cells.

Cells are treated with 1 µM FITC-PNA S or FITC-PNA in the presence of lipofectamine and analysed by flow cytometric assay (A) and immunofluorescence analysis (B). In panel A, black curve represent not transfected cells, blue curve cells transfected with FITC-PNA, red curve cells treated with FITC-PNA S.

Figure 8. qPCR of ErbB2 and GAPDH genes.

RNA was obtained after transfection of SKBR3 cells with PNA S or PNA at 5 and 10 µM concentration. The experiments were performed in triplicate and repeated at least 3 times. Data indicate the relative gene expression of cells treated with PNA S or PNA versus not treated cells, error bars represent the standard deviation of the mean. Statistical significance was carried out by means of the two tailed paired Student’s t test, *p = 0.02; **p = 0.0006.

Figure 9. Flow cytometric analysis of ErbB2 expression on SKBR3 cells.

Cells are treated with different amounts of PNA S and PNA in the presence of lipofectamine. (A) Single result representative of three similar experiments. Not transfected cells (black curve), not transfected and treated cells with an antiErbB2 antibody (green curve), transfected with 10 µM PNA S and treated with an antiErbB2 antibody (red curve) and transfected with 10 µM PNA and treated with an antiErbB2 antibody (blue curve). (B) Histograms were obtained from at least three independent experiments. Data are expressed as percentage of decrease versus control (cells not transfected and treated with an antiErbB2 antibody) ± SE. Statistical significance was carried out by means of the two tailed paired Student’s t test, **p<0.01, *p<0.05.