Performance of PNA probes in solution and on surface: a comparison

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3. Experimental Part

4.4 Performance of PNA probes in solution and on surface: a comparison

As shown in described experiments, the behaviour of tested PNA models deeply differs between the binding properties in solution and on surface. 5L-chiral box PNA seemed to be the best model for the DNA binding in solution, followed by fully achiral PNA and 2D -chiral box PNA (Table 1). This situation is diametrically opposed to the PNA behavior on surface. Here, the best binding properties seemed to belong to the achiral PNA while the 5L-chiral box PNA gave very less intense signals (Figg 10a and 11a). On the other side, the high stability of achiral PNA showed on surface was detrimental for the mismatch recognition. Indeed, while in solution all the three PNA structures showed similar good SNP discrimination properties, with 2D-chiral box PNA slightly better performing, the hybridization on array revealed 2D-chiral box PNA as the best structure in terms of selectivity in mismatch recognition, while the achiral PNA still gave a strong signal when the hybridization with mismatch DNA was performed. These results suggest that the surface could play a meaningful role during the hybridization step. It may be speculated that the nature of the solid support and the presence of positive charges on chiral PNA probes are more likely responsible for the reduced binding properties on surface, since they could promote the aggregation of the PNA, lowering the efficiency of the hybridization. Concerning the mismatch recognition properties, it has be noted that there is a major difference between the two types of modifications in chiral PNAs: while the side chains in the 2D model are pointing toward the major groove, in the 5L model they are directed toward the minor groove.11 The higher sequence selectivity observed in the case of 2D chiral box can be due to the position of the side chain, which is attached to the α-carbon of the more rigid glycine moiety of the PNA backbone, whereas the side chain in the 5L derivative is placed in the more flexible aminoethyl group. Distortion of the former will likely generate a conformation in which the side chains collide with each other, whereas in the case of the 5L derivative, they can be rearranged and the eventual repulsive interactions are compensated by the electrostatic interactions with the negative potential of the minor groove. Since the side chains in the 5L model are closer to the DNA backbone, however, eventual surface-PNA interactions on this side will strongly hamper the interaction with DNA, while the same effect occurring on the major groove side would be better tolerated, thus leading to a better performance on the microarray system.26

5. Conclusions

Since several works had showed the importance of chirality of PNAs in affecting in a positive way DNA recognition and enhancing sequence selectivity, in this chapter the performance of three different PNA probes, fully achiral PNA, 2D-arginine-based chiral PNA and 5L- arginine-based chiral PNA, has been investigated, in terms of DNA binding affinity and mismatch recognition, both in solution and on solid surface.

The results showed that the 5L-chiral box PNA was superior in binding affinity in solution, whereas the 2D-chiral box PNA model was superior in performances when recognition of single nucleotides was considered both in solution and in the microarray format. These differences could be mainly addressed to the displacement of the chiral monomers in the PNA-DNA duplex three-dimensional structure and, therefore, to their

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interaction with the solid surface. This interfacing, in turns, highly differs according to the chemistry of the stereogenic centres and is enhanced by the presence of several chiral monomers used to form chiral-box PNAs.

The information achieved here can be very precious in the design of a PNA-based microarray for the recognition of single nucleotide polymorphisms in food analysis.

6. References

1 Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science, 1991, 254:1497–

1500.

2 Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules. Nature, 1993, 365:566–568.

3 Wittung P, Nielsen PE, Buchardt O, Egholm M, Norden B. DNA like double helix formed by peptide nucleic acid. Nature, 1994, 368:561–563.

4 Paulasova P, Pellestor F. The peptide nucleic acids (PNAs): a new generation of probes for genetic and cytogenetic analyses. Annales de Génétique, 2004, 47:349-358.

5 Yilmaz LS, Okten HE, Noguera DR. Making all parts of the 16s rRNA of Escherichia coli accessible in situ to single dna oligonucleotides. Applied and Environmental Microbiology, 2006, 72:733–744.

6 Demidov VA, Potaman VN, Frank-Kamenetskil MD, Egholm M, Buchardt O, Sonnichsen SH, Nielsen PE. Stability of peptide nucleic acids in human serum and cellular extracts. Biochemical Pharmacology, 1994, 48:1310–1313.

7 Corradini R, Sforza S, Tedeschi T, Totsingan F, Marchelli R. Peptide nucleic acids with a structurally biased backbone: effects of conformational constraints and stereochemistry. Current Topics in Medicinal Chemistry, 2007, 7:681-694.

8 Giesen U, Kleider W, Berding C, Geiger A, Orum H, Nielsen PE. A formula for thermal stability (Tm) prediction of PNA/DNA duplexes. Nucleic Acids Research, 1998, 26:5004–5006.

9 Ren B, Zhou JM, Komiyama M. Straightforward detection of SNPs in double‐stranded DNA by using exonuclease III/nuclease S1/PNA system. Nucleic Acids Research, 2004, 32:e42.

10 Corradini R, Sforza S, Tedeschi T, Marchelli R. Chirality as a tool in nucleic acid recognition: principles and relevance in biotechnology and in medicinal chemistry.

Chirality, 2007, 19:269–294.

11 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. Proceedings of the National Academy of Sciences USA, 2003, 100:12021–12026.

12 Sforza S, Tedeschi T, Corradini, Marchelli R. Induction of helical handedness and DNA binding properties of peptide nucleic acids (PNAs) with two stereogenic centres.

European Journal of Organic Chemistry, 2007, 2007:5879– 5885.

13 Sforza S, Corradini R, Ghirardi S, Dossena A, Marchelli R. DNA binding of a D -lysine-based chiral PNA: direction control and mismatch recognition. European Journal of Organic Chemistry, 2000, 2000:2905–2913.

174

14 Tedeschi T, Sforza S, Dossena A, Corradini R, Marchelli R. Lysine-based peptide nucleic acids (PNAs) with strong chiral constraint: control of helix handedness and DNA binding by chirality. Chirality, 2005, 17:S196–S204.

15 Duheolm KL, Petersen KH, Jensen DK, Egholm M, Nielsen PE, Buchardt O. Peptide nucleic acid (PNA) with a chiral backbone based on alanine. Bioorganic & Medicinal Chemistry Letters, 1994, 4:1077-1080.

16 Nielsen PE, Haaima G, Lohse A, Buchardt. Peptide nucleic acids (PNAs) containing thymine monomers derived from chiral amino acids: hybridization and solubility properties of D-lysine PNA. Angewandte Chemie International Edition in English, 1996, 35:1939-1942.

17 Calabretta A, Tedeschi T, Di Cola G, Corradini R, Sforza S, Marchelli R. Arginine-based PNA microarrays for APOE genotyping. Molecular BioSystem, 2009, 5:1323-1330.

18 Sforza S, Haaima G, Marchelli R, Nielsen PE. Chiral peptide nucleic acids (PNAs): helix handedness and DNA recognition. European Journal of Organic Chemistry, 1999, 1:197-204.

19 Germini A, Rossi S, Zanetti A, Corradini R, Fogher C, Marchelli R. Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food. Journal of Agricultural and Food Chemistry, 2005, 53:3958-3962.

20 Rossi S, Scaravelli E, Germini A, Corradini R, Fogher C, Marchelli R. A PNA-array platform for the detection of hidden allergens in foodstuffs. European Food Research and Technologies, 2006, 223:1-6.

21 Germini A, Scaravelli E, Lesignoli F, Sforza S, Corradini R, Marchelli R. Polymerase chain reaction coupled with peptide nucleic acid high-performance liquid chromatography for the sensitive detection of traces of potentially allergenic hazelnut in foodstuff. European Food Research and Technologies, 2005, 220:619-624.

22 Kalendar R, Lee D, Schulman AH. FastPCR software for PCR primer and probe design and repeat search. Genes, Genomes and Genomics, 2009, 3:1-14.

23 http://www6.appliedbiosystems.com/support/pnadesigner.cfm.

24 http://eu.idtdna.com/ analyzer/ Applications/OligoAnalyzer

25 Sforza S, Corradini R, Galaverna G, Dossena A, Marchelli R. The chemistry of peptide nucleic acids. Minerva Biotecnologica, 1999, 11:163-174.

26 Manicardi A, Calabretta A, Bencivenni M, Tedeschi T, Sforza S, Marchelli R. Affinity and selectivity of C2- and C5-substituted “chiral-box” PNA in solution and on microarrays. Chirality, 2010, 22:E161- E172.

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