New peptide nucleic acids for biotechnological applications
Castiglione, Mariangela (2010) New peptide nucleic acids for biotechnological applications. [Tesi di dottorato] (Inedito)
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In the present study was designed and realized a new similar to the PNA. It is a chiral nucleo-γ-peptide, dabPNA, isomer of the aegPNA unit and characterized by a 2,4-diaminobutyric acid (DABA)-based backbone carrying the carboxymethylated nucleobase on its α amino group. Differently from aegPNA monomers, dabPNA presents a shorter backbone (3C between the nitrogen atom and the carbonyl) and an enhanced distance between the nucleobase and the backbone, i.e. 3 atoms (1N+2C) instead of 2C in aegPNAs. Furthermore, in comparison with ornPNA that binds to RNA forming a stable triplex, daba-based PNA has a methylene group less in the backbone. To achieve dabPNA oligomers, the new monomers tL-dab, tD-dab, aL-dab and aD-dab were synthesized in good yield using suitable protected building blocks and characterized by NMR and ESI-MS techniques. Initially, the homothymine oligomers (tL-dab)12 and (tD-dab)6 were synthesized, using a synthetic strategy that ensured the maintenance of chirality during the coupling steps, and tested for hybridization towards natural nucleic acids. No binding evidence with both DNA (dA12) and RNA (A12) was revealed by CD and UV experiments. Furthermore, the insertion of a single tL-dab unit in the middle or at N-terminus of a homothymine aegPNA chain leads to a decreased binding efficiency to the target DNA in comparison to full aegPNA.Since dabPNAs based on L- and D-DABA don’t bind natural nucleic acids, we explored the possibility that complementary nucleopeptides based on D or L-DABA could bind between themselves. This property would be interesting in order to develop novel DABA-based dendrimeric systems, as new materials, and also to realize new diagnostic tools, as for example new molecular beacon probe. To verify this interesting possibility, the monomers aL-dab and aD-dab were oligomerized to the homoadenine hexamers (aL-dab)6 and (aD,L-dab)6. In order to find the correct combination of chirality suitable for obtaining the binding, also the (tD,L-dab)6 oligomer were realized to perform the hybridization studies. From CD and UV experiments, binding evidence was revealed in the case of the complementary oligomers with alternate chirality, (aD,L-dab)6 and (tD,L-dab)6, even if the stability of the complex formed was not so high (Tm 11°C). Furthermore, some interesting properties relative to the self-complementary oligomer (aL−dab-tD−dab)3 emerged from preliminary DLS experiments that evidenced the formation of multimeric aggregates for this system. These results, together with the high serum stability of the DABA-based oligomers, suggest further studies on dabPNAs as new self-recognizing bio-inspired polymers, with the potentiality to develop new nanomaterials or new biotechnological tools in bioengineering and biomedical applications.
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