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Chapter 6
6.1 Introduction
The use of PNAs in diagnostic applications has been presented in the previous chapters.
Although PNAs are useful tools in diagnostics, this is not the only field in which these
molecules can be usefully applied. As reported in the first chapter, application of PNAs in
gene therapy is a very hot topic, since they have features suited to most requirements for
selectively targeting and blocking a DNA or RNA sequence 1 . Enzymatic and chemical
resistance, in fact, make these molecules suitable for in-vivo applications, since these features
increase their life in a biological system 2 , whereas selectivity and affinity for the
complementary sequences allow specific and strong binding of the sequences of interest.
Nevertheless, some drawbacks still limits their extensive application; the low solubility due to
the apolar structure is a problem, since it limits the concentrations to be used in therapy, and
also cellular and nuclear uptake 3 might be a limiting factor, since, in order to have a
significant effect, these molecules have to selectively concentrate in the cell region where the
targeting is required (the cytoplasm if the target is RNA, the nucleus when the target is DNA).
Although it has been demonstrated that, in some cases, unmodified PNAs are able to enter
some types of cell without any modification or transfection approach 4 , usually there is the
necessity to develop a strategy aimed at increasing cellular and/or nuclear uptake of these
molecules. Some approaches involved the techniques developed for DNA uptake 4 , such as
electroporation 5 or co-transfection with lyposomes 6 . Nevertheless, the necessity to develop
more general and efficient delivery methods led to the design of modified PNAs for enhanced
uptake. The most exploited strategy has been the conjugation of PNAs with molecules able to
cross cellular or nuclear membrane, thus carrying the linked probe; a great variety of moieties
have been linked to PNAs for this purpose. The conjugation of PNAs to terpyridine units
promoted the cellular and nuclear delivery of such molecules. The possibility for terpyridine
to chelate the zinc ion, turned out in a preferential uptake by zinc-rich tissues 7 . The synthesis
of a PNA linked to a neamine unit allowed to obtain a molecule able to cross the cellular
membrane and hydrolyze RNA within the cytoplasm 8 . Lipophilic units such as adamantane or
triphenylphosphonium cation have been bound as well to PNAs in order to improve the
uptake, but this approach showed always good results in terms of uptake and targeting
efficience only in some cases 4,9 . Other groups linked to PNAs aimed at interacting with
specific cell membrane receptors. For example, PNAs linked to a lactose unit were selectively
internalized into hepatic cells that possessed a receptor for this sugar 10 . In another experiment,
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