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Chapter 1 1.1 Introduction One of the most challenging target for the scientists in the last years is to understand what takes place in the cells and the ability to specifically control cell behavior, directing it towards desired functions. In particular, one of the most important and studied process is the storage, replication and use of all the information encoded in DNA because of its involvement, together with other factors, in the processes that regulate the whole cell life. How the genetic information is stored in this molecule, how it is transferred to RNA and how it is used in all aspects of cell life have been the subject of extensive studies. Nowadays many of these processes have been understood, but still some are unknown. What is clear is that the ability to control and manipulate nucleic acids in a cell might open the possibility to control the cell itself. Targeting a particular gene directly in the nucleus or the messenger RNA produced in the cytoplasm can selectively suppress or enhance the presence of a certain protein in a cell, changing its fate in terms of development, replicative ability, toxicity, or killing it. This ability could be exploited in many fields, but one of the most important is undoubtedly medicine, where selective killing or selective growth of particular cells can make the difference between health or illness. Anyway, gene regulation is only part of the goals that can be achieved by techniques aimed at specific gene targeting, since also diagnostic applications are of paramount importance. A specific and selective detection of particular sequences may be useful in order to better understand cellular processes in which those sequences are involved. Alternatively, when it is already known that a particular sequence is involved in the onset of a disease, its fast recognition can be extremely useful in the early diagnosis. Finally, the recognition of DNA sequences typical of bacteria or contaminant organisms, or RNA sequences typical of viruses, can give very important information about their presence or absence in air, water or food. 1.2 Synthetic DNA Analogues It is clear that nucleic acid recognition is a very hot topic. In order to achieve this task, highly performing molecular tools, able to form stable complexes only with the desired sequences are needed. As a matter of fact, given a particular DNA sequence in a given genome, there always is a huge number of other sequences, only differing for few bases or even for only 6
PNAs: from birth to adulthood one, that can also be recognized by the same probe due to the similarity with the target sequence. For this reason, the molecular probe used to target a given sequence has to be very selective and, at the same time, it has to form very stable complexes, given the low amount of DNA to be recognized usually present in the biological matrices. Synthetic DNA oligonucleotides complementary to the target sequence are among the most common probes used for this purpose. Nowadays, DNA oligonucleotides are commercially available and quite cheap, but several drawbacks are associated with them. Synthetic nucleotides identical to natural ones can obviously be recognized and cleaved by DNAse enzymes, a limitation for in vivo applications, and a potential problem also for in vitro applications, when contaminations occur. Moreover, in order to obtain stable complexes, long oligonucleotides are often required (20 nuclobases or more), and this can lead to a decreased selectivity that may disturb target recognition in diagnostic applications or affect unselectively many different cells during in vivo applications. 1 A further problem is given by the high ionic strength required for overcoming the electrostatic repulsion between the two negatively charged strands (the target DNA and the oligonucleotide probe), limiting diagnostic applications in a low ionic strength environment. Finally, in vivo applications of DNA oligonucleotides are usually limited by the low cellular uptake, since simple oligonucleotides are not able to enter the cells 2 . The answers to these problems might come from the synthesis of new molecules obtained by suitable modifications of the DNA structure 3 . Many modifications have been introduced, and among those which gave good results, some presented only minor changes (for example the bonds between each DNA monomer), others showed major structural changes within the backbone (Figure 1-1). Phosphorothioate oligonucleotides (PS-DNA) can be obtained by substitution of one non linking oxygen atom from the phosphate group with a sulfur 4 . These molecules are more chemically and enzimatically resistant, and they have been used as antisense drugs, and are usually classified as first generation antisense oligonucleotides. However, they still presented some problems, such as a lower binding efficiency towards RNA, aspecific interactions with some proteins, and some toxicity when used in living organisms. N3’-P5’ phosphoroamidates (NPs) 5 , are another structure obtained by the substitution of an oxygen atom from the phosphodiester group with a nitrogen atom. These analogues were synthesized and tested in binding assays, showing increased binding towards complementary sequences if compared to DNA oligonucleotides as well as enhanced enzyme resistance. Evolution towards better modifications led to 2’-O-methyl (OMe) and 2’-O-methoxy-ethyl RNA (MOE), usually 7
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Arginine-PNAs monomers in the middl
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Arginine-PNAs The synthesis of the
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Arginine-PNAs yield in this reactio
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Arginine-PNAs (increased electrosta
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Arginine-PNAs chain + Boc methyl gr
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Arginine-PNAs foam. Yield: 30%. 1 H
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Arginine-PNAs was allowed to stir f
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Arginine-PNAs PNAs was carried out
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Chapter 4 Chiral PNA application on
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Arg-PNA Microarrays a surface. Alth
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Arg-PNA Microarrays 4.2 Results and
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Arg-PNA Microarrays Figure 4-2: HPL
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Arg-PNA Microarrays PNA-microarray
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Arg-PNA Microarrays Table 4-2. Geno
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Arg-PNA Microarrays complex mixture
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Arg-PNA Microarrays hybridisation e
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Arg-PNA Microarrays 30 T. Tedeschi,
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Chapter 5 5.1 Introduction The incr
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Chapter 5 be printed, in order to m
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Chapter 5 Table 5-1. PNA sequences
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Chapter 5 printing the PNA on a gla
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Chapter 5 22.4°C 24.0°C 25.5°C 2
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Chapter 5 PNA 3 PNA 5 PNA 6 Slide 1
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Chapter 5 times. The substrates wer
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Chapter 5 chemicals were used as re
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Chapter 5 5.4.6 Microcontact Printi
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Chapter 5 5.5 References 1 A. L. Be
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Chapter 6 6.1 Introduction The use
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Chapter 6 Among all the peptides us
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Chapter 6 Figure 6-2: A standard PN
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Chapter 6 peptide 1 and the PNA tri
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Chapter 6 6.2.2 Uptake experiments
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Chapter 6 (HBTU), N-[(dimethylamino
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Chapter 6 1.41 (s, 9H, Boc methyl g
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Chapter 6 Arg + CHα Lys), 4.0-4.6
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Chapter 6 12 B. P. Gangamani, V. A.
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