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Chapter 1 that may compete with the formation of the duplex. PNAs can also form stable PNA-PNA duplexes, still based on standard Watson-Crick hydrogen bonds. Recognition experiments demonstrated that PNA-PNA duplexes are more stable than PNA-RNA and PNA-DNA complexes. Although natural nucleic acids form duplexes only in the antiparallel form, PNAs have the peculiar property to bind complementary sequences either in antiparallel and parallel orientation, albeit antiparallel binding is usually strongly preferred, giving more stable duplexes. The antiparallel is conventionally defined as the one in which the amino terminus of the PNA faces the 3’ end of the DNA 19 (and obviously parallel complexation is defined in the other way round) (Figure 1-3) One of the most important features of PNAs is their ability to selectively recognize a complementary sequence and to discriminate it from other sequences only differing for one nucleobase 20 . Recognition experiments done on PNA-DNA duplexes showed a selectivity, expressed as difference in Tm between fullmatch and mismatch duplexes, which is more than 5°C higher than that measured for the corresponding DNA-DNA duplexes. This binding specificity makes PNAs suitable for all the diagnostic and therapeutic applications where mismatch recognition is required. Figure 1-4: Watson-Crick and Hoogstein interaction in triplex formation. Another interesting feature of PNAs is their ability to form complexes other than duplexes. Homo-pyrimidine PNAs bind to complementary DNA sequences to form (PNA) 2 -DNA triplexes exploiting Watson-Crick interaction for one PNA strand and Hoogstein for the binding of the second strand. Since a protonated cytosine is involved in the base triplet formation, this is most stable in the pH range of 5.0 –5.5 21 (Figure 1-4). These complexes are characterized by a very high stability: a poly-T PNA decamer hybridized with a poly-A DNA decamer showed a melting temperature of 72°C, while only 23°C was observed for the DNA- DNA complex. The formation of analogous (PNA) 2 -RNA complexes has been investigated as 12
PNAs: from birth to adulthood well, and their stability has been found to be similar to that of the triplexes formed with DNA. Modified nucleobases, such as isocytosine, which is permanently protonated in one Hoogsteen hydrogen bond donor site, independently from the pH, make the triplex formation easier 22 . The PNA ability to form very stable triplexes can be exploited when the probes are targeted to double stranded DNA: when the target sequence is homopurinic, homopyrimidinic PNAs show the ability to open and invade the double helix, performing strand invasion, to form (PNA) 2 -DNA triplex (triplex invasion) 23 . Moreover, purine-rich PNAs, are also able to invade DNA-DNA helices by forming PNA-DNA duplexes (duplex invasion) 24 , while C rich PNAs triplex forming ability can be exploited in double stranded DNA to form PNA-(DNA) 2 triplex complexes 22 (Figure 1-5). Figure 1-5: Scheme of the complexes formed by PNA and double-stranded DNA. PNA oligomers are drawn in bold. (1) Standard duplex invasion complex formed with some homopurine PNAs. (2) Double-duplex invasion complex, possible with PNAs containing modified nucleobases. (3) Conventional triplex formed with cytosine-rich homopyrimidine PNAs (4) Triplex invasion complex, leading to the displacement of the second DNA strand into a ‘D-loop’. (F. Pellestor, P. Paulasova, European Journal of Human Genetics, 2004, 12, 694) This ability can be exploited in vivo, because it allows to easily target the desired nucleic acid sequence. Unfortunately, the necessity to have homo-pyrimidine or homo-purine sequences for efficient DNA invasion is a strong limitation, because it limits the choice of the target sequence. In order to overcome this problem, new methods have been developed to perform strand invasion. One of the most efficient strategy is the so-called double duplex strand invasion, which is possible to achieve by using two PNA sequences targeted to complementary sequences of the double strand DNA; in order to avoid the formation of a PNA-PNA duplexes (the two PNAs are obviously complementary to each other), PNAs should contain 13
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Page 3 and 4: 2.4.4 Fluorescence measurements ...
Page 5: Chapter 6 The double nature of Pept
Page 8 and 9: Preface In Chapter 2 a particular t
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Page 25 and 26: PNAs: from birth to adulthood 1.6 M
Page 27 and 28: PNAs: from birth to adulthood amino
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Page 59 and 60: PNA Molecular Beacons The mastermix
Page 61 and 62: Chapter 3 A new class of Chiral PNA
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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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Contributions Publications includin
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