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Chapter 4 were linked to the submonomers on resin by using DIC/DhBtOH coupling protocol. Free PNAs were cleaved from the resin by using a 1:3 TFMSA/TFA mixture (10% thioanisole + 10% m-cresol) and precipitated by Et 2 O. HPLC analysis for all PNAs was carried out by LC- MS by using an XTerra anlytical C18 column (3x250mm, 5µm, flow 0.5ml/min), gradient elution from 100% H 2 O (0.2% HCOOH, eluent A) to 60% H 2 O and 40% CH 3 CN (0.2%HCOOH, eluent B) in 30 min. MS detector set in the positive ion mode, capillary voltage 3kV, cone voltage 30V, full scan acquisition from 150 to 1500 m/z. HPLC purification for all PNAs was carried out on a semipreparative Jupiter (Phenomenex) C18 column (10x300mm, 5µm, flow 4ml/min); eluent A: 100% H 2 O (0.1% TFA), eluent B: 60% H 2 O and 40% CH 3 CN (0.1%TFA). Specific preparative elution conditions for every PNA are given below. PNA 1:H-(AEEA) 2 GCCGCA (2D,5L-Arg) CACGT-NH 2 . The synthesis was performed on 25mg of a preloaded Boc-T-MBHA-PS resin (loading 0.2mmol/g). Crude yield: 82%. The crude products were purified by semipreparative RP-HPLC, in gradient elution: from 100% A to 100% B in 25 min ESI-MS: calcd m/z: 691.5 (MH 5+ 5 ), 576.4 (MH 6+ 6 ), 494.2 (MH 7+ 7 ) found m/z 691.8, 576.5, 494.4. PNA 2 H-(AEEA) 2 CAGGCA (2D,5L-Arg) CTTCT-NH 2 . Crude yield: 80%. The crude product was purified by RP-HPLC in gradient elution: from 100% A to 100%B in 30 minutes. ESI-MS calcd m/z: 689.5 (MH 5+ 5 ), 574.7 (MH 6+ 6 ), 492.8 (MH 7+ 7 ) found m/z: 689.7, 574.7, 492.6. PNA 3. H-(AEEA) 2 GCCGCG (2D,5L-Arg) CACGT-NH 2 . The manual synthesis was performed on 25mg of a preloaded Boc-T-MBHA-PS resin (loading 0.2mmol/g). Crude yield: 80%. The crude product was purified by RP-HPLC in gradient elution : from 100% A to 100% B in 25 min. ESI calcd m/z: 694.7, 579.1, 496.5 found m/z: : 694.7, 579.0, 496.5. PNA 4 H-(AEEA) 2 CAGGCG (2D,5L-Arg) CTTCT-NH 2 Crude yield: 85%. The crude product was purified by RP-HPLC in gradient elution: from 100% A to 100%B in 30 minutes. ESI-MS calcd m/z: 1153.8 (MH 3+ 3 ) 865.6 (MH 4+ 4 ), 692.7 (MH 5+ 5 ), 577.4 (MH 6+ 6 ) found m/z: 1154.0, 865.8, 692.8, 577.5. 4.4.3 Melting temperature measurements Stock solutions of PNA 1, 2, 3, 4 and of oligonucleotides were prepared in doubly distilled water and their actual concentration calculated by UV absorbance using the following ε260 (M -1 cm -1 ) for the nucleobases: T 8600, C 6600, A 13700, G 11700. By using these concentrations, hybrid solutions containing 5µM PNA-DNA duplexes were prepared. All the 86
Arg-PNA Microarrays hybridisation experiments were carried out in 10mM phosphate buffer, 100mM NaCl, 0.1mM EDTA, pH= 7. All the hybrid samples reported were first incubated at 90°C for 5 minutes, then slowly cooled to room temperature. The samples were heated (1°C/min) and the UV signal variation at 260nm was recorded. Melting temperatures were measured as the maximum of the first derivatives of the melting curves. 4.4.4 PNA spotting on microarray slides Each PNA was dissolved in a solution containing carbonate buffer (0.1M, pH= 9), SDS 0.001% at a concentration of 50 µM and spotted by using a pin system spotter SpoArray 24 (Perkin-Elmer, Waltham, USA) onto commercial slides (CodeLink, Amersham Bioscences, New Jersey, USA). Then the following washing steps were performed: washing solution 1: carbonate 0.1M and SDS 0.2%; washing solution 2: carbonate 0.1M and SDS 1%; washing solution 3: carbonate 0.1M and SDS 0.001%. Fixing was performed overnight at a controlled moisture ( ∼ 75%). Capping of the unreacted sites was done by using a solution of Tris 0.1M, ethanolamine 50mM, pH=9, at 50°C for 1h. Conditioning of the slides was performed at 50°C with SSC buffer (0.6M NaCl, 0.06M sodium citrate, pH= 7) and SDS 1% solution for 1h. Final washings were carried out with doubly distilled water for 5 minutes. 4.4.5 Oligonucleotide hybridization on microarray slides Each Cy5 labeled oligonucletide was dissolved in a solution containing SSC buffer (0.3M NaCl, 0.03M sodium citrate, pH= 7), SDS 0.1% at a concentration of 100nM. Before hybridization the slides were hydrated with a solution of SSC buffer (0.3M NaCl, 0.03M sodium citrate, pH= 7), SDS 0.1% at 40°C for 30 minutes. Then, oligonucleotide solutions were deposited on the slides by using a hybridization chamber and left at 50°c for 2 hours. After hybridization, washing steps were performed at 50°C for 5 minutes with a solution of SSC buffer (0.3M NaCl, 0.03M sodium citrate, pH= 7), SDS 0.1%. Final washing was carried out for 1 minute at room temperature with a two different SSC buffer solutions: 0.03M NaCl, 0.003M sodium citrate, pH= 7 and 0.01M NaCl, 0.001M sodium citrate, pH= 7. 87
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Università degli studi di Parma Do
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2.4.4 Fluorescence measurements ...
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Chapter 6 The double nature of Pept
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Preface In Chapter 2 a particular t
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Chapter 1 PNAs: From birth to adult
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PNAs: from birth to adulthood one,
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PNAs: from birth to adulthood betwe
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PNAs: from birth to adulthood explo
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PNAs: from birth to adulthood well,
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PNAs: from birth to adulthood + Res
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PNAs: from birth to adulthood repet
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PNAs: from birth to adulthood 1.6 M
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PNAs: from birth to adulthood amino
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PNAs: from birth to adulthood Cycli
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PNAs: from birth to adulthood DNA a
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PNAs: from birth to adulthood 1.7 C
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PNAs: from birth to adulthood Submo
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PNAs: from birth to adulthood beaco
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PNAs: from birth to adulthood DNA s
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Page 55 and 56: PNA Molecular Beacons The sample th
Page 57 and 58: PNA Molecular Beacons pipetting to
Page 59 and 60: PNA Molecular Beacons The mastermix
Page 61 and 62: Chapter 3 A new class of Chiral PNA
Page 63 and 64: Arginine-PNAs monomers in the middl
Page 65 and 66: Arginine-PNAs The synthesis of the
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Page 69 and 70: Arginine-PNAs (increased electrosta
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Page 73 and 74: Arginine-PNAs foam. Yield: 30%. 1 H
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Page 77 and 78: Arginine-PNAs PNAs was carried out
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Page 83 and 84: Arg-PNA Microarrays 4.2 Results and
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Page 87 and 88: Arg-PNA Microarrays PNA-microarray
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Page 102 and 103: Chapter 5 Table 5-1. PNA sequences
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Page 106 and 107: Chapter 5 22.4°C 24.0°C 25.5°C 2
Page 108 and 109: Chapter 5 PNA 3 PNA 5 PNA 6 Slide 1
Page 110 and 111: Chapter 5 times. The substrates wer
Page 112 and 113: Chapter 5 chemicals were used as re
Page 114 and 115: Chapter 5 5.4.6 Microcontact Printi
Page 116 and 117: Chapter 5 5.5 References 1 A. L. Be
Page 118 and 119: Chapter 6 6.1 Introduction The use
Page 120 and 121: Chapter 6 Among all the peptides us
Page 122 and 123: Chapter 6 Figure 6-2: A standard PN
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Page 126 and 127: Chapter 6 6.2.2 Uptake experiments
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Page 130 and 131: Chapter 6 1.41 (s, 9H, Boc methyl g
Page 132 and 133: Chapter 6 Arg + CHα Lys), 4.0-4.6
Page 134 and 135: Chapter 6 12 B. P. Gangamani, V. A.
Page 136 and 137: Contributions Publications includin
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