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Title: DNA triplexes in chemistry, biology and medicine
Author: Tailor, Radha
ISNI:       0000 0004 2708 0960
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2011
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The formation of DNA triple helices offers the possibility of selectively targeting specific genes to control their expression in vivo. This anti-gene strategy provides powerful tools for the development of therapeutics (anti-cancer drugs, drugs for viral infections) at the transcriptional level. DNA triplexes are formed when an oligonucleotide binds to the major groove of double helical DNA; the third strand can bind in either a parallel motif, or an anti-parallel motif. The requirement of low pH for the protonation of cytosine in the parallel binding motif makes the formation of triple helices difficult under physiological conditions. Described in this thesis is a novel method for the synthesis of the deoxycytidine analogue, 2-amino-3-methyl-5-(2’-deoxy-β-D-ribofuranosyl)pyridine (MeP). The phosphoramidite monomer of MeP was synthesised and incorporated as a “protonated” cytidine analogue into triplex forming oligonucleotides (TFOs). It was compared with other cytosine analogues, 5-methyl-(2’-deoxy-β-D-ribofuranosyl)cytosine (MeC), 2’-Omethyl MeP (MePOMe), and 2’-O-aminoethyl MeC (MeCAE). Triplex stability studies indicate that over the pH range 6.2-8.0, the general trend observed in terms of melting temperature (Tm) was as follows: MeP > MeC > MePOMe > MeCAE. DNase I footprinting studies indicate that at pH 7.5, MeP, when incorporated into the TFO, enhances the stability of the triplex by three-fold relative to MeC. In addition, UV melting, DNase I footprinting, and gel electrophoresis studies were carried out on a triplex formed by the binding of a TFO containing MeP and a 5’-Psoralen to a target duplex. This revealed the benefits of the combined modifications on the stability of the resultant triplex. “Soaking” experiments (in vivo) were also performed with this TFO on the organism C. elegans (the worms were soaked in solutions of the TFO for TFO delivery), to observe whether the TFO would induce loss-of-function phenotypes. Tm measurements indicated that in the pH range 6.6-8.0, photo-crosslinking of the TFO to the duplex created a shift in the triplex Tm of ~ + 26 °C when compared to the un-crosslinked triplex
Supervisor: Brown, Tom Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry