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DNA Triplexes in chemistry biology and medicine

DNA Triplexes in chemistry biology and medicine
DNA Triplexes in chemistry biology and medicine
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-?-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-?-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
Tailor, Radha
976ab24e-8cb7-4908-8e49-bd0ac9a74eac
Tailor, Radha
976ab24e-8cb7-4908-8e49-bd0ac9a74eac
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89

Tailor, Radha (2011) DNA Triplexes in chemistry biology and medicine. University of Southampton, Chemistry, Doctoral Thesis, 323pp.

Record type: Thesis (Doctoral)

Abstract

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-?-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-?-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

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Submitted date: 25 March 2011
Organisations: University of Southampton

Identifiers

Local EPrints ID: 192831
URI: http://eprints.soton.ac.uk/id/eprint/192831
PURE UUID: ea03fc44-cfc5-4594-9e3a-4e0a07271944

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Date deposited: 08 Jul 2011 12:16
Last modified: 14 Mar 2024 03:52

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Contributors

Author: Radha Tailor
Thesis advisor: Tom Brown

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