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Factors affecting DNA Triplex formation

Factors affecting DNA Triplex formation
Factors affecting DNA Triplex formation
Triplex-forming oligonucleotides (TFOs) can be used to target DNA in a sequence-specific fashion, and have a number of potential therapeutic and biotechnological applications. TFOs bind within the DNA major groove where they form sequence-specific contacts with exposed groups on the target duplex. Pyrimidine-rich TFOs bind parallel to the target purine strand forming C+.GC and T.AT triplets and usually require conditions of low pH, which are needed for protonation of the third strand cytosines. In contrast, purine-rich TFOs bind antiparallel to the target and form triplexes containing G.GC and A.AT triplets. DNase I footprinting studies with parallel triplexes often reveal enhanced cleavage at the triplex-duplex junction at the 3’-end of the duplex purine strand. This study systematically investigated how this enhanced cleavage is affected by the nature of the base pairs that flank the TFO-binding site. For this we have used the well-characterised TFO-binding site in the tyrT(43-59) fragment and have changed the base at the 3’-end of the homopurine strand from cytosine to each of the other three bases in turn. In each case the footprints were accompanied by enhanced DNase I cleavage at the 3’-triplex-duplex junction on the purine strand, which is thought to be due to local structural changes that render the DNA to be more susceptible to cleavage by the enzyme. The enhancements were generally greater for flanking pyrimidines than purines. Similar experiments investigated the effect of changing the terminal triplet from T.AT to C+.GC, again flanked by each base in turn. Although there were no significant differences in the concentration dependence of the footprints, fluorescence melting experiments showed that triplexes flanked by G and A are more stable than those flanked by C and T. We also used diethylpyrocabonate (DEPC) to probe the reactivity of adenines at the triplex-duplex junction and find that some, but not all, sequence combinations generate enhanced reactivity, suggesting that triplex formation has altered the stacking pattern of adenines on the 3’-side of the TFO binding site. For antiparallel triplex formation, DNase I enhancements were also observed at a number of bands beyond the 5’-end of each TFO’s binding site. This is also attributed to the TFO-induced DNA structural changes that increase the accessibility of the enzyme to the target site. The results of concentration dependence of the footprints are similar to the parallel ones though fragment AC with 17-mer-G TFO had a much lower C50.
Sayoh, Ibrahim
aaea482c-fbe4-498c-8006-5efa3ac23313
Sayoh, Ibrahim
aaea482c-fbe4-498c-8006-5efa3ac23313
Fox, Keith
9da5debc-4e45-473e-ab8c-550d1104659f

Sayoh, Ibrahim (2016) Factors affecting DNA Triplex formation. University of Southampton, Centre for Biological Sciences, Doctoral Thesis, 220pp.

Record type: Thesis (Doctoral)

Abstract

Triplex-forming oligonucleotides (TFOs) can be used to target DNA in a sequence-specific fashion, and have a number of potential therapeutic and biotechnological applications. TFOs bind within the DNA major groove where they form sequence-specific contacts with exposed groups on the target duplex. Pyrimidine-rich TFOs bind parallel to the target purine strand forming C+.GC and T.AT triplets and usually require conditions of low pH, which are needed for protonation of the third strand cytosines. In contrast, purine-rich TFOs bind antiparallel to the target and form triplexes containing G.GC and A.AT triplets. DNase I footprinting studies with parallel triplexes often reveal enhanced cleavage at the triplex-duplex junction at the 3’-end of the duplex purine strand. This study systematically investigated how this enhanced cleavage is affected by the nature of the base pairs that flank the TFO-binding site. For this we have used the well-characterised TFO-binding site in the tyrT(43-59) fragment and have changed the base at the 3’-end of the homopurine strand from cytosine to each of the other three bases in turn. In each case the footprints were accompanied by enhanced DNase I cleavage at the 3’-triplex-duplex junction on the purine strand, which is thought to be due to local structural changes that render the DNA to be more susceptible to cleavage by the enzyme. The enhancements were generally greater for flanking pyrimidines than purines. Similar experiments investigated the effect of changing the terminal triplet from T.AT to C+.GC, again flanked by each base in turn. Although there were no significant differences in the concentration dependence of the footprints, fluorescence melting experiments showed that triplexes flanked by G and A are more stable than those flanked by C and T. We also used diethylpyrocabonate (DEPC) to probe the reactivity of adenines at the triplex-duplex junction and find that some, but not all, sequence combinations generate enhanced reactivity, suggesting that triplex formation has altered the stacking pattern of adenines on the 3’-side of the TFO binding site. For antiparallel triplex formation, DNase I enhancements were also observed at a number of bands beyond the 5’-end of each TFO’s binding site. This is also attributed to the TFO-induced DNA structural changes that increase the accessibility of the enzyme to the target site. The results of concentration dependence of the footprints are similar to the parallel ones though fragment AC with 17-mer-G TFO had a much lower C50.

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Ibrahim Sayoy.Final thesis after viva.pdf - Other
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More information

Published date: June 2016
Organisations: University of Southampton, Centre for Biological Sciences

Identifiers

Local EPrints ID: 403876
URI: http://eprints.soton.ac.uk/id/eprint/403876
PURE UUID: a6915bdb-22c9-47fd-8df1-149ace32d289
ORCID for Keith Fox: ORCID iD orcid.org/0000-0002-2925-7315

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Date deposited: 15 Dec 2016 13:57
Last modified: 06 Jun 2018 13:16

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