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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
2
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pubmed:dateCreated |
1993-4-8
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pubmed:abstractText |
The ability of pyrimidine-rich and purine-rich oligodeoxynucleotides to form stable triple-helical structures with imperfect R.Y target sites, containing C interruptions in the purine strand (CG inversions) and located within Bluescript KS+, a plasmid of 2959 bp, has been investigated by electrophoresis, ultraviolet absorbance and cleavage-protection experiments. First, we synthesized double-stranded oligonucleotides corresponding to the plasmid sites and studied their interaction with oligopyrimidines which oppose either G or T to CG inversions. The resulting imperfect DNA triplexes were detected by gel-mobility shift. Their melting profiles were found to be biphasic, and the triplex-to-duplex plus single-strand transition was affected by hysteresis. The 21-nucleotide triplex containing three GC.G mismatched triads had tm = 45 degrees C, while the same triplex, but with three GC.T triads, had tm = 31 degrees C. Moreover, replacing C with 5-methylcytosine in the third strand resulted in a significant stabilization of the defective triplex, tm = 49 degrees C. The potential of the 21-nucleotide oligopyrimidines to recognize and bind in a sequence-specific manner to imperfect R.Y sequences in Bluescript KS+ has been investigated by means of a restriction-endonuclease-protection assay, taking advantage of the fact that one R.Y sequence of Bluescript KS+ was partially overlapped with a HaeII site. Effective endonuclease inhibition was observed with oligopyrimidines opposing G-to-GC inversions, at 10-50 microM. By contrast, the oligopyrimidine opposing T-to-CG inversions did not exhibit any interference with endonuclease activity in our standard conditions. Finally, we have tested the ability of purine-rich strands to bind the R.Y sites of Bluescript KS+. A very weak cleavage protection was observed by using an oligomer (130 microM) with a polarity antiparallel to the purine strand of the target site. The resulting Y.R.R triplex was stabilized by CG.G (GC.G) and TA.A base triplets. This triplex denatures with a low cooperative melting profile suggesting the absence of strong interactions between the third strand and the target site.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0014-2956
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
1
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pubmed:volume |
212
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
395-401
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pubmed:dateRevised |
2007-7-23
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pubmed:meshHeading |
pubmed-meshheading:8444176-Base Sequence,
pubmed-meshheading:8444176-Binding Sites,
pubmed-meshheading:8444176-DNA,
pubmed-meshheading:8444176-Molecular Sequence Data,
pubmed-meshheading:8444176-Nucleic Acid Conformation,
pubmed-meshheading:8444176-Oligodeoxyribonucleotides,
pubmed-meshheading:8444176-Plasmids
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pubmed:year |
1993
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pubmed:articleTitle |
Sequence-specific DNA-triplex formation at imperfect homopurine-homopyrimidine sequences within a DNA plasmid.
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pubmed:affiliation |
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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