Abstract
Triple helix formation of nucleic acids is the most rational approach to designing site-specific transcription inhibitors. To increase their efficiency, reactive moieties such as psoralen or ethenocytosine have been introduced on the third strand. In transfected cells, these compounds induce a site-specific covalent binding of the third strand to the targeted sequence and efficiently block RNA polymerases. However, the stability of this transcription inhibition has never been checked. We have designed a plasmid containing a triple helix binding site in the coding region of the beta-galactosidase reporter gene and a polymerase chain reaction assay to follow quantitatively the cross-link of a psoralen-derivatized third strand in transfected cells. This assay has revealed that the cross-link was removed within a few hours, leading only to a transitory inhibition of gene expression. Control experiments in DNA repair-deficient cells suggest the implication of repair enzymes in this process.
Highlights
Triple helix formation of nucleic acidsis the most ra- otides and theirbiological effects (8, 9)
Howexpression.Controlexperimentsin DNA repair-defi- ever, the stabilityof the lesionsinduced by reactive triplehelix cient cells suggest the implicatioonf repair enzymes in has never yet been investigated in eukaryotic systems and is this process
We provide evidence that the cross-links of the psoralen-derivatized oligonucleotide are removed in intact cells; DNA repair occurs in spite of the steric hindranceinduced by the hybridizationof the third strand near the damaged site
Summary
Modification of pCHllOTH by 5-Hydroxypsoralenor Psoralen-linked Oligonucleotides pg(4 pmol) of plasmid were incubatedat 20 "C for 30 min with a300-fold molar excess of OTH or OTHC oligonucleotides in 1 ml of 20 mM HEPES, pH 6.5, 10 mM MgCl,. PCR Assay-A PCR assay was developed to follow the hybridization of the psoralen oligonucleotide at its target sequence and the behavior of the cross-link in transfected cells. The FCT fragment was processed in the same temperature and concentration conditions and submitted to 28 cycles This assay provideaslinear response to plasmid concentration between 10 and400 ngipoint. The same protocol was used as described above, except that the FTH fragment was amplified for 26 cycles and the FCT fragment for 34 cycles. This assay providesa linear response to plasmid concentration betwee0n.5 and 40 &point. PCHllOTH induced by psoralen alone or by a third strand linked to psoralen was evaluated by a PCR assay as described under "Experimental Procedures."
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