Peptide nucleic acid (PNA) binding and its effect on in vitro transcription in friedreich's ataxia triplet repeats.
Peptide nucleic acids (PNAs) are DNA mimics in which peptide-like linkages are substituted for the phosphodiester backbone. Homopyrimidine PNAs can invade double-stranded DNA containing the homologous sequence by displacing the homopyrimidine strand from the DNA duplex and forming a PNA/DNA/PNA triplex with the complementary homopurine strand. Among biologically interesting targets for triplex-forming PNA are (GAA/CTT)(n) repeats. Expansion of these repeats results in partial inhibition of transcription in the frataxin gene, causing Friedreich's ataxia. We have studied PNA binding and its effect on T7 RNA polymerase transcription in vitro for short repeats (n = 3) and for long repeats (n = 39), placed in both possible orientations relative to the T7 promoter such that either the GAA-strand, or the CTT-strand serves as the template for transcription. In all cases PNA bound specifically and efficiently to its target sequence. For the short insert, PNA binding to the template strand caused partial transcription blockage with well-defined sites of RNA product truncation in the region of the PNA-binding sequence, whereas binding to the nontemplate strand did not block transcription. However, PNA binding to long repeats, whether in the template or the nontemplate strand, resulted in a dramatic reduction of the amount of full-length transcription product, although in the case of the nontemplate strand there were no predominant truncation sites. Biological implications of these results are discussed.
Belotserkovskii
Boris P
BP
Department of Biology, Stanford University, Stanford, California.
Liu
Richard
R
Hanawalt
Philip C
PC
D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005621:Friedreich Ataxia / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic; D018911:Trinucleotide Repeats / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism
D020135:Peptide Nucleic Acids; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases