In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide nucleic acid.
Peptide nucleic acids (PNAs) complementary to the 15 bases around the fusion point of both genomic DNA and cDNA of the promyelocytic leukemia/retinoic acid receptor alpha (PML/ RAR alpha; P/R) hybrid gene present in acute promyelocytic leukemia cells were synthesized and shown by gel retardation experiments to specifically bind oligonucleotides corresponding to the fusion region of the P/R molecule. PNA was also able to successfully compete with anti-P/R DNA for duplex formation with P/R DNA and to displace the anti-P/R DNA from dsDNA. In vitro transcribed P/R RNA from two inserts of approximately 350 to approximately 700 bp were tested in gel acceleration experiments with fluorescein-conjugated PNA and showed stable binding (resistant to denaturing conditions) of PNA to the newly transcribed RNA. Control RNA or transcripts from the noncoding strand did not bind PNA. However, this PNA, although able to specifically clamp polymerase chain reaction, was incapable of inhibiting in vitro translation of the PML/RAR alpha mRNA, even when a bis-PNA was used. Therefore, a PNA was targeted against the start region of the P/R cDNA and against poly-purine regions of the gene. Specific inhibition of in vitro translation and transcription was shown, starting at concentrations as low as 100 nmol/L. When oligonucleotides presenting the same sequence were compared, PNA proved to be approximately 40 times more active. In conclusion, in vitro inhibition of translation and transcription of the P/R gene can be obtained with PNA; however, it is still necessary to target the ATG start region or poly-purine regions of the gene.
Gambacorti-Passerini
C
C
Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
Mologni
L
L
Bertazzoli
C
C
le Coutre
P
P
Marchesi
E
E
Grignani
F
F
Nielsen
P E
PE
D001483:Base Sequence; D002474:Cell-Free System; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000494:pharmacology; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D010455:Peptides; D000071156:Promyelocytic Leukemia Protein; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D025521:Tumor Suppressor Proteins
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D015514:Oncogene Proteins, Fusion; D010455:Peptides; D000071156:Promyelocytic Leukemia Protein; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; C070576:PML protein, human