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 leukemi a/retinoic acid receptor alpha (PML/RAR alpha; P/R) hybrid gene presen t in acute promyelocytic leukemia cells were synthesized and shown by gel retardation experiments to specifically bind oligonucleotides corr esponding 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 transcribe d P/R RNA from two inserts of approximate to 350 and approximate to 70 0 bp were tested in gel acceleration experiments with fluorescein-conj ugated PNA and showed stable binding (resistant to denaturing conditio ns) of PNA to the newly transcribed RNA, Control RNA or transcripts fr om 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 re gion of the P/R cDNA and against poly-purine regions of the gene. Spec ific inhibition of in vitro translation and transcription was shown, s tarting at concentrations as low as 100 nmol/L. When oligonucleotides presenting the same sequence were compared, PNA proved to be approxima tely 40 times more active. In conclusion, in vitro inhibition of trans lation and transcription of the P/R gene can be obtained with PNA; how ever, it is still necessary to target the ATG start region or poly-pur ine regions of the gene. (C) 1996 by The American Society of Hematolog y.