In vitro catalytic activities of DNA/RNA chimeric hammerhead ribozymes against AML1-MTG8 mRNA, a fused gene transcript in acute myeloid leukemia with t(8;21).
In order to design the best construct for therapeutic hammerhead ribozymes against AML1-MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia, we synthesized DNA/RNA chimeric ribozymes directed to the area adjacent to the fusion point between AML1 and MTG8. Catalytic efficiency and fusion gene specificity of ribozymes were examined by kinetic studies of the cleavage reactions of AML1-MTG8, AML1, and MTG8 RNAs transcribed in vitro. Ribozyme 2 (Rz2) specifically cleaved AML1-MTG8 RNA at three nucleotides downstream of the fusion junction with high efficiency. The highest cleavage efficiency was achieved by Rz4.3, which targeted non-contiguous sequences and cleaved at 19 nucleotides downstream of the fusion junction. Rz4.3 also cleaved MTG8 RNA but the cleavage efficiency was three orders of magnitude lower than that for AML1-MTG8 RNA. Therefore, Rz4.3 and Rz2 are the proper ribozymes for in vivo application to modulate gene expression of the AML1-MTG8.
Kozu
T
T
Department of Immunology and Virology, Saitama Cancer Center Research Institute, Japan.
Sueoka
E
E
Okabe
S
S
Sueoka
N
N
Komori
A
A
Fujiki
H
H
D000208:Acute Disease; D001483:Base Sequence; D002891:Chromosomes, Human, Pair 21; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D050676:Core Binding Factor Alpha 2 Subunit; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000075142:RUNX1 Translocation Partner 1 Protein; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D014178:Translocation, Genetic
D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D000075142:RUNX1 Translocation Partner 1 Protein; C493728:RUNX1 protein, human; C082222:RUNX1T1 protein, human; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA