Increased efficacy of antileishmanial antisense phosphorothioate oligonucleotides in Leishmania amazonensis overexpressing ribonuclease H.
Ribonuclease H (RNase H), an enzyme that cleaves an RNA sequence base-paired with a complementary DNA sequence, is proposed to be the mediator of antisense phosphorothioate oligonucleotide (S-oligo) lethality in a cell. To understand the role of RNase H in the killing of the parasitic protozoan Leishmania by antisense S-oligos, we expressed an episomal copy of the Trypanosoma brucei RNase H1 gene inside L. amazonensis promastigotes and amastigotes that constitutively express firefly luciferase. Our hypothesis was that S-oligo-directed degradation of target mRNA is facilitated in a cell that has higher RNase H activity. Increased inhibition of luciferase mRNA expression by anti-luciferase S-oligo and by anti-miniexon S-oligo in these stably transfected promastigotes overexpressing RNase H1 was correlated to the higher activity of RNase H in these cells. The efficiency of killing of the RNase H overexpressing amastigotes inside L. amazonensis-infected macrophages by anti-miniexon S-oligo was higher than in the control cells. Thus, RNase H appears to play an important role in the antisense S-oligo-mediated killing of Leishmania. Chemical modification of S-oligos that stimulate RNase H and/or co-treatment of cells with an activator of RNase H may be useful for developing an antisense approach against leishmaniasis. The transgenic Leishmania cells overexpressing RNase H should be a good model system for the antisense-mediated gene expression ablation studies in these parasites.
Department of Microbiology, Meharry Medical College, 1005 D. B. Todd Jr. Blvd., 37208, Nashville, TN, USA.
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M
Mishra
J R
JR
Bennett
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G
Chaudhuri
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D001483:Base Sequence; D016054:DNA, Protozoan / Q000032:analysis; D004353:Drug Evaluation, Preclinical; D015870:Gene Expression / Q000187:drug effects; D007891:Leishmania / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D016914:Ribonuclease H / Q000096:biosynthesis / Q000502:physiology*; D013872:Thionucleosides / Q000494:pharmacology*; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism
D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013872:Thionucleosides; D008156:Luciferases; D016914:Ribonuclease H