Abstract
We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.
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- Bertrand J. R., Rayner B., Imbach J. L., Paoletti C., Malvy C. Comparative activity of alpha- and beta-anomeric oligonucleotides on rabbit beta globin synthesis: inhibitory effect of cap targeted alpha-oligonucleotides. Biochem Biophys Res Commun. 1989 Oct 16;164(1):311–318. doi: 10.1016/0006-291x(89)91719-1. [DOI] [PubMed] [Google Scholar]
- Boiziau C., Kurfurst R., Cazenave C., Roig V., Thuong N. T., Toulmé J. J. Inhibition of translation initiation by antisense oligonucleotides via an RNase-H independent mechanism. Nucleic Acids Res. 1991 Mar 11;19(5):1113–1119. doi: 10.1093/nar/19.5.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
- Bruzik J. P., Van Doren K., Hirsh D., Steitz J. A. Trans splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature. 1988 Oct 6;335(6190):559–562. doi: 10.1038/335559a0. [DOI] [PubMed] [Google Scholar]
- Cazenave C., Chevrier M., Nguyen T. T., Hélène C. Rate of degradation of [alpha]- and [beta]-oligodeoxynucleotides in Xenopus oocytes. Implications for anti-messenger strategies. Nucleic Acids Res. 1987 Dec 23;15(24):10507–10521. doi: 10.1093/nar/15.24.10507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cazenave C., Stein C. A., Loreau N., Thuong N. T., Neckers L. M., Subasinghe C., Hélène C., Cohen J. S., Toulmé J. J. Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides. Nucleic Acids Res. 1989 Jun 12;17(11):4255–4273. doi: 10.1093/nar/17.11.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cornelissen A. W., Verspieren M. P., Toulmé J. J., Swinkels B. W., Borst P. The common 5' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides. Nucleic Acids Res. 1986 Jul 25;14(14):5605–5614. doi: 10.1093/nar/14.14.5605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gagnor C., Bertrand J. R., Thenet S., Lemaître M., Morvan F., Rayner B., Malvy C., Lebleu B., Imbach J. L., Paoletti C. alpha-DNA. VI: Comparative study of alpha- and beta-anomeric oligodeoxyribonucleotides in hybridization to mRNA and in cell free translation inhibition. Nucleic Acids Res. 1987 Dec 23;15(24):10419–10436. doi: 10.1093/nar/15.24.10419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hélène C., Toulmé J. J. Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta. 1990 Jun 21;1049(2):99–125. doi: 10.1016/0167-4781(90)90031-v. [DOI] [PubMed] [Google Scholar]
- Krieg A. M., Tonkinson J., Matson S., Zhao Q., Saxon M., Zhang L. M., Bhanja U., Yakubov L., Stein C. A. Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1048–1052. doi: 10.1073/pnas.90.3.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mauel J., Behin R., Biroum-Noerjasin Quantitative release of live microorganisms from infected macrophages by sodium dodecyl sulphate. Nat New Biol. 1973 Jul 18;244(133):93–94. doi: 10.1038/newbio244093a0. [DOI] [PubMed] [Google Scholar]
- Miller S. I., Landfear S. M., Wirth D. F. Cloning and characterization of a Leishmania gene encoding a RNA spliced leader sequence. Nucleic Acids Res. 1986 Sep 25;14(18):7341–7360. doi: 10.1093/nar/14.18.7341. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Minshull J., Hunt T. The use of single-stranded DNA and RNase H to promote quantitative 'hybrid arrest of translation' of mRNA/DNA hybrids in reticulocyte lysate cell-free translations. Nucleic Acids Res. 1986 Aug 26;14(16):6433–6451. doi: 10.1093/nar/14.16.6433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Monia B. P., Lesnik E. A., Gonzalez C., Lima W. F., McGee D., Guinosso C. J., Kawasaki A. M., Cook P. D., Freier S. M. Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J Biol Chem. 1993 Jul 5;268(19):14514–14522. [PubMed] [Google Scholar]
- New R. R., Chance M. L., Thomas S. C., Peters W. Antileishmanial activity of antimonials entrapped in liposomes. Nature. 1978 Mar 2;272(5648):55–56. doi: 10.1038/272055a0. [DOI] [PubMed] [Google Scholar]
- Pascolo E., Blonski C., Shire D., Toulmé J. J. Antisense effect of oligodeoxynucleotides complementary to the mini-exon sequence of the protozoan parasite Leishmania amazonensis. Biochimie. 1993;75(1-2):43–47. doi: 10.1016/0300-9084(93)90023-l. [DOI] [PubMed] [Google Scholar]
- Rabinovitch M., Zilberfarb V., Ramazeilles C. Destruction of Leishmania mexicana amazonensis amastigotes within macrophages by lysosomotropic amino acid esters. J Exp Med. 1986 Mar 1;163(3):520–535. doi: 10.1084/jem.163.3.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rapaport E., Misiura K., Agrawal S., Zamecnik P. Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8577–8580. doi: 10.1073/pnas.89.18.8577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rizzuto R., Sandonà D., Capaldi R. A., Bisson R. Nucleotide sequence of the cDNA encoding subunit VIIe of cytochrome c oxidase from the slime mold Dictyostelium discoideum. Nucleic Acids Res. 1990 Nov 25;18(22):6711–6711. doi: 10.1093/nar/18.22.6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stein C. A., Cheng Y. C. Antisense oligonucleotides as therapeutic agents--is the bullet really magical? Science. 1993 Aug 20;261(5124):1004–1012. doi: 10.1126/science.8351515. [DOI] [PubMed] [Google Scholar]
- Teichman-Weinberg A., Littauer U. Z., Ginzburg I. The inhibition of neurite outgrowth in PC12 cells by tubulin antisense oligodeoxyribonucleotides. Gene. 1988 Dec 10;72(1-2):297–307. doi: 10.1016/0378-1119(88)90155-2. [DOI] [PubMed] [Google Scholar]
- Ullu E., Tschudi C. 2'-O-methyl RNA oligonucleotides identify two functional elements in the trypanosome spliced leader ribonucleoprotein particle. J Biol Chem. 1993 Jun 25;268(18):13068–13073. [PubMed] [Google Scholar]
- Verspieren P., Cornelissen A. W., Thuong N. T., Hélène C., Toulmé J. J. An acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome mRNAs kills cultured parasites. Gene. 1987;61(3):307–315. doi: 10.1016/0378-1119(87)90194-6. [DOI] [PubMed] [Google Scholar]
- Wachter L., Jablonski J. A., Ramachandran K. L. A simple and efficient procedure for the synthesis of 5'-aminoalkyl oligodeoxynucleotides. Nucleic Acids Res. 1986 Oct 24;14(20):7985–7994. doi: 10.1093/nar/14.20.7985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walder J. A., Eder P. S., Engman D. M., Brentano S. T., Walder R. Y., Knutzon D. S., Dorfman D. M., Donelson J. E. The 35-nucleotide spliced leader sequence is common to all trypanosome messenger RNA's. Science. 1986 Aug 1;233(4763):569–571. doi: 10.1126/science.3523758. [DOI] [PubMed] [Google Scholar]
- de Smidt P. C., Le Doan T., de Falco S., van Berkel T. J. Association of antisense oligonucleotides with lipoproteins prolongs the plasma half-life and modifies the tissue distribution. Nucleic Acids Res. 1991 Sep 11;19(17):4695–4700. doi: 10.1093/nar/19.17.4695. [DOI] [PMC free article] [PubMed] [Google Scholar]