Antisense Oligonucleotides Containing Modified Bases Inhibit in Vitro Translation of Leishmania amazonensis mRNAs by Invading the Mini-exon Hairpin
1999; Elsevier BV; Volume: 274; Issue: 12 Linguagem: Inglês
10.1074/jbc.274.12.8191
ISSN1083-351X
AutoresDaniel Compagno, Jed N. Lampe, Chantal Bourget, Igor V. Kutyavin, Ludmila Yurchenko, Eugeny A. Lukhtanov, Vladimir V. Gorn, Howard Gamper, Jean‐Jacques Toulmé,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoComplementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170–11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs. Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170–11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs. mini-exon derived pre-RNA oligodeoxynucleotide selectively binding complementary normal base methylpropidium-EDTA phosphodiester phosphorothioate Trypanosomatids are attractive targets for the antisense approach, as every mature transcript contains a common species-specific mini-exon sequence spliced onto its 5′ end (1Toulmé J.J. Murray J.A.H. Antisense RNA and DNA. Wiley Inc., New York1992: 175-194Google Scholar, 2Toulmé J.J. Bourget C. Compagno D. Yurchenko L. Parasitology. 1997; 114: S45-S59Crossref PubMed Scopus (45) Google Scholar). Maturation of mRNAs includes a trans-splicing event, which transfers a 39-nucleotide segment of the mini-exon derived pre-RNA (the medRNA)1 to the 5′ end of every message (3Borst P. Annu. Rev. Biochem. 1986; 55: 701-732Crossref PubMed Scopus (286) Google Scholar). Hybridization of an antisense oligonucleotide (ODN) to this sequence can potentially inhibit translation of all transcripts. Indeed, it was demonstrated that anti-mini-exon oligonucleotides were able to prevent translation ofTrypanosoma and Leishmania mRNAs in cell-free extracts (4Cornelissen A.W.C.A. Verspieren P. Toulmé J.J. Swinkels B.W. Borst P. Nucleic Acids Res. 1986; 14: 5605-5614Crossref PubMed Scopus (63) Google Scholar, 5Walder J.A. Eder P.S. Engman D.M. Brentano S.T. Walder R.Y. Knutzon D.S. Dorfman D.M. Donelson J.E. Science. 1986; 233: 569-571Crossref PubMed Scopus (110) Google Scholar, 6Pascolo E. Blonski C. Shire D. Toulmé J.-J. Biochimie. 1993; 75: 43-47Crossref PubMed Scopus (13) Google Scholar), to kill procyclic forms of T. brucei (7Verspieren P. Cornelissen A.W.C.A. Thuong N.T. Hélène C. Toulmé J.J. Gene (Amst .). 1987; 61: 307-315Crossref PubMed Scopus (77) Google Scholar), and to cure L. amazonensis-infected macrophages in vitro (Refs. 8Ramazeilles C. Mishra R.K. Moreau S. Pascolo E. Toulmé J.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7859-7863Crossref PubMed Scopus (38) Google Scholar and 9Mishra R.K. Moreau C. Ramazeilles C. Moreau S. Bonnet J. Toulmé J.J. Biochim. Biophys. Acta. 1995; 1264: 229-237Crossref PubMed Scopus (22) Google Scholar; see Ref. 2Toulmé J.J. Bourget C. Compagno D. Yurchenko L. Parasitology. 1997; 114: S45-S59Crossref PubMed Scopus (45) Google Scholar for a review). The trypanosomatid medRNA was proposed to adopt a secondary structure based upon conservation of folding pattern for different RNA (10Bruzik J.P. Van Doren K. Hirsh D. Steitz J.A. Nature. 1988; 335: 559-562Crossref PubMed Scopus (145) Google Scholar). Recently the Leptomonas collosoma medRNA was shown to switch between two alternate structures (11Lecuyer K. Crothers D.M. Biochemistry. 1993; 32: 5301-5311Crossref PubMed Scopus (77) Google Scholar, 12Harris K.A. Crothers D.M. Ullu E. RNA. 1995; 1: 351-362PubMed Google Scholar). One form leads to a base pairing pattern conserved for all of the trypanosome medRNA, suggesting critical functional interactions for splicing (11Lecuyer K. Crothers D.M. Biochemistry. 1993; 32: 5301-5311Crossref PubMed Scopus (77) Google Scholar). In L. amazonensis the mini-exon was shown to fold into a structure that interferes with the hybridization of antisense ODNs (13Verspieren P. Loreau N. Thuong N.T. Shire D. Toulmé J.J. Nucleic Acids Res. 1990; 18: 4711-4717Crossref PubMed Scopus (27) Google Scholar, 14Pascolo E. Hudrisier D. Sproat B. Thuong N.T. Toulmé J.-J. Biochim. Biophys. Acta. 1994; 1219: 98-106Crossref PubMed Scopus (16) Google Scholar). RNA intramolecular structures that prevent the formation of oligonucleotide-RNA intermolecular complexes weaken antisense effects. This limitation has prompted the design of oligonucleotides able to overcome the mini-exon structure (for a review, see Ref. 15Toulmé J.J. Le Tinévez R. Brossalina E. Biochimie. 1996; 78: 663-673Crossref PubMed Scopus (24) Google Scholar). In a recent report, the L. amazonensis mini-exon sequence was efficiently complexed by an ODN capable of folding back on itself to form a triple strand with the putative hairpin element (16Pascolo E. Toulmé J.-J. J. Biol. Chem. 1996; 271: 24187-24192Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). This "double hairpin" complex readily formed at pH 6.0 using a pyrimidine motif for triplexing. However, this oligomer did not show selective inhibitory properties in cell-free translation experiments, probably due to pH conditions that are not appropriate for the formation of a triple helix involving C-G*C+ triplets. Alternatively, the disruption of the secondary structure by oligomers of high affinity can be considered. We have been investigating selectively binding complementary (SBC) ODNs as an alternative for targeting structured nucleic acids (17Kutyavin I.V. Rhinehart R.L. Lukhtanov E.A. Gorn V.V. Meyer R.B. Gamper H.B. Biochemistry. 1996; 35: 11170-11176Crossref PubMed Scopus (117) Google Scholar, 18Woo J. Meyer R. Gamper H.B. Nucleic Acids Res. 1996; 24: 2470-2475Crossref PubMed Scopus (70) Google Scholar). These ODNs are intended to be used as complementary pairs or as a single self-complementary agent. Due to the presence of modified bases, they are unable to form stable hybrids with one another but should hybridize to normal DNA or RNA complements. We have shown that SBC ODNs containing 2-aminoadenine and 2-thiothymine bases can strand invade the end of double-stranded DNA in a process that is favored both kinetically and thermodynamically (17Kutyavin I.V. Rhinehart R.L. Lukhtanov E.A. Gorn V.V. Meyer R.B. Gamper H.B. Biochemistry. 1996; 35: 11170-11176Crossref PubMed Scopus (117) Google Scholar). The A/U-rich hairpin proposed for the L. amazonensis mini-exon sequence presents an ideal target for testing whether SBC ODNs can strand invade an RNA stem loop by hybridizing to every base of the element. In this study we experimentally confirmed the hairpin structure of theL. amazonensis mini-exon sequence and demonstrated that antisense ODNs with SBC character are more effective than normal base (NB) ODNs in addressing this hairpin. Our results show that SBC ODNs form very stable hybrids with the entire hairpin sequence, and that the heteroduplexes are substrates for Escherichia coli RNase H. We demonstrated that SBC ODN-RNA hybrids inhibit translation ofL. amazonensis mRNA in a cell-free extract when RNase H is present. The successful targeting of a simple hairpin by SBC ODNs suggests that other secondary structure features in RNA should also be accessible to these ODNs. Preparation of protected phosphoramidite precursors of 2-thiothymidine and 2-aminoadenosine and synthesis of SBC ODNs using these reagents have been described (17Kutyavin I.V. Rhinehart R.L. Lukhtanov E.A. Gorn V.V. Meyer R.B. Gamper H.B. Biochemistry. 1996; 35: 11170-11176Crossref PubMed Scopus (117) Google Scholar). NB ODNs with DNA, RNA, or 2′-O-methyl backbones were synthesized by routine procedures using commercially available chemicals. The Beaucage reagent was used to prepare NB and SBC ODNs with a phosphorothioate backbone (19Iyer P.I. Lawrence R.P. Egan W. Regan J.B. Beaucage S.L. J. Org. Chem. 1990; 55: 4693-4698Crossref Scopus (265) Google Scholar). All antisense ODNs contained a 3′-hexanol end group as a consequence of using a modified hexanol primer controlled pore glass support (20Gamper H.B. Reed M.W. Cox T. Virosco J.S. Adams A.D. Gall A.A. Scholler J.K. Meyer Jr., R.B. Nucleic Acids Res. 1993; 21: 145-150Crossref PubMed Scopus (137) Google Scholar). Capillary gel electrophoresis indicated that the SBC ODNs were at least 85% pure, and hydrolysates of SBC ODNs prepared as described previously (17Kutyavin I.V. Rhinehart R.L. Lukhtanov E.A. Gorn V.V. Meyer R.B. Gamper H.B. Biochemistry. 1996; 35: 11170-11176Crossref PubMed Scopus (117) Google Scholar) gave the expected ratios of nucleosides. Extinction coefficients of ODNs were calculated using a nearest neighbor model (21Cantor C. Warshaw M.M. Shapiro H. Biopolymers. 1970; 9: 1059-1077Crossref PubMed Scopus (879) Google Scholar) and employed values of 9,800 m−1 cm−1 for 2-thiothymidine (22Cheong C. Tinoco I. Chollet A. Nucleic Acids Res. 1988; 16: 5115-5122Crossref PubMed Scopus (76) Google Scholar) and 6,800 m−1cm−1 for 2-aminoadenosine (23Connolly B.A. Newman P.C. Nucleic Acids Res. 1989; 17: 4957-4974Crossref PubMed Scopus (117) Google Scholar) at 260 nm. The mini-exon RNA of L. amazonensis was prepared by in vitro transcription of pBluescriptIIKS in which the mini-exon sequence was cloned downstream of the T7 promoter, using Ampliscribe T7 transcription kit (Tebu). In the resulting transcript, the original mini-exon sequence was flanked by sequences derived from the vector, both on the 5′ (5′-GGGCGAAUUGGAGCUC) and on the 3′ sides (5′-GAUC). The RNA was purified by electrophoresis on a 12% polyacrylamide denaturing gel. 5′-End-labeled mini-exon RNA (100 pmol) was incubated for 20 min at 37 °C in the presence of 1 unit of S1 nuclease (Boehringer Mannheim), in a 50 mm sodium acetate buffer (pH 4.5) containing 28 mm NaCl and 4.5 mmZnSO4. RNases T1 and V1 (Boehringer Mannheim) digestions were performed for 10 and 20 min, respectively, in 20 mmHEPES buffer (pH 7.4) containing 140 mm KCl, 20 mm sodium acetate, and 3 mm MgCl2(S buffer). Cleavage by 15 μm methyl propidium-EDTA-Fe(II) complexes was carried out for 10 min at 37 °C in S buffer. RNase T1 digestion under denaturing conditions was achieved for 10 min at 50 °C in 20 mm sodium acetate and 5 m urea. Digestion products were analyzed by electrophoresis on a 12% polyacrylamide denaturing gel followed by autoradiography. Complementary ODNs were diluted in 20 mm HEPES, pH 7.2, 10 mmMgCl2, and 140 mm KCl to give 2 μm of each ODN. Hybridization was assured by rapid heat-cooling of the samples. A 260 was recorded as a function of temperature in a Lambda 2 (Perkin-Elmer) spectrophotometer equipped with a PTP-6 automatic multicell temperature programmer. Samples were heated at the rate of 0.5 °C/min. Melting temperatures (Tm values) were determined from the derivative maxima. The mini-exon RNA prepared by in vitro transcription as described above was labeled by incorporating [α-32P]ATP (37.5 MBq/mmol). RNA and oligonucleotide were heated separately for 5 min at 65 °C and cooled down on ice. One pmol of RNA was mixed with the desired ODN in 15 μl of 50 mm Tris acetate buffer (pH 7.0) containing 10 mm magnesium acetate. The mixture was incubated for 15 min at 4 °C. The samples were then run in the same Tris buffer at 10 V/cm for about 15 h, on a 15% nondenaturing polyacrylamide gel at 4 °C. The activity in the bands corresponding to the free and bound RNA species was evaluated by PhosphorImager analysis or by Cerenkov counting. The dissociation constant was taken as the ODN concentration at which 50% of the target RNA was retarded. Total RNA was isolated from L. amazonensispromastigotes by the guanidine chloride method (6Pascolo E. Blonski C. Shire D. Toulmé J.-J. Biochimie. 1993; 75: 43-47Crossref PubMed Scopus (13) Google Scholar). In vitrotranslation of this RNA (1 μg) was catalyzed by wheat germ extract (25 μl; Promega) or rabbit reticulocyte lysate (35 μl; Promega) in a total volume of 50 μl. Antisense ODN was added to the RNA on ice, immediately before the initiation of translation, and reactions were conducted for 1 h, at 25 °C (wheat germ extract) or 30 °C (rabbit reticulocyte lysate) in the presence of [35S]methionine (37 TBq/mmol; Amersham). Some incubations were supplemented with 2.5 units of E. coli RNase H (Boehringer Mannheim). Reaction aliquots were analyzed for labeled proteins by precipitation with trichloroacetic acid onto Whatman GF/A glass fiber filters and counting in a liquid scintillation counter. Relative levels of protein synthesis were calculated as described previously (13Verspieren P. Loreau N. Thuong N.T. Shire D. Toulmé J.J. Nucleic Acids Res. 1990; 18: 4711-4717Crossref PubMed Scopus (27) Google Scholar). A synthetic RNA 35-mer was used for RNase H mapping of L. amazonensis mini-exon-ODN complexes. Prior to incubation with RNase H, the oligonucleotide and the RNA were treated as described for electrophoretic mobility shift assay. 5′-End-labeled RNA was then mixed with the desired ODN at a final concentration of 2 and 50 μm, respectively, in a 20 mm HEPES buffer, pH 7.8, containing 50 mm KCl, 10 mm MgCl2, and 1 mmdithiothreitol. The mixture was kept at 4 °C for 15 min prior to incubation with 0–4 units of E. coli RNase H (Promega) for 15 min at 4 °C. The reaction was stopped by adding one volume of 8m urea. Samples were analyzed by electrophoresis on a 20% polyacrylamide gel containing 7 m urea. Preparation of macrophages and parasites was carried out as described (8Ramazeilles C. Mishra R.K. Moreau S. Pascolo E. Toulmé J.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7859-7863Crossref PubMed Scopus (38) Google Scholar). Leishmania amazonensis (LV79 strain), prepared from infected BALB/c mice, was used to infect adherent macrophages at a multiplicity of 5 parasites/cell, resulting in more than 70% infected cells. This was normalized to 100% for comparison between different experiments. Infected macrophages were incubated at 34 °C with the desired oligonucleotide concentration for 48 h in RPMI/HEPES medium containing 10% fetal calf serum. The cultures were then washed, fixed in methanol, and stained with Giemsa. Cells were observed by microscopy to determine the level of infection. About 500 macrophages were scored for each oligonucleotide concentration; a cell was identified as infected when it contained at least one recognizable parasite. The mini-exon region is known to fold into secondary structures (11Lecuyer K. Crothers D.M. Biochemistry. 1993; 32: 5301-5311Crossref PubMed Scopus (77) Google Scholar, 12Harris K.A. Crothers D.M. Ullu E. RNA. 1995; 1: 351-362PubMed Google Scholar). We investigated the structure of a 35-nucleotide-long mini-exon sequence in which the four modified nucleotides (24Bangs J.D. Crain P.F. Hashizume T. McCloskey J.A. Boothroyd J.C. J. Biol. Chem. 1992; 267: 9805-9815Abstract Full Text PDF PubMed Google Scholar) at the 5′ end of the natural sequence were omitted (Figs.1 and 2). Indirect evidence favors the existence of a stable structure in the mini-exon sequence of L. amazonensis. Anomalous electrophoretic mobility of this oligomer as well as low binding efficiency of complementary oligonucleotides have been observed previously (6Pascolo E. Blonski C. Shire D. Toulmé J.-J. Biochimie. 1993; 75: 43-47Crossref PubMed Scopus (13) Google Scholar, 13Verspieren P. Loreau N. Thuong N.T. Shire D. Toulmé J.J. Nucleic Acids Res. 1990; 18: 4711-4717Crossref PubMed Scopus (27) Google Scholar). Prior to initiating the targeting of this structure with NB and SBC ODNs, we confirmed its existence by footprinting. The cleavage pattern obtained with nuclease S1, RNases T1 and V1, and methylpropidium-EDTA (MPE) led to the secondary structure shown in Fig.1. Positions 23–26, which are cleaved by S1 nuclease (lane 4), correspond to the apical loop of the imperfect hairpin. Whereas G23 was a cleavage site for RNase T1, no band corresponding to G17 and G29 was observed suggesting a structured region. This was further confirmed by RNase V1 and MPE, which preferentially cleave double-stranded structures. The reduced cleavage observed with RNase V1 compared with MPE in the upper part of the stem might be related to the bulged U20. The double-stranded stem likely extends to the U35-U37/A13-A11 region as indicated both by RNase V1 and MPE sensitivity, whereas both G8 and G39,G40 have a clear single-strandedness character. The structure of the upper part of the stem is in fair agreement with previous models (11Lecuyer K. Crothers D.M. Biochemistry. 1993; 32: 5301-5311Crossref PubMed Scopus (77) Google Scholar); the differences at the bottom of the stem region are related to the different sequences used.Figure 2Antisense ODNs used to target the mini-exon sequence of L. amazonensis. The mini-exon sequence, with paired bases underlined, is at thetop (39Miller S.I. Landfear S.M. Wirth D.F. Nucleic Acids Res. 1986; 14: 7341-7360Crossref PubMed Scopus (68) Google Scholar). Four antisense ODNs 15Le-I, 15Le-II, 25Le, and 16Le are aligned below together with DNA (DNA-I/II) and RNA (RNA-I/II) complements to 15Le-I and 15Le-II. 15Le-I, 15Le-II, and 25Le were synthesized with PO and PS backbones and contained adenine and thymine bases (NB) or 2-aminoadenine and 2-thiothymine bases (SBC). 15Le-I and 15Le-II were also synthesized with a 2′-O-methyl backbone and contained uracil in place of thymine. 16Le has been previously used to target the mini-exon sequence (8Ramazeilles C. Mishra R.K. Moreau S. Pascolo E. Toulmé J.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7859-7863Crossref PubMed Scopus (38) Google Scholar) and contained a NB sequence and either a PO or PS backbone. The 3′-hexanol group present on every antisense ODN except 16Le is not shown.View Large Image Figure ViewerDownload (PPT) The mini-exon sequence of L. amazonensis is an attractive target for evaluating SBC ODNs as antisense agents. The high A/U content (77%) of the hairpin and its flanking arms should favor the use of SBC ODNs that contain 2-aminoadenine and 2-thiothymine bases in place of adenine and thymine. These base analogs cannot hydrogen-bond to one another, due to steric clash, but can form good pairs with complementary unmodified bases (17Kutyavin I.V. Rhinehart R.L. Lukhtanov E.A. Gorn V.V. Meyer R.B. Gamper H.B. Biochemistry. 1996; 35: 11170-11176Crossref PubMed Scopus (117) Google Scholar). As a consequence, an SBC ODN complementary to the hairpin element of the mini-exon should be single-stranded and yet capable of strand invading the stem-loop structure. In this case the upper 8-base pair stem of the hairpin would be replaced by 20 base pairs formed between the ODN and the RNA. Moreover, 9 of these new base pairs would be highly stable 2-aminoadenine-uracil doublets with three hydrogen bonds (25Howard F.B. Miles H.T. Biochemistry. 1984; 23: 6723-6732Crossref PubMed Scopus (47) Google Scholar). To promote annealing, both NB and SBC ODNs were synthesized as 25-mers (25Le; see Fig. 2) with the 5 extra bases complementary to the 5′ arm at the base of the hairpin. SBC ODN pairs were also compared with NB ODN pairs as agents to target the mini-exon hairpin. These 15-mer ODNs were complementary to the 5′ or 3′ half of the hairpin (15Le-I or 15Le-II; see Fig. 2). Annealing was again promoted by making a 5-base-long segment of each ODN complementary to one of the overhangs at the base of the hairpin. Hybridization of both ODNs to the hairpin generated a 30-base pair DNA-RNA hybrid with a nick separating the ODNs. By employing two paired ODNs instead of a single self-complementary ODN, the likelihood of mutual interaction between the NB ODNs was significantly reduced and the potential advantage of the SBC pair accordingly diminished. NB and SBC versions of 25Le or 15Le-I + 15Le-II were synthesized with phosphodiester (PO) or phosphorothioate (PS) backbones. Properties of these ODNs were compared relative to 16Le, a NB 16-mer (Fig. 2) used in a previous study (8Ramazeilles C. Mishra R.K. Moreau S. Pascolo E. Toulmé J.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7859-7863Crossref PubMed Scopus (38) Google Scholar). Since the entire sequence of a stable hairpin was being targeted, the various antisense ODNs were prone to hairpin formation as well. Self-association of these ODNs was examined by UV-absorption thermal denaturation. While none of the SBC ODNs gave a melting transition, PO and PS versions of NB 25Le gave melting temperatures (Tm) of 46 and 31 °C, respectively (TableI). The NB combination of 15Le-I + 15Le-II formed a very weak hybrid when both ODNs had a PO backbone (Tm = 13 °C) and no hybrid at all when they possessed a PS backbone. The results obtained with antisense sequences are in fair agreement with the expected pairing properties of the ODNs; NB-containing oligomers give rise to more stable antisense structures than SBC ODNs and are therefore less likely to hybridize with the target sense sequence.Table IStability of hybrids formed by antisense Le ODNs with each other and with complementary DNA or RNA targetsODNs and hybridsT mNB/POaDenotes bases and backbone composition of antisense ODNs.SBC/PONB/PSSBC/PSNB/2′-OMe°C15Le-I + 15 Le-II13—bND, not determined; —, no transition was detected.——ND25Le46—31—ND15Le-I + DNA-I46ND35494615Le-I + RNA-I41ND29576315Le-II + DNA-II485940ND5015Le-II + RNA-II375828ND62a Denotes bases and backbone composition of antisense ODNs.b ND, not determined; —, no transition was detected. Open table in a new tab Absorption thermal denaturation was also used to compare the relative stabilities of hybrids formed by 15Le-I or 15Le-II with complementary DNA or RNA oligomers. Our results show that SBC ODNs with PO or PS backbones form more stable hybrids with DNA and RNA than the analogous NB ODNs (Table I). While the enhanced stability of SBC ODN-DNA hybrids (ΔTm = 11–14 °C) is probably attributable to the additional hydrogen bond present in 2-aminoadenine/thymine base pairs, the even greater stability of SBC ODN-RNA hybrids (ΔTm = 21–28 °C) requires further explanation. One possibility is that SBC ODNs favor the formation of an A-motif duplex with RNA. Indeed, the SBC/PO 15Le-II hybrid with RNA-II is similar in stability to the hybrid formed using an ODN with a 2′-O-methyl backbone, a combination that favors an A-type duplex (Table I). Electrophoretic mobility shift analysis was used to compare hybridization of NB and SBC ODNs to the RNA mini-exon hairpin. Fig. 3 shows representative autoradiographs from which K d values were extracted (Table II). NB 15Le-I and NB 15Le-II were poor binding agents, with the PS analogs worse than the PO analogs. Dissociation constants for 15Le-II were in the high micromolar range: 20 and 150 μm for NB/PO and NB/PS analogs, respectively. SBC versions of the same ODNs were much more effective binding agents. For example, the K d values of hybrids formed by PO or PS versions of SBC 15Le-II were about 50- or 200-fold smaller than the K d values of the corresponding complexes formed by NB 15Le-II ODNs. This agrees fairly well with theTm values obtained with complementary RNA (see above); SBC ODNs were better able to open the hairpin structure of the mini-exon. For comparison we evaluated the binding of a standard 16-mer used in a previous study (8Ramazeilles C. Mishra R.K. Moreau S. Pascolo E. Toulmé J.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7859-7863Crossref PubMed Scopus (38) Google Scholar); the PO and PS 16Le were characterized byK d of 0.7 and 5 μm, respectively.Table IIEquilibrium dissociation constants for the binding of NB and SBC antisense ODNs to the mini-exon RNA of L. amazonensisODNsK dNB/POSBC/PONB/PSSBC/PSμm15Le-I>>10.3>>20.215Le-II200.41500.815Le-I + 15Le-II40.52500.625Le200.25500.7 Open table in a new tab Binding of both 15Le-I and 15Le-II to RNA created a duplex twice as long as that formed by either ODN alone. The two SBC ODN-RNA hybrids had K d values of about 5 × 10−7m, while the NB ODN-RNA hybrids had K dvalues at least 10-fold higher. This is probably related both to the increased affinity of the SBC oligomers for RNA and to the reduced ability of SBC 15-mers to pair with each other, in comparison to NB ODNs. The partially self-complementary 25Le ODNs provided an opportunity to compare the binding efficiencies of single-stranded SBC ODNs to that of structured NB ODNs. In each instance, the SBC ODN was the most effective binding agent (about 70-fold reduction inK d). Hybrids that contained an SBC 25Le ODN were as stable as hybrids that contained paired SBC 15Le ODNs, indicating a minor contribution of duplex length (25 versus 30 base pairs) and molecularity of the hybridization reaction (bimolecularversus trimolecular). The NB and SBC ODNs with a PO backbone were tested for antisense activity against total mRNA from L. amazonensis in a cell-free translation system catalyzed by wheat germ extract. In this model system, SBC 25Le was the most effective antisense agent (Fig. 4). It elicited half-maximal inhibition at 0.08 μm (the C1/2 value). These parameters compare favorably to those of reference ODN 16Le (which has a PO backbone and contains NB bases), which has a C1/2 of about 1 μm. In repeated tests, NB 25Le failed to significantly inhibit translation (Fig. 4 D). The dramatic difference in functionality between the SBC and NB versions of 25Le reflects the physical binding properties of these two ODNs (Table II). The paired 15Le-I + 15Le-II ODNs were also effective antisense agents, and here again the SBC pair was more potent than the NB pair. C1/2 of 0.5 μm was determined for the SBC ODN pair. When each 15-mer was tested alone for antisense activity, potency was reduced and no advantage of SBC over NB ODNs was detected. None of the anti-mini-exon ODNs interfered with translation of brome mosaic virus RNA (<5% inhibition at 0.5 μm ODN). Conversely, inverted antisense ODNs (both NB and SBC) did not inhibit translation of L. amazonensis RNA (<5% inhibition at 1 μm ODN). Phosphorothioate NB and SBC antisense ODNs did not inhibit translation at concentrations below 0.5 μm (data not shown). Adsorption of these ODNs by proteins in the extract may explain why they failed to elicit an antisense effect. Following incubation with wheat germ extract, the PO SBC 25Le ODN ran identically to an untreated control on a nondenaturing polyacrylamide gel while the PS ODN ran as a slow moving smear attributable to the association with proteins from the extract (data not shown). Such binding has been reported by others for NB ODNs with a PS backbone (26Brown D.A. Kang S.H. Gryaznov S.M. De Dionisio L. Heidenreich O. Sullivan S. Xu X. Nerenberg M.I. J. Biol. Chem. 1994; 269: 26801-26805Abstract Full Text PDF PubMed Google Scholar). Moreover, upon binding to RNA, these PS sequences formed poorer substrates for RNase H than PO counterparts (see below). Two different mechanisms account for the inhibition of translation by antisense ODNs: RNase H-independent (translation arrest) and RNase H-dependent cleavage of target RNA (27Cazenave C. Loreau N. Thuong N.T. Toulmé J.J. Hélène C. Nucleic Acids Res. 1987; 15: 4717-4736Crossref PubMed Scopus (130) Google Scholar, 28Boiziau C. Kurfurst R. Cazenave C. Roig V. Thuong N.T. Toulmé J.J. Nucleic Acids Res. 1991; 19: 1113-1119Crossref PubMed Scopus (126) Google Scholar). To investigate the mechanism by which SBC ODNs inhibit L. amazonensis on RNA translation, we carried out translation in rabbit reticulocyte lysate, which has a low (if any) class I RNase H activity under translation conditions (29Cazenave C. Frank P. Büsen W. Biochimie. 1993; 75: 113-122Crossref PubMed Scopus (36) Google Scholar). In Fig. 4 F the effect of SBC 25Le (with a PO backbone) on translation was monitored both in the presence and absence of added E. coli RNase H. The results show that inhibition of protein synthesis in this medium occurs via an RNase H-dependent pathway. To confirm that
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