Proc. Natl. Acad. Sci. USA
Vol. 88, pp. 10023-10026, November 1991 Biochemistry
Triple helix formation inhibits transcription elongation in vitro SHARON L. YOUNG, STEVEN H. KRAWCZYK, MARK D. MATrEUCCI,
AND
JOHN J. TOOLEt
Gilead Sciences, Inc., 346 Lakeside Drive, Foster City, CA 94404
Communicated by Harold Weintraub, August 9, 1991 (received for review July 5, 1991)
thereby affords more potential targets, our goal was to determine whether ODNs designed to form a triple helix can disrupt the elongation ofeukaryotic transcription in vitro. We have found that regions of triple helix cause stalling of the RNA polymerase II transcription complex; when covalently formed, triple helix causes a stable block to transcription.
ABSTRACT We have identified a 15-nucleotide site within a G-free transcription cassette that forms triple helix with sequence-specific oligodeoxyribonucleotides. When oligodeoxynucleotides were added to template DNA prior to in vitro transcription, a significant fraction of transcripts were truncated at a site corresponding to the region of triple helix formation. Kinetic analysis of the transcription products demonstrated that these truncated transcripts could be elongated to full length upon prolonged incubation. When an alkylating base was incorporated into the oligodeoxynucleotide to form covalent triple helix, most of the transcripts remained truncated. We conclude that triple helix formation can stall or, in the case of covalent crosslinking, can block RNA polymerase II and thus may provide a method for the specific inhibition of gene expression.
MATERIALS AND METHODS Reagents. Sonicated salmon sperm DNA, dimethyl sulfate (DMS), pyrrolidine, spermine, and acid-washed glass beads (200 ,um) were purchased from Sigma. The Klenow fragment of DNA polymerase I and the endonuclease Sma I were purchased from Boehringer Mannheim. ODNs were synthesized on a DNA synthesizer (model 8750, MilliGen/ Biosearch, Novato, CA) using H-phosphonate chemistry. Radioisotopes were purchased from Amersham. 3'-OMethyl-GTP, rNTPs, and Sephadex G-50 medium were purchased from Pharmacia. The T cells (Jurkat cell line) and the G-free cassette plasmid were generously provided by Gerry Crabtree (Stanford University). DMS Footprint Assay. The coding strand of a 400-base-pair DNA fragment from the G-free cassette was radiolabeled using [a-32P]dGTP as substrate for the Klenow fragment of DNA polymerase I. Approximately 4 nM radiolabeled DNA was annealed to various concentrations of 6-methyl-8-oxo2'-deoxyadenosine (MODA)/thymidine ODNs in 50 mM KCI/5 mM MgCl2/1 mM spermine/50 mM Hepes, pH 7.2, containing salmon sperm DNA (50 ,g/ml) at 23°C for 30 min. Samples were then reacted with DMS and analyzed by autoradiography as described by Maxam and Gilbert (13). In Vitro Transcription. G-free cassette DNA was linearized using Sma I and annealed with ODNs as described for the DMS footprint assay. After annealing, 500 ng of the G-free DNA was used as template in an in vitro transcription reaction using human T-cell (Jurkat) extracts prepared as described by Gorski et al. (14). Transcription reactions were performed as described by Sawadogo and Roeder (15), and the products were visualized by autoradiography after electrophoresis through a 6% polyacrylamide gel containing 90 mM Tris borate pH 8.3/8.3 M urea/2.5 mM EDTA. For pulse-chase experiments, linear G-free cassette DNA was annealed with triple helix-forming ODN (10 ,uM) and then used to direct in vitro transcription as described above. After the transcription had proceeded for 15 min, UTP was added to a final concentration of 1 mM, and the reaction mixtures were incubated an additional 15 or 30 min. The RNA products were analyzed as described above. Triple-Helix-Mediated Alkylation. The template strand of the G-free cassette was annealed with N4, N4-ethano-2'deoxycytidine (C*)-containing ODN (10 ,uM) in 50 mM KCI/5
Oligodeoxyribonucleotides (ODNs) can form a sequencespecific triple helix by hydrogen bonding to polypurine tracts within duplex DNA. Two structural motifs by which these triple helices form have been described. The more widely studied motif is based on Hoogsteen hydrogen bonding of a pyrimidine oligonucleotide wherein T recognizes A-T base pairs and protonated C (C+) recognizes G-C base pairs (1-6). The ODNs bind in the major groove of DNA such that the pyrimidine ODN is parallel to the purine strand of the duplex. Because of the requirement for cytosine to be protonated, ODNs containing C form a triple helix in a pH-dependent manner since the pKa of cytosine is 4.5 (7-9). Evidence for another structural class of triple helix was described by Cooney et al. (10), who showed that G-rich ODNs can specifically bind to polypurine tracts of duplex DNA wherein G recognizes G-C base pairs and A recognizes A-T base pairs. Although these ODNs were originally proposed to bind in a parallel manner, subsequent work by Beal and Dervan (11) has demonstrated that third-strand binding occurs in an antiparallel manner, and furthermore, T could also recognize A-T base pairs. Our interest in triple helices has focused on the potential biological applications of these structures. Maher et al. (12) utilized triple helix formation to site-specifically inhibit restriction enzyme cleavage of duplex DNA when the triple helix partially overlapped the restriction site. In addition, they showed that the eukaryotic transcription factor Spl was inhibited from binding to DNA when a triple helix region overlapped part of the Spl recognition site. Cooney et al. (10) demonstrated the repression of c-myc transcription in vitro using a G-rich ODN that specifically formed a triple helix within a region of the c-myc P1 promoter. Subsequently, Postel et al.t reported that these same triple helix-forming ODNs could inhibit transcription from the c-myc promoter, specifically and up to 90%o, by direct addition to HeLa cells in culture. Since the transcribed region is generally much larger than the associated regulatory sequences of a given gene, and
Abbreviations: ODN, oligodeoxyribonucleotide; DMS, dimethyl sulfate; MODA, 6-methyl-8-oxo-2'-deoxyadenosine; C*, N4,N4ethano-2'-deoxycytidine; nt, nucleotide(s). tTo whom reprint requests should be addressed. tPostel, E. H., Flint, S. J. & Hogan, M. E., Paper Presentation, 19th Annual Symposium on Molecular and Cellular Biology, January 27-February 3, 1990, Keystone, CO.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10023
10024
Biochemistry: Young et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
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Vol. 88, pp. 10023-10026, November 1991 Biochemistry
Triple helix formation inhibits transcription elongation in vitro SHARON L. YOUNG, STEVEN H. KRAWCZYK, MARK D. MATrEUCCI,
AND
JOHN J. TOOLEt
Gilead Sciences, Inc., 346 Lakeside Drive, Foster City, CA 94404
Communicated by Harold Weintraub, August 9, 1991 (received for review July 5, 1991)
thereby affords more potential targets, our goal was to determine whether ODNs designed to form a triple helix can disrupt the elongation ofeukaryotic transcription in vitro. We have found that regions of triple helix cause stalling of the RNA polymerase II transcription complex; when covalently formed, triple helix causes a stable block to transcription.
ABSTRACT We have identified a 15-nucleotide site within a G-free transcription cassette that forms triple helix with sequence-specific oligodeoxyribonucleotides. When oligodeoxynucleotides were added to template DNA prior to in vitro transcription, a significant fraction of transcripts were truncated at a site corresponding to the region of triple helix formation. Kinetic analysis of the transcription products demonstrated that these truncated transcripts could be elongated to full length upon prolonged incubation. When an alkylating base was incorporated into the oligodeoxynucleotide to form covalent triple helix, most of the transcripts remained truncated. We conclude that triple helix formation can stall or, in the case of covalent crosslinking, can block RNA polymerase II and thus may provide a method for the specific inhibition of gene expression.
MATERIALS AND METHODS Reagents. Sonicated salmon sperm DNA, dimethyl sulfate (DMS), pyrrolidine, spermine, and acid-washed glass beads (200 ,um) were purchased from Sigma. The Klenow fragment of DNA polymerase I and the endonuclease Sma I were purchased from Boehringer Mannheim. ODNs were synthesized on a DNA synthesizer (model 8750, MilliGen/ Biosearch, Novato, CA) using H-phosphonate chemistry. Radioisotopes were purchased from Amersham. 3'-OMethyl-GTP, rNTPs, and Sephadex G-50 medium were purchased from Pharmacia. The T cells (Jurkat cell line) and the G-free cassette plasmid were generously provided by Gerry Crabtree (Stanford University). DMS Footprint Assay. The coding strand of a 400-base-pair DNA fragment from the G-free cassette was radiolabeled using [a-32P]dGTP as substrate for the Klenow fragment of DNA polymerase I. Approximately 4 nM radiolabeled DNA was annealed to various concentrations of 6-methyl-8-oxo2'-deoxyadenosine (MODA)/thymidine ODNs in 50 mM KCI/5 mM MgCl2/1 mM spermine/50 mM Hepes, pH 7.2, containing salmon sperm DNA (50 ,g/ml) at 23°C for 30 min. Samples were then reacted with DMS and analyzed by autoradiography as described by Maxam and Gilbert (13). In Vitro Transcription. G-free cassette DNA was linearized using Sma I and annealed with ODNs as described for the DMS footprint assay. After annealing, 500 ng of the G-free DNA was used as template in an in vitro transcription reaction using human T-cell (Jurkat) extracts prepared as described by Gorski et al. (14). Transcription reactions were performed as described by Sawadogo and Roeder (15), and the products were visualized by autoradiography after electrophoresis through a 6% polyacrylamide gel containing 90 mM Tris borate pH 8.3/8.3 M urea/2.5 mM EDTA. For pulse-chase experiments, linear G-free cassette DNA was annealed with triple helix-forming ODN (10 ,uM) and then used to direct in vitro transcription as described above. After the transcription had proceeded for 15 min, UTP was added to a final concentration of 1 mM, and the reaction mixtures were incubated an additional 15 or 30 min. The RNA products were analyzed as described above. Triple-Helix-Mediated Alkylation. The template strand of the G-free cassette was annealed with N4, N4-ethano-2'deoxycytidine (C*)-containing ODN (10 ,uM) in 50 mM KCI/5
Oligodeoxyribonucleotides (ODNs) can form a sequencespecific triple helix by hydrogen bonding to polypurine tracts within duplex DNA. Two structural motifs by which these triple helices form have been described. The more widely studied motif is based on Hoogsteen hydrogen bonding of a pyrimidine oligonucleotide wherein T recognizes A-T base pairs and protonated C (C+) recognizes G-C base pairs (1-6). The ODNs bind in the major groove of DNA such that the pyrimidine ODN is parallel to the purine strand of the duplex. Because of the requirement for cytosine to be protonated, ODNs containing C form a triple helix in a pH-dependent manner since the pKa of cytosine is 4.5 (7-9). Evidence for another structural class of triple helix was described by Cooney et al. (10), who showed that G-rich ODNs can specifically bind to polypurine tracts of duplex DNA wherein G recognizes G-C base pairs and A recognizes A-T base pairs. Although these ODNs were originally proposed to bind in a parallel manner, subsequent work by Beal and Dervan (11) has demonstrated that third-strand binding occurs in an antiparallel manner, and furthermore, T could also recognize A-T base pairs. Our interest in triple helices has focused on the potential biological applications of these structures. Maher et al. (12) utilized triple helix formation to site-specifically inhibit restriction enzyme cleavage of duplex DNA when the triple helix partially overlapped the restriction site. In addition, they showed that the eukaryotic transcription factor Spl was inhibited from binding to DNA when a triple helix region overlapped part of the Spl recognition site. Cooney et al. (10) demonstrated the repression of c-myc transcription in vitro using a G-rich ODN that specifically formed a triple helix within a region of the c-myc P1 promoter. Subsequently, Postel et al.t reported that these same triple helix-forming ODNs could inhibit transcription from the c-myc promoter, specifically and up to 90%o, by direct addition to HeLa cells in culture. Since the transcribed region is generally much larger than the associated regulatory sequences of a given gene, and
Abbreviations: ODN, oligodeoxyribonucleotide; DMS, dimethyl sulfate; MODA, 6-methyl-8-oxo-2'-deoxyadenosine; C*, N4,N4ethano-2'-deoxycytidine; nt, nucleotide(s). tTo whom reprint requests should be addressed. tPostel, E. H., Flint, S. J. & Hogan, M. E., Paper Presentation, 19th Annual Symposium on Molecular and Cellular Biology, January 27-February 3, 1990, Keystone, CO.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10023
10024
Biochemistry: Young et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
a
a MO0DA
W-0~
, r
4 "o
