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Oligonucleotide Clamps Arrest DNA Synthesis on a Single-Stranded DNA Target (Triplex/Psoralen/Replication) CARINE GIOVANNANGELI*, NGUYEN T

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Oligonucleotide Clamps Arrest DNA Synthesis on a Single-Stranded DNA Target (Triplex/Psoralen/Replication) CARINE GIOVANNANGELI*, NGUYEN T Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10013-10017, November 1993 Biochemistry Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target (triplex/psoralen/replication) CARINE GIOVANNANGELI*, NGUYEN T. THUONGt, AND CLAUDE HtLtNE* *Laboratoire de Biophysique, Institut National de la Sante et de la Recherche Mddicale Unite 201, Centre National de la Recherche Scientifique Unit6 Associde 481, Museum National d'Histoire Naturelle, 43, Rue Cuvier, 75231 Paris Cedex 05, France; and tCentre de Biophysique Moldculaire, 45071 Orleans Cedex 02, France Communicated by I. Tinoco, June 1, 1993 ABSTRACT Triple helices can be formed on single- bound to a polypurine sequence (5, 6). Here we show that stranded oligopurine target sequences by composite oligonu- different linkers can be used to tether the Watson-Crick and cleotides consisting oftwo oligonucleotides covalently linked by Hoogsteen base-pair-forming portions of the OLO without either a hexaethylene glycol linker or an oligonucleotide se- loss of stability of the resultant triple helix. A psoralen quence. The first oligomer forms Watson-Crick base pairs with derivative attached to the 5' end of the OLO may be the target, while the second oligomer engages in Hoogsteen base crosslinked to its target sequence in such a way that the three pairing, thereby acting as a molecular clamp. The triple-helical strands of the resultant triplex become covalently linked to complex formed by such an oligonucleotide clamp, or "oligo- one another. These complexes act as strong stop signals, nucleotide4oop-oligonucleotide" (OLO), is more stable than blocking chain elongation during replication. This inhibition either the corresponding trimolecular triple helix or the double is much more efficient than that obtained with an antisense helix formed upon binding of the oligopyrimidine complement oligonucleotide carrying a reactive psoralen group. to the same oligopurine target. Attaching a psoralen derivative to the 5' end of the OLO allowed us to photoinduce a covalent MATERIALS AND METHODS linkage to the target sequence. The psoralen moiety became Oligonucleotide Synthesis. The unmodified oligonucleo- covalently linked to all three portions of the triplex, thereby tides were obtained from the Pasteur Institute and purified by making the oligonucleotide clamp irreversible. These crosslink- reverse-phase HPLC. The 16L18-mer OLO (see sequence in ing reactions introduced strong stop signals during DNA rep- Fig. 1) was synthesized on a Pharmacia automated synthe- lication, as shown on a plasmid containing a portion of the HIV sizer using phosphoramidite chemistry (3, 7). The psoralen- proviral sequence ofhuman immunodeficiency virus. A 16-mer substituted oligopyrimidines Pso-16-mer(p) and Pso-16L18- oligopurine sequence corresponding to the "polypurine tract" mer were synthesized from 5-(F-iodohexyloxy)psoralen and of human immunodeficiency virus was chosen as a target for a the corresponding unsubstituted oligomer carrying a 5'- psoralen-OLO conjugate. Three different stop signals for DNA thiophosphate group (8). polymerase were observed, corresponding to different sites of Plasmid Construction. The plasmid pLTR (a gift from the polymerase arrest on its template. Even in the absence of late H. Hirel, Rhone-Poulenc-Rorer) was constructed by photoinduced crosslinking, the psoralen-OLO coijugate was insertion of human immunodeficiency virus (HIV) BRUCG able to arrest DNA replication. The formation of triple-helical provirus restriction fragments (BamHI-HindIII and HindIlI- structures on single-stranded targets may provide an alterna- Cla I) into pBR328 by standard procedures. pLTR contains tive to the antisense strategy for the control of gene expression. 1440 bp of HIV proviral DNA carrying a 16-bp oligopurine-oligopyrimidine sequence. The HIV genome con- Oligonucleotides have been used to inhibit the biological tains two repeats of the 16-nt oligopurine sequence 5'- activity of RNA molecules in the so-called "antisense" AAAAGAAAAGGGGGGA-3'. One oligopurine stretch is strategy (for review, see ref. 1). In this strategy, an oligonu- present on the 5' side of the U3 sequence, within the nefgene cleotide binds to a complementary RNA sequence and in- (the so-called polypurine tract, positions 8662-8677 in HIV hibits protein synthesis or viral RNA replication. Oligonu- BRUCG or 3526-3541 in pLTR), and the second, which is cleotides can also bind to the major groove of double- absent from pLTR, is located in the 3' region of the pol gene stranded DNA, thereby forming triple helices. They are thus (positions 4367-4382 in HIV BRUCG) (9). able to inhibit replication or transcription of specific genes, in Irradiation Studies. Two targets, a 29R-mer single-stranded what is termed the "antigene" strategy (for review, see ref. oligonucleotide and a double-stranded fragment (abbreviated 2). In both the antisense and antigene strategies, covalent D) consisting of the 29R-mer plus a complementary 18-mer attachment of an activatable reagent at one or both end(s) of (see sequences in Figs. 1 and 3), were used as substrates for the oligonucleotide allows irreversible reactions to occur at psoralen-induced photo-crosslinking. The 29R-mer was pu- the target site (1, 2). rified by gel electrophoresis and 5'-end labeled with We previously showed that a dimeric oligonucleotide ("oli- [y-32P]ATP and polynucleotide kinase (Ozyme). The dena- gonucleotide-loop-oligonucleotide," or OLO) displayed tured linearized plasmid (pLTRs.s.) was also chosen as a strong affinity for single-stranded DNA by forming both target to form psoralen photoadducts. A Xenon lamp (150 W) Watson-Crick and Hoogsteen hydrogen bonds with a single- in a Cunow housing system provided the light source. The stranded oligopurine target (3). A 24-mer pyrimidine oligo- light was filtered through a Pyrex filter in water to remove nucleotide was also shown to bind an 11-mer purine oligo- radiation below 310 nm. Electrophoresis was carried out in nucleotide by forming a triplex structure (4). Circular oligo- either 10% or 8% polyacrylamide gels containing 7 M urea. nucleotides can also form triple-helical structures when Quantification of gel autoradiograms was carried out by densitometry. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HIV, human immunodeficiency virus; OLO, oligo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. nucleotide-loop-oligonucleotide. 10013 Downloaded by guest on September 28, 2021 10014 Biochemistry: Giovannangeli et al. Proc. Natl. Acad. Sci. USA 90 (1993) Hoogsteen Portion 1....... Loop r'lll 111111111 lLIII IIIIII II1I1111 Watson-Crick Portion Intercalator 29R-mer CCACTTTTT AAAAGAAAAGGGGGGA CTGG 5' 3' Oligonucleotide Hoogsteen Portion Linker Watson-Crick Portion 5' 3' 18-mer T C6T4 CT4 A2 16-mer T4 C T4 C6 T 16 L 18-mer T4 C T4 C6 T -O-(CH2-CH2-0)6- T C6 T4 C T4 A, 16 Ts 18-mer T4CT4C6T TTTTT TC6T4 CT4 A- 16 T4 18-mer T4CT4C6T TTTT TC6T4 CT4A, 16 T3 19-merI T4CT4C6T TTT GTC6T4 CT4A2 FIG. 1. (Upper) Representation of the triple helix formed on a single-stranded nucleic acid containing an oligopurine stretch with an oligonucleotide clamp formed by a Watson-Crick portion and a Hoogsteen portion linked together (OLO). The nature of the linker is indicated in Lower. The scheme at left represents an OLO whose Watson-Crick and Hoogsteen parts have the same length. The scheme at right shows an intercalator-OLO conjugate whose Watson-Crick part is two bases longer than the Hoogsteen part to allow for intercalation at the triplex-duplex junction. The intercalator is covalently attached to the 5' end of the Hoogsteen part. (Lower) Sequence of the 29R-mer single-stranded DNA fragment used as a substrate for binding of the oligonucleotides shown below. The 16-nt oligopurine target sequence is indicated by larger letters. Various composite oligonucleotides consisting of two portions were synthesized. The first portion is either 18 or 19 nt long and can form Watson-Crick hydrogen bonds (Watson-Crick portion), while the second is 16 nt long and can form Hoogsteen hydrogen bonds (Hoogsteen portion) with the oligopurine target sequence. Different linkers were used: a hexa(ethylene glycol) linker or a stretch of n thymines (n = 3, 4, or 5). Two separate oligonucleotides corresponding to each of the two portions (18-mer for the Watson-Crick portion and 16-mer for the Hoogsteen portion) were used as controls. Replication Experiments. pLTR was digested with Bsu36I cleotide. The transition in the lower temperature range orEcoRV and denatured with 0.2 M NaOH for 15 min at 30°C (around 30°C) reflects dissociation of the psoralen- to form pLTRs.s. The denatured plasmid (10 nM) was incubated at 30°C in a 40 mM Tris-HCl, pH 7.5/50 mM 1.1 NaCl/20 mM MgCl2 in the presence of a 5'-32P-labeled primer. Two primers were used to replicate each of the two U000000 strands. Primer locations are indicated in Fig. 4. This incu- 1.0 EWE 0AAAA bation was carried out in either the absence or the presence EU AA, of oligonucleotides able to form a complex with the 16-nt 0.9 A target sequence. Some of the samples were irradiated, while o 0000 AA A- others were not. Dithiothreitol (4 mM) and T7 DNA poly- 00 merase 0) (Sequenase version 2.0, 0.13 unit/,ul; United States co 0.8 00 Biochemical) were then added and synthesis was initiated by 00 00 A,&AA addition of37.5 ,tM dNTPs. The reactions were stopped after ..00AA0S 50 min by addition of EDTA (50 mM). Sequence analysis 0.7- made use ofthe same primers, and elongation was carried out in the presence of A &I dNTPs and one ddNTP to allow for chain u .bi . -. termination. c 10 20 30 40 50 60 Spectroscopic Methods. Absorption spectra were recorded on a Uvikon 820 spectrophotometer.
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