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. Melting curves were temperature(OC) obtained by increasing the temperature of at 500-,l samples FIG. 2. Transition curves obtained by measuring A258 as a a rate of 0.15°C/min. function of temperature in a buffer containing 10 mM sodium cacodylate (pH 6.0) and 100 mM NaCl. A slight excess of 29R-mer strand was used (1.8 AM) to ensure complete binding of the 18-mer RESULTS AND DISCUSSION (1.5 ,uM) and of the OLO (1.3 ,uM) (see Fig. 1 for abbreviations). Oligonucleotide clamps (Fig. 1) were synthesized with a Triplex formation ofthe 16-mer (1.2 ,IM) with a duplex solution at 1.5 Watson-Crick oligonucleotide 2 (or 3) nt longer than the ,uM concentration was also followed at 258 nm. The triple-helix- Hoogsteen part in order to create a triplex-duplex junction forming oligonucleotide (16-mer) and the 16L18-mer were substi- when both parts were tuted by psoralen at the 5' end, as described in Fig. 3. x, 29R-mer hydrogen-bonded to the target 16-nt plus 18-mer (mixture abbreviated as D below) (Tm = 480C); o, D plus oligopurine sequence (corresponding to the polypurine tract Pso-16-mer (Tm = 31°C and 48°C); A, 29R-mer plus Pso-16L18-mer ofHIV proviral DNA). An intercalator can insert its aromatic ("Tm" = 460C); *, 29R-mer + 16T319-mer (Tm = 380C and 50°C). ring at this junction (8, 10, 11). When two transitions are observed the first Tm value corresponds to Triple-Helix Formation on Single-Stranded DNA with an 50% dissociation of the Hoogsteen portion from the duplex; the OLO. Complex formation between the target 29R-mer and second Tm value corresponds to melting ofthe duplex. When a single the oligonucleotide clamps shown in Fig. 1 was followed by transition is observed an apparent "T." is given which corresponds measuring the temperature of to 50% of the optical transition. The melting curve of 29R-mer with dependence A258 (Fig. 2). Two 16L18-mer ("Tm" = 420C), as well as that of D plus unsubstituted transitions were observed when the 18-mer duplex obtained 16-mer (Tm = 20°C and 48°C) can be found in figure 2 of ref. 3. by mixing the 29R-mer target with the complementary 18-mer Oligonucleotides 16T418-mer and 16T518-mer gave melting curves was combined with the triple-helix-forming 16-mer oligonu- superimposable with that of 16L18-mer (data not shown). Downloaded by guest on September 28, 2021 Biochemistry: Giovannangeli et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10015

substituted 16-mer (Pso-16-mer; see Fig. 3 for nomenclature) a from its double-stranded target, while the other, occurring in e ( 50- CH2)6 POligo the upper temperature range (around 50°C), was observed in 0 the absence of the 16-mer and was attributed to thermal dissociation of the 18 Watson-Crick base pairs of the duplex, k- 334 0~ as described (10, 12). 9 20 5'1 1 .,1 3' When the single-stranded 29R-mer target was mixed with CCACTTTTT AAA} GAAAGGGGGGA CTGG 29R-mer either of the OLOs, the triplex-to-duplex transition was -TTTTTTCCCCCCTDoo shifted toward higher temperatures, as compared with the AA TTTTCTTTTCCCCCCT)LOOP 3' _ _ dissociation of the Hoogsteen portion observed when the 2 16W.C. 16-mer was not covalently linked to the 18-mer. A single m transition comprised this profile, whose hyperchromicity approximately equaled the sum of those for dissociation of A the 16-mer from the duplex and dissociation of the 18 B Watson-Crick base pairs. Therefore dissociation of both C Watson-Crick and Hoogsteen hydrogen bonds formed be- D tween the purine stretch of the 29R-mer target and the OLOs E occurred in the same temperature range. The same melting profile was obtained independent of the linker (hexaethylene m 20 glycol, T4, or T5) used to connect the 16-mer to the 18-mer. 9 A 1:1 OLO/target complex was formed as determined from titration experiments. 16H A dimeric oligonucleotide, with a 19-nt Watson-Crick 18w.c, portion and a 16-nt Hoogsteen portion, 16T319-mer, was 16w.dc tested; with the additional base at the 5' end of the Watson- FIG. 3. Photoinduced reactions of oligonucleotide-psoralen con- Crick portion (as compared with the 18-mer oligonucleotide) jugate with the 29R-mer. (Upper) Representation of Pso-16L18-mer the distance to the 3' end of the Hoogsteen portion became bound to its target 29R-mer with psoralen (rectangle) covalently minimal and a linker with three thymines was sufficient. This attached (chemically) to the 5'-end of Pso-16L18-mer. 5-Methylcy- 16T319-mer (Fig. 1) was tested for complex formation with tosine was introduced in place of cytosine in the Hoogsteen portion the 29-nt single-stranded target (29R-mer). The 16T319-mer to enhance triple helix stability (10). Two thymines at the triplex- formed 19 Watson-Crick base pairs as opposed to the 18 duplex boundary can undergo photoaddition reactions with psoralen (Pso). One is located on the 29R-mer, and the other on the Watson- formed by the 16T418- and 16T518-mers. The additional CG Crick portion of Pso-16L18-mer. The open rectangle refers to the base pair on the 3' side of the oligopurine target sequence psoralen ring attached via its C-S atom (represented at the center of stabilized the Watson-Crick duplex and facilitated separate the rectangle) to the 16-mers or 16L18-mer. As shown in the box the analysis of the dissociations of the two portions of the reactive 4'-5' and 3-4 double bonds of the psoralen ring are positioned 16T319-mer. Dissociation of the 16-nt Hoogsteen portion at the top and the bottom of the rectangle, respectively. (Lower) Time took place around 40°C, compared with 20°C for that corre- course of photoinduced reaction of Pso-16L18-mer. The 29R-mer (10 sponding to dissociation of the unsubstituted 16-mer from the nM) wasS'-labeled and incubated at 30°C in presence of 1.5,&M oligonucleotide-psoralen conjugate in 40 mM TrisHCl, pH 7.5/50 duplex (result not shown). mM NaCl/20 mM MgCl2. Irradiation was performed at 30°C with the Chemical Modification of the Target by Oligonucleotide- 150-W xenon lamp for 3, 10, 30, 90, or 210 sec (lanes A-E, Psoralen Conjugates. Psoralen attached to antisense oligonu- respectively) and samples were electrophoresed in a denaturing 10% cleotides was shown to enhance their biological activity, polyacrylamide gel (the top of the gel is to the right). The nature of following UV irradiation (13, 14). A psoralen derivative the photoproducts (m, monoadduct; b, bisadduct) is represented attached to the 5' end of antigene oligonucleotides, was below the gel. The scheme of the photoadducted products is not shown to induce efficient covalent crosslinking between the meant to represent the actual structure but only to represent the two strands of DNA (8, 11), provided that there was a oligonucleotides to which the psoralen ring is attached in the different 5'-TpA-3'/3'-ApT-5' step at the triplex-duplex photoproducts. Numbers refer to the length of oligonucleotides on junction. each side of the crosslinked thymines. On the 29R-mer the 4'-5' When Pso-16L18-mer was incubated and irradiated in the double bond of psoralen reacts with the 5-6 double bond of the presence of the labeled 29R-mer for increasing periods of thymine at position 9 from the 5' end; on the 18-mer W.C. portion the time, two slowly migrating species were observed upon 3-4 double bond reacts with the thymine at position 16 from the 5' end electrophoresis (Fig. 3). One slowly migrating species formed (see ref. 8). H. and W.C. refer to Hoogsteen and Watson-Crick at short irradiation times and was converted to the other interactions, respectively, with the 16-nt-long oligopurine sequence during longer irradiation periods. of the 29R-mer. The 29R-mer is represented by a thin line. Psoralen can form two types of monoadducts with thy- mine, involving either the 3-4 (pyrone) double bond or the The photochemical reactions of different oligonucleotide- 4'-5' (furan) double bond (15, 16) (see Fig. 3 for nomencla- psoralen conjugates were studied by using the 29R-mer DNA ture). Only the 4'-5' (furan) monoadduct can absorb light at target. Pso-16L18-mer achieved the highest percentage of wavelengths longer that 310 nm and form the resulting photoadducts with the labeled 29R-mer. At a concentration of bisadduct. Fig. 3 indicates that the slowest of the two slowly 1.5,uM the percentage of crosslinks reached a plateau above migrating bands corresponds to the monoadduct (m) formed 60%, compared with either 35% for the intermolecular triplex on the 29R-mer, involving the 4',5' double bond of psoralen. formed by the 29R-mer, 18-mer, and Pso-16-mer(p) (synthe- The more compact structure of the bisadduct (b) is probably sized with 5-methylcytosine) or 15% for the 29R-mer bound responsible for its slightly faster migration under denaturing to the complementary (antisense) oligomer, 16-mer-Pso(ap), conditions (Fig. 3). In this product, the psoralen is linked at under the same irradiation conditions. The antisense 16-mer- three different positions to three oligonucleotide sequences. Pso(ap) was synthesized in an antiparallel (ap) orientation It is chemically linked to the 16-mer Hoogsteen portion of the with respect to the 16-nt oligopurine sequence. Psoralen was OLO and photochemically adducted to the thymines on both attached to the 3' end of 16-mer-Pso(ap) so that it was brought the 29R-mer and the 18-mer Watson-Crick portion of the in close proximity to the same bases of the 29R-mer as those OLO. The oligonucleotide clamp is thus made irreversible. reached by psoralen linked to either the 5' end of the Downloaded by guest on September 28, 2021 10016 Biochemistry: Giovannangeli et al. Proc. Natl. Acad Sci. USA 90 (1993) Hoogsteen portion of the OLO or to the triplex-forming Watson-Crick complementary oligomer (18-mer) and Pso- 16-mer, Pso-16-mer(p), which binds in a parallel (p) orienta- 16-mer(p) forming Hoogsteen hydrogen bonds (Fig. 4, lane tion with respect to the 29R-mer. 3). The 18-mer formed a duplex with the strand of the Inhibition of Single-Stranded DNA Replication by OLO- denatured plasmid containing the 16-nt oligopurine target Psoralen Conjugates: In Vitro Experiments with T7 DNA Poly- sequence (Fig. 4). Upon addition of Pso-16-mer(p), a triple merase. Formation ofpsoralen photoproducts (15-17), as well helix was formed. After irradiation, psoralen was crosslinked as a stable triplex on a duplex target, has been shown to inhibit to the duplex-triplex junction, as described (8, 11). Elonga- transcription (18, 19). On a single-stranded nucleic acid, tri- tion of the 20R-mer primer stopped at two sites (stops 1 and plex formation might be expected to be more efficient in 2 in Fig. 4, lane 3). Stop 1 was identical to that observed with arresting biological processes than formation ofa double helix the antisense 16-mer-Pso(ap) bound to its complementary with an antisense oligonucleotide. To test this hypothesis, 16-nt oligopurine target sequence, except that a single band denatured, linearized pLTR plasmid (pLTRs.s.) was used as a was observed, instead oftwo bands as seen with the antisense target for binding by OLO-psoralen conjugates. The resulting oligonucleotide. This result reflects the higher selectivity of complexes were then irradiated, thus crosslinking OLO- the crosslinking reaction for the 5'-TpA-3' step located at the psoralen conjugates to the target strand. Primers were used to duplex-triplex junction, compared with the single-strand- initiate replication on either strand after linearization and duplex junction when the antisense oligomer is used. Stop 2 denaturation of the plasmid as described in Fig. 4. is 7-8 nt upstream from stop 1. The active site of the DNA With the antisense oligomer 16-mer-Pso(ap), bands corre- polymerase, at which chain elongation occurs, is 7-8 nt from sponding to chain termination (Fig. 4, lane 2) appeared at the the edge of the enzyme moving along the template (20, 21). bases preceding the thymines which had photoreacted on the So the simplest explanation is that DNA polymerase is template strand. This result indicates that psoralen monoad- physically arrested at the crosslinked psoralen during its ducts formed at this site prevented read-through by DNA progression along the template. The positions of DNA poly- polymerase. The most important photoadducts were ob- merase corresponding to stops 1 and 2 are schematically tained at the first two thymines on the 5' side of the oligopu- depicted in Fig. 4 (Center). rine stretch in the target. The hexamethylene linker between The 29R-mer can hybridize to the strand of the denatured psoralen and 16-mer(ap), as well as the dynamics of the plasmid which contains the 16-nt oligopyrimidine sequence single-stranded template, can explain the two sites of pho- (pLTRs.s. EcoRV) (Fig. 4). The binding of Pso-16-mer(p) to toadduct formation and so the different sites oftermination of this duplex was followed by photoinduced crosslinking of DNA synthesis observed in Fig. 4 (lane 2). Under the psoralen at the triplex-duplex junction. When the 20Y-mer conditions used in these studies, the polymerase efficiently primer was used, two efficient stops were observed during displaced the complementary antisense oligonucleotide when replication (Fig. 4, lane 6). These two stops have the same it was not crosslinked to the template strand, and full-length origin as those observed for the plasmid strand containing the replication products were observed. oligopurine sequence under the conditions described above. A "standard" (trimolecular) triplex was formed on single- DNA polymerase can be physically arrested when its active stranded linearized plasmid (pLTRs.s. Bsu36I), with the site reaches the psoralen adduct (stop 1) and when its

[LTRIs.s. PLTRs.s. (Bsu36 1) (EcoR V) Comllplele 1 2 6 +±A cloigauion 3 A 4 5 IBuO6 w- 110f)-r',, (1- FcoR V (8(0),)) X|-~ 142-pte'er IN 41 a- S T1O 1 (8713) 16

;, (77) --'~, 5 *

16 C dineI' 1 _,,A662] I(11 2_ .. r STOI' 2 *.

(86)26 A4 FE ~ mm..._ -ks roi'} 1 A4 bpurinej A4 -._ G6 (Ro'-7) L. .'~~~~~~~~~~ 5 '*

16 20Y-mer (idla 3)le t I'ol+ (I XT l : 61X (RI+-1.11 6r. _--STOP'3 ()X!~~~~~~C ti'oI+ dXI ' 2 1 _ 4 -, 5'*

FIG. 4. Inhibition of DNA synthesis by oligonucleotide-psoralen conjugates. Alkaline-denatured linear pLTR was incubated at 30°C in 40 mM Tris HCl, pH 7.5/50 mM NaCl/20 mM MgCl2 in the absence (lane 1) or presence of 16-mer-Pso(ap) (lane 2), 18-mer plus Pso-16-mer(p) (lane 3), Pso-16L18-mer with (lane 4) or without (lane 5) irradiation, and 29R-mer plus Pso-16-mer(p) (lane 6). 5-Methylcytosine was introduced in place of cytosine in the Hoogsteen portion. After irradiation (30°C, 150-W xenon lamp) for 5 min (lanes 1-4 and 6; in lane 5 the sample was not irradiated), elongation was performed in standard conditions (see Materials and Methods). In lanes 1-5 the strand containing the 16-nt oligopurine sequence was used as a template (Left). In lane 6 (Right) the strand containing the 16-nt oligopyrimidine sequence was used as a template. Sequences were also obtained by primer extension, and lanes A and G + A correspond to an elongation experiment performed in the presence of ddTTP and of ddCTP plus ddTTP, respectively. Samples were analyzed in a denaturing 8% polyacrylamide gel. The mechanisms for site-specific arrest of DNA synthesis are schematically represented (Center). In the upper two schemes, the Watson-Crick and Hoogsteen parts are not connected by a loop. They correspond to the results shown in lane 3. The same stops are observed when the Watson-Crick and Hoogsteen parts are linked to each other (lane 4). Pol, polymerase. Downloaded by guest on September 28, 2021 Biochemistry: Giovannangeli et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10017 forefront contacts the crosslink, but its active site is 7-8 nt with the antisense oligomer. The efficacy ofreplication arrest upstream (stop 2). was strongly enhanced when an OLO-psoralen conjugate The intensity of stop 1 is higher than that of stop 2 on both was crosslinked to its target site. The OLO-psoralen conju- strands of the denatured plasmid (Fig. 4, lanes 3 and 6). This gate was much more efficient than the corresponding an- result indicates that the front of DNA polymerase can effi- tisense oligonucleotide-psoralen conjugate. ciently move across the crosslinked psoralen until its active Antisense oligonucleotides have been successfully used to site is facing the psoralen adduct, which prevents incorpo- bind complementary sequences of messenger or viral RNAs, ration of any deoxynucleotide into the growing chain. thereby inhibiting the biological function of the latter (1). It A triplex was then formed with the oligonucleotide clamp is possible to form triple helices involving an oligopurine Pso-16L18-mer on the top strand of the plasmid containing RNA sequence as one of the Watson-Crick strands (22) and the oligopurine target sequence. After irradiation, the two to bind an oligopyrimidine RNA sequence to a double helix previously described stops were present, but a third stop (23, 24). Formation of a triple-stranded structure on a single- appeared outside the triplex site, 12-13 nt upstream from the stranded template might prove to be more efficient in block- 3' side of the purine stretch in the template (Fig. 4, lane 4). ing translation, splicing, and reverse transcription than du- This new termination site (stop 3) is consistent with a physical plex formation via an antisense arrest of DNA polymerase by the loop of the triple-stranded oligomer. complex as depicted in Fig. 4 Center. When the polymerase We thank Dr G. Duval-Valentin for helpful discussions. This work is arrested at this position, chain elongation should stop 7-8 was supported in part by Rh6ne-Poulenc-Rorer, by the Ligue Na- nt upstream from the site ofarrest (as observed for stop 2 with tionale Francaise contre le Cancer, and by the Agence Nationale de respect to stop 1). The four additional bases accounting for Recherche contre le SIDA. the position of the arrest site may be explained by the steric effect of the linker (loop) used to tether the two oligonucle- 1. Helene, C. & Toulme, J. J. (1990) Biochim. Biophys. Acta 1049, otides in Pso-16L18-mer. 99-125. The concentration of oligonucleotide-psoralen conjugate 2. Helene, C. (1991) Anti-Cancer Drug Design 6, 569-584. which, after irradiation, blocked 50% of the elongation by 3. Giovannangeli, C., Montenay-Garestier, T., Rougee, M., Chas- DNA polymerase was determined from densitometric anal- signol, M., Thuong, N. T. & Helene, C. (1991) J. Am. Chem. ysis of the autoradiograms: S ,uM was necessary with the Soc. 113, 7775-7777. antisense oligonucleotide 16-mer-Pso (ap), whereas 0.1 4. Xodo, L. E., Manzani, G. & Quadrifoglio, F. (1990) Nucleic AM Acids Res. 18, 3557-3564. was sufficient with Pso-16L18-mer. This difference reflects 5. Kool, E. T. (1991) J. Am. Chem. Soc. 113, 6265-6266. the enhanced thermodynamic stability of the 16L18-mer 6. Prakash, G. & Kool, E. T. (1992) J. Am. Chem. Soc. 114, triple-stranded complex and the efficiency of psoralen pho- 3523-3527. toaddition in producing mono- and bisadducts, which de- 7. Durand, M., Chevrier, M., Chassignol, M., Thuong, N. T. & pends in turn on the stability and stereochemistry of the Maurizot, J. C. (1990) Nucleic Acids Res. 18, 6353-6359. complexes. 8. Takasugi, M., Guendouz, A., Chassignol, M., Decout, J. L., Moreover, in contrast to the unmodified OLO, the OLO- Lhomme, J., Thuong, N. T. & Helene, C. (1991) Proc. Natl. psoralen conjugate, even without irradiation, was able to Acad. Sci. USA 88, 5602-5606. form a sufficiently strong, noncovalent complex to arrest the 9. Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S. & Alizon, M. DNA polymerase (Fig. 4, lane 5). Intercalation ofpsoralen at (1985) Cell 40, 9-17. 10. Sun, J. S., Franqois, J. C., Montenay-Garestier, T., Saison- the duplex-triplex junction was efficient enough to block the Behmoaras, T., Roig, V., Thuong, N. T. & Helene, C. (1989) progression ofthe enzyme even in the absence ofphotoprod- Proc. Natl. Acad. Sci. USA 86, 9198-9202. ucts with thymine bases. Only stop 2 was observed, in 11. Giovannangeli, C., Thuong, N. T. & Helne, C. (1992) Nucleic agreement with the physical arrest of the front of the enzyme Acids Res. 16, 4275-4281. at the strong duplex-triplex intercalation site. Stop 1 was not 12. Sun, J. S., Giovannangeli, C., Franqois, J. C., Kurfurst, R., observed, since there was no chemically modified base to Montenay-Garestier, T., Asseline, U., Saison-Behmoaras, T., inhibit deoxynucleotide incorporation at the active site ofthe Thuong, N. T. & Helene, C. (1991) Proc. Natl. Acad. Sci. USA enzyme. 88, 6023-6027. Conclusion. This study shows that it is possible to 13. Kulka, M., Smith, C. C., Aurelian, L., Meade, K., Miller, P. design & Ts'o, P. 0. P. (1989) Proc. Natl. Acad. Sci. USA 86, oligonucleotides capable of forming both Watson-Crick and 6868-6872. Hoogsteen hydrogen bonds with a single-stranded nucleic 14. Chang, E. H., Miller, P. S., Cushman, C., Devadas, K., Pirolo, acid containing an oligopurine sequence. These oligonucle- K. F., Ts'o, P. 0. P. & Yu, Z. P. (1991) Biochemistry 30, otide clamps have higher binding affinities than do standard 8283-8286. antisense Watson-Crick oligomers. These properties may 15. Shi, Y., Gamper, H. & Hearst, J. E. (1987) Nucleic Acids Res. prove useful in the design of more efficient ligands of single- 15, 6843-6854. stranded nucleic acids. 16. Shi, Y., Gamper, H., Van Houten, B. & Hearst, J. E. (1988) J. Circular oligonucleotides have been described (5, 6) which Mol. Biol. 199, 277-293. can bind to an more 17. Sastry, S. S. & Hearst, J. E. (1991) J. Mol. Biol. 221, 1091- oligopurine sequence strongly than 1110. OLOs, probably for entropic reasons. However, one of the 18. Young, S. L., Krawczyc, S. H., Matteucci, M. D. & Toole, advantages of OLOs, besides the ease of synthesis, is that J. J. (1991) Proc. Natl. Acad. Sci. USA 88, 10023-10026. reactive groups may be attached to either end. The Watson- 19. Duval-Valentin, G., Thuong, N. T. & Helene, C. (1992) Proc. Crick part of the OLO can be extended by one, two, or more Natl. Acad. Sci. USA 89, 504-508. base pairs in order to create a site for an intercalating agent 20. Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G. & Steitz, attached to the 5' end of its Hoogsteen part. Triple-helix T. A. (1985) Nature (London) 313, 762-766. formation with an OLO-intercalator conjugate on a single- 21. Joyce, C. M., Ollis, D. L., Rush, J., Steitz, T. A., Konigsberg, stranded target was sufficient to arrest DNA replication in the W. H. & Grindley, N. D. F. (1986) UCLA Symposia on Mo- absence of any irreversible reaction. lecular and Cellular Biology, ed. Oxender, D. (Liss, New 5' York), pp. 197-205. Attachment of a psoralen moiety to the end of an OLO 22. Roberts, R. W. & Crothers, D. (1992) Science 258, 1463-1466. improves binding and brings the photoreactive agent into an 23. Shimizu, M., Konishi, A., Shimada, Y., Inoue, H. & Ohtsuka, appropriate position for photoadduct formation with both E. (1992) FEBS Lett. 302, 155-158. strands ofthe Watson-Crick part. Formation ofthe bisadduct 24. Escude, C., Sun, J. S., Rougee, M., Montenay-Garestier, T. & was much more efficient than formation of the monoadduct HdlEne, C. (1992) C.R. Acad. Sci. Paris Serie III 315, 521-525. Downloaded by guest on September 28, 2021