Oligonucleotide-directed triple-helix formation

Oligonucleotide-directed

triple-helix

formation

Jian-Sheng Sun and Claude Hkkne Mu&urn

National d’Histoire Naturelle, Paris, France

Oligonucleotide-directed triple-helix formation has introduced new areas of investigation, including the specific control of gene expression, the development of site-specific artificial nucleases and new diagnostic procedures. During the past year, new developments in the structural analysis of triple-helical complexes have appeared. NMR and vibrational spectroscopies together with molecular dynamics and energy-minimization calculations have led to a model of triple-helical DNA which is markedly different from that deduced from previous fiber-diffraction data on homopolymers. Replacement of deoxyribose by ribose or its 2’-0-alkyl derivatives in one or several of the three strands has dramatic effects on triplex stability. New backbones and non-natural bases have been designed to improve triple-helix stability and extend the range of recognition sequences for oligonucleotide binding to double-helical DNA. These recent advances raise new possibilities for the development of biochemical and pharmacological applications based on oligonucleotide-directed triple-helix formation.

Current Opinion

in Structural

Introduction

represent a unique class of ligands that recognize the major groove of double-helical DNA by forming hydrogen bonds with Watson-Crick base pairs. Although triple helices were described as early as 1957 [l], it is only recently that oligonucleotidedirected triple-helix formation has been developed as one of the most versatile approaches to the sequencespecific recognition of double-helical DNA j2-41 (for recent reviews, see [ 5-71). Target sequenceswere originally limited to polypurinepolypyrimidine sequencesof DNA, but recent experiments have shown that a much broader range of DNA sequencescan be recognized by oligonucleotides forming a triple helix. This approach to DNA recognition has introduced new areas of investigations aimed at controlling transcription of a single gene within a living organism and at cleaving a single site within human chromosomes. This review will highlight some of the past year’s developments in the field of oligonucleotide-directed triple-helix formation, which were aimed at designing new oligonucleotide derivatives that could bind tightly and selectively to DNA base sequences,at understanding the structural aspectsof triplehelical complexes, and at developing their applications in both sequence-specificcleavageand the control of gene transcription.

Biology 1993, 3:345-356

Structure of triple-helical natural bases

complexes

involving

Oligonucleotides

Three classesof triple helices have been described for oligonucleotides containing only naturai nucleotide units. They differ according to the nucleotide composition of the triple-helix-forming oligonucleotide which binds to a polypurine~polypyrimidine sequence of double-helical DNA All three classes involve Hoogsteen- or reverse Hoogsteen-like hydrogen-bonding interactions between the triple-helix-forming oligonucleotide and the purines of Watson-Crick base pairs (Fig. 1).

Oligonucleotides (C,T) motif

containing

cytosine

and thymine:

the

The two canonicai triplets T.A x T and C.G x C + are isomorphous in the Hoogsteen configuration (Fig. 2). In this review, base triplets will be referred to as X.Y X 2 where the symbols . and x indicate Watson-Crick and Hoogsteen (or reverse Hoogsteen) hydrogen bonds, respectively. The third strand binds in a parallel orientation with respect to the polypurine strand of the duplex, with all nucleotides in the anriglycosidic torsional conformation. Cytosine has to. be protonated to form two hydrogen bonds with a CG base pair; therefore, the sta-

Abbreviations

AP-2-aminopurine; BPI-benzopyridoindole; D-DNA; D3-l-O-deoxy-P-o-ribofuranosyl)-4-(3-benzimidophenyl)imidazole; M-8-oxo-adenine; mC--5-methylcytosine; MEA-(methoxyethyl)phosphoramidate; MP-methylphosphonate; P1-l-O-deoxy-~-o-ribofuranosyl~-3-methyl-5-amino-lH-pyrazolo~4,3,dl-pyrimidine-7-one; PNA-polyamide backbone; $-2’-0-methyl-pseudo-isocytidine; Pu-purine; Py-pyrimidine; R- RNA; X-7-diaza-2’-deoxyxanthosine. @ Current

Biology

Ltd ISSN 0959440X

345

346

Nucleic acids Fig. 1. Hydrogen-bonding interactions in base triplets described in the text. The left column corresponds to Hoogsteen-like configurations and the right column to reverse Hoogsteen-like configurations. These configurations correspond to different po‘sitions of the glycosidic bond of the third strand with respect to that of the polypurine strand. $, pseudb-isocytosine [57,581; M, 8-oxoadenine 159,601; Pl, 3-methyl-5-amino-lH-pyrazolo-[4,3,dlpyrimidine-7-one I611; AP, 2-amiiopurine [631; i, .7-diaza-2’-deoxvxanthosine [621; I, inosine. The orientation of the third strand wiih respect to the polypurine strand is indicated by a dot (parallel orientation) or a cross (antiparallel) at the position of the Cl’ atom of the third strand. All nucleotides adopt the anti glycosidic conformation except for nucleotide M, which is assumed to be in the syn conformation. The reverse Hoogsteen configuration has not been experimentally described for triple-helical complexes involving CC X C+, CG X I/I and CC X Pl triplets with anti conformation, nor for CG x M triplet with syn conformation.

bility of the triplet decreasesas pH increases. Until recently, the only structural information on a DNA triple helix was based on fiber-diffraction data of the triple helix poly(dT).poly(dA) x poly(dT) reported nearly two decades ago, which suggested an A-like conformation [8]. Recent proton NMR studies have revealed that the majority of sugars adopt a conformation close to C2’endo puckering, except for some cytosines, especially those in the third strand, which exhibit a higher proportion of C3’-endo conformation [9-111. Vibrational spectroscopy (infrared and Raman) has also provided evidence that triple helices with ‘canonical’ base triplets T.A x T and C.G x C + are structurally and conformationaIIy more similar to the B-form than to the A-form of DNA [ 12-141. Infrared studies of a (T.A x T>” [where n is any digit] triple helix have shown that its sugar puckers are in the C2’-endo region and not C3’-endo. From Raman and infrared studies of the (C-G x C+ >, triple helix it was concluded that sugars in the purine (dG) strand adopt a C2’-endo type pucker whereas those in the pyrimidine strands (dC) have sugar puckers in the C3’-endo region [141. A model for a (T.A x T>” triple helix compatible with infrared spectral data has been proposed recently [14,15]. An axial rise per residue of - 3.3A was obtained with 12 residues per turn, in agreement with X-ray fiber diffraction data. The pseudo-dyad between the Watson-Crick base-paired adenine and thymine strands is preserved and a pseudo-rotational symmetry relates the Hoogsteenpaired adenine and thymine strands. These symmetries give rise to a dyad between the two thymine strands which is perpendicular to the helix axis and result in identical conformations for all three strands with sugar pucker in the C2’-endo region, as in B-DNA. Similarly, a model for a (C*GX C+ >,, triple helix compatible with the data obtained from infrared and Ramanvibrational spectroscopyhas been proposed [ 141.The rise per base triplet (3.9& is larger than that of (T.A x T>” triple helix and of B-DNA double helices, becauseof electrostatic repulsion between successivepositively charged C.G x C+ base triplets. The helical twist is slightly reduced. It is worth noticing that only the guanine strand has a B-like glycosidic angle (boo), whereas the cytosine strands have an A-like glycosidic angle (207, consistent with C2’-endo

Oligonucleotide-directed

type pucker in the guanine strand and C3’-endo in the cytosine strands. Recentinvestigations have shown that a third-strand RNA containing uracil and cytosine binds more strongly to double-helical DNA than a third-strand DNA [16-181. Additional stabilization is obtained with the 2’-O-methyl derivatives [16,18]. The observation that 2’.deoxy-2’fluororibose (C3’-endo) in the third strand does not lead to enhanced stability relative to 2’-deoxyribose [ I61 shows that parameters other than sugar puckering play important roles in triple-helix stability. Molecular modeling studies have suggestedeither hydrogen-bonding interactions of the 2’-OH groups with phosphates of the polypurine strand (C Escude et al, unpublished data) or hydrophobic interactions of the 2’-O-methyl groups within the narrow groove located between the backbones of the polypurine strand and the oligopyrimidine third strand (C Escude et al, unpublished data).

Oligonucleotides (CJA) motif

containing

guanine and adenine: the

The base triplet T.A x A can only adopt the reverseHoogSteenconfiguration (Fig. 1). Triple helices can form when oligonucleotides containing guanines and adenines recognize purines of Watson-Crick base pairs [ 19,201.The oligonucleotide binds in an antiparallel orientation with respect to the target polypurine sequence with both T+Ax A and C.G x G base triplets in the reverse HoogSteen coniiguration. These two triplets are not isomorphous (Fig. 2). Therefore, backbone distortion of the third strand is expected to occur at each step between a T.A x A and a CG X G triplet. This might explain why such triple helices are difficult to observe for some sequences. Recentinfrared and Ramanstudies of (dCdG x dG), and (dC.dG x rG>” triple helices have provided evidence for anti glycosidic torsional conformation and the preference for C2’-endo sugar pucker of the (dC), strand and C3’-endo pucker of the WatsonXrick (dG), strand, with the third strand exhibiting either C3’-erido [(IG),] or C2’-endo [(dG),] sugarpucker [21]. As the C.G x G base triplet can adopt both Hoogsteen and reverse Hoogsteen conligurations, studies on homopolymers do not give direct information on the orientation of the third strand in (CG i G>” triple helices. Molecular modeling combined with results from vibrational spectroscopy suggest that the orientation of the third strand is parallel with respect to the (dG), strand of the Watson-Crick double helix, with Hoogsteen hydrogen-bonding interactions and anti glycosidic torsion angle. An opposite (antiparallel) orientation is expected when a (dG), strand folds back on a (dC), sequence to form a triple helix, as observed in intramolecular triple helices. In triple helices involving guanine and adenine in the third strand, an antiparallel orientation with respect to the target polypurine sequenceis imposed by the fact that T.A X A base triplets are restricted to the reverse Hoogsteen con&uration when nucleotides are in the anti conformation. In recent energy-minimization and molecular dynamics calculations on (CG X G>” triple helices, the parallel ori-

triple-helix

formation

Sun and H&he

entation was not considered [22] or was discarded on the basis of its much less negative solvation free energy [ 231. Further experimental studies are required to determine the preferred orientation of the third strand in a (C.G x G)r, intermolecular triple helix.

Oligonucleotides (G,T) motif

containing

guanine

and thymine:

the

Oligonucleotides containing guanines and thymines were first used by Cooney et al. [24] to inhibit transcription from the c-myc gene promoter. Band-shift and footprinting assays have revealed that the orientation of (G,T) oligonucleotides is antiparallel to the purine-rich strand of the target double strand, rather than parallel as originally suggested [25]. But a recent study has shown that the orientation of the third strand depends on its sequence [26]. As shown in Fig. 1, both T*Ax T and C.G x G base triplets can adopt either Hoogsteen or reverse Hoogsteen configurations. In both cases,the two triplets are not isomorphous, hence an expected distortion of the third-strand backbone which will be more pronounced in the Hoogsteen than in the reverse HoogSteenconIiguration (Fig. 2). Energy-minimization studies suggest that the Hoogsteen configuration is energetically preferred in both (T.A x T>” and (CG x G), homopolymeric triple helices, in agreement with spectroscopic studies (see above). But the energy penalty associated with backbone distortion for mixed (G,T) sequences is higher for Hoogsteen than for reverse Hoogsteen coniigurations. Therefore, the orientation of the (G,T)containing third strand is expected to shift from parallel to antiparallel orientation when the number of s’ApG3’ and/or sGpA3 steps increases in the target sequence; indeed, this ‘has been experimentally observed [26]. Energy-minimization studies suggest that the antiparallel orientation is preferred for a lo-mer composed of 50% T.A X T -and 50% C-G x G base triplets when there are more than three (ApG + GpA) steps in the target sequence, but that the parallel orientation is preferred otherwise (J-SSun, unpublished data). Recent NMR data on an intramolecular triple helix with a (G,T) third strand have shown that all nucleotides adopt anti glycosidic torsion angles with reverse HoogSteenhydrogen bonding of the CG x G and T.A x T base triplets (I Radhakrishnan, C de 10s Santos, DJ Patel, personal communication) [27]. The intrastrand nuclear Overhauser eifect connectivities between adjacent nucleotides reveal that the two Watson-Crick strands form an unperturbed stacked right-handed duplex. In contrast, the structure of the third strand exhibits considerable distortions. The rise and twist parameters for GpG steps are similar to those observed for the duplex segment. By contrast, the helical rise increases and the axial twist is reduced for the s’TpG3’ steps whereas the reverse is observed for s’GpT3’ steps. Connectivities were not detected at TpG steps. No base stacking was observed in the GpTpG sequence of the third strand. These distortions of the third-strand conformation clearly result from the absence of isomorphism of the C.G X G and T.A x T base triplets (Fig. 2).

347

348

Nucleic

Cl’

acids

Cl’ THIRD STRAND REVERSE-HOOGSTEEN

THIND STRAND HOOGSTEEN

AP,G-.

r, c: k M--r

Pl-or

Cl’

PY

Pu

a-T I 0 rG M

o-p,

0

l

0

Cl’

n -G,X

A-0

0-l

*

I

l

Cl’

Cl’

PY

Pu

1

Fig. 2. Positions of the Cl’ atoms of third strands with respect to the Cl’ atoms of the Watson-Crick PyPu base pairs fT.A or C.G). In the Hoogsteen configuration (see Fig. 11, the following symbols are used: A: T, C+, jr, M; 3: PI ; 0: I; n : C, AP. In the reverse Hoogsteen configurations (right) :A: T; A: C+, $, M; 0 : PI ; 0: A ; n : G, X. lsomorphism is observed for T-A X T, CG X C+, CG X \Ir, CG x M Hoogsteen-like base triplets (CC X Pl is nearly isomorphous to T.A Xl?, for T.A X AP and CG X C Hoogsteenlike base triplets, ar(d for T.A X X and CC X G base triplets in the reverse Hoogsteen configuration.

Oligonucleotides and CG,Tl motifs

containing

three

bases: mixed

(C,Tl

Because protonation of cytosines is required to form C.Gx C+ base triplets, runs of contiguous cytoslnes in the third strand are very detrimental to triplex stability in the (CT) motif, even when cytoslne is methylated at the C5 position. We have shown that a more stable triple helix is formed if a stretch of cytosines in the third strand is replaced by guanines [Z&29]. The ollgonucleotide still binds parallel to the polypurine sequence. In contrast, replacing an isolated cytosine within a stretch of thymines by guanines decreasestriplex stability as a result of the non-isomorphlsm of T.A x T and CG X G base triplets. The sequence investigated in this study (5’T&T*G$‘) provides a first example of an oligonucleotide containing three natural baseswhich can recognize a polypurinepolypyrimidine sequencewith good stability under physiological conditions. It should be noted that guanine can also be introduced ln a (C,T)containing oligonucleotide to recognize a single thymine interrupting a polypurlne sequence in the double helix [30]. This A*TX G triplet is stablewhen flanked by canonical T.A x T triplets but its stability drops when CG x C+ neighbors are present [ 311.

Selectivity formation

of oligonucleotide-directed

triplex

Severalstudies have been devoted recently to the selectivity of triplex formation [31361. Ollgonucleotldes that contain cytoslnes and thymlnes discriminate mlsmatchesarising from changesin the base-pairsequence

of a double-helical target DNA as well as, lf not better than, mismatches arising from single base changes ln a single-stranded target. Some mismatches have received -more attention as a result of an earlier report by Griffin and Dervan [30], which showed that a thymlne interrupting a polyputine sequence ln double-helical DNA could be recognized by guanlne in the third strand (an A.T x G triplet) with stability comparable to that obtained when thymine in the third strand recognized an TA base pair (a canonical T.A X T base triplet). It was later shown that the stability of the A.T x G triplet was dependent on the neighboring ilanking triplets [31]. NMR studies have shown that the NH2 group of guanine is not only hydrogen bonded to the 04 carbonyl of thymine interrupting the polypurine strand but also forms a hydrogen bond to the 04 carbonyl of thymine of one of the neighboring T.A base pairs [37,38]. The structure deduced from the NMR data is identical to that predicted on the basis of energyminimization studies [33]. The structure of C-G X X (where X represents any nucleotide) mismatcheswithin a (C,T) motif has also been investigated by NMR [39]. The less destabilizing mismatched trlplet involves a single hydrogen bond between the NH2 group of guanine in the third strand and O-6 of guanine in the polypurine strand, in agreement with energy-minimization and molecular-modeling studies [33], As shown in Fig. 1, guanine can recognize a CG base pair by forming two hydrogen bonds (C-G x G base triplet). The structure observed when guanine interrupts a (C,T) motif reflects the compromise between triplet stability and backbone distortion, as the CG x G triplet is isomorphous with neither T.A x T nor C*GX C + triplets (Fig. 2). Free energy losses associatedwith mismatches in trlplehelical structures have been determined for several types of mismatches. They usually range from -2 to - 6 kcal mol- l. The free energy diEerence between a canonical and a non-canonical triplet in a triple-helical structure is expected to depend on several parameters, including the flanking sequences, pH (for third-strand oligonucleotides containing cytosines), ionic conditions, etc. More data are needed before a prediction can be made of the impact that a mismatch will have on triplex stabiity, e.g. under physiological conditions. This is especially true for the (GA) and (G,T) motifs for which less experimental data on mismatch destabilization are available than for the (C,T) motif (see above).

Extension of the range of recognition sequences for oligonucleotide-directed triple-helix formation

TWOpolypurinepolypyrimidlne sequencesseparated by two base pairs can be recognized by two (C,T)-contaming oligonucleotides that bind to each other by Watson-Crick hydrogen bonds [40]. The two trlplexforming sequencesare thus maintained in the appropriate position to bind simultaneously and cooperatively to

Oligonucleotide-directed

their respectivetarget sites.The dimerization domain can be stabilized by sequence-specificligand binding (41 I. The three motifs for oligonucleotide-directed triple-helix formation that we have discussed in the preceding paragraphs involve the recognition of polypurinepolypyrimidine sequences of double-helical DNA Interruption of the target polypurine sequence by one pyrimidine base markedly decreasestriple-helix stability, except for one casediscussedearlier (an A.T X G triplet flanked by two canonical T.A x T triplets). It is however possible to recognize a polypurine sequence adjacent to a polypyrimidlne sequence by alternate strand triple-helix formation. The third strand will switch from the oligopurine sequence on one strand to the oligopurine sequence on the other strand. When the third strand contains only cytosine and thymine, the two pyrimidine oligonucleotides bind with opposite orientations on the two adjacent polypurinepolypyrimidine sequences. Therefore they should be linked via either their 3’-ends or their 5’-ends, depending on whether the target sequence is 5’-(Pu),-(Py),3’ or 5’-(Py),-(Pu),-3’ (Pu, purine; Py, pyrimidine), respectively.A semi-rigid xylose linker was recently used to attach the 3’-ends of two oligopyrimidines [421.The 3’-3’linked oligomer binds cooperatively to two triple-helix domains separated by two base pairs. In order to recognize oligopurine sequenceson two different strands, one can take advantage of the different orientations that oligonucleotides adopt, depending on their base composition. As described above, the (CT)containing oligonucleotides bind in a parallel orientation with respect to the target polypurine sequence whereas (GA)-containing oligonucleotides bind in the opposite orientation. It is therefore possible to design an oligonucleotide containing only natural 3’-5’phosphodiester linkagesthat will switch from one strand of DNA to the other one [43]. A sequence 5’-(Pu),-(Py),-3’ (m, n, any digit) can be targeted with an oligonucleotide consisting of a pyrimidine domain and a purine domain linked by a 3’-5’ phosphodiester. If the target sequence reads 5’(Py),-(Pu)“-3’, the third strand may require additional nucleotides to connect the two binding domains at the site of crossover in the major groove [43], although this might depend on the sequence at the junction between the two domains [44]. Sun et al. [26] have shown that the orientation of (G,T)containing oligonucleotides depends on the number of GpT or TpG steps. Consequently, an oligonucleotide containing only guanines, thymines and natural 3’-5’ phosphodiester linkages can be designed to recognize adjacent polypurine tracts on alternate strands of the duplex. The (G,T) motif could also be combined with a (G&d and/or a (CT) motif to build oligonucleotides that recognize double-helical DNA by alternate strand triple-helix formation.

Triple-helix-specific

ligands

Ethidium bromide has been shown to bind the triple helix poly(dT)poly(dA) x poly(dT) better than the double

triple-helix

formation

Sun and HCkne

helix poly(dT)apoly(dA) [451. Energy-transfer experiments have suggested that ethidium does intercalate between base triplets. But the double-helical structure of poly(dA).poly(dT) has unusual characteristics which are unfavorable to intercalation. The binding of ethidium bromide to this structure is highly cooperative as a result of the conformational change Induced by intercalation. This is not observed with the triple-helical * structure. Therefore, the higher a.Rlnity observed for the triple-helical structure poly(dT).poly(dA) x poly(dT) compared with double-helical poly(dT).poly(dA) reflects the low affinity of ethidium for the latter rather than high affinity for a triple helix. In triple helices of mixed T.A x T/CG x C + sequences, ethidium binds better to the double helix than to the triple helix as expected, because the positive charge of ethidium is not favorable to intercalation in the vicinity of positively charged C.G x C+ triplets [46]. This holds true for other positively charged intercalators. We have recently found however that benzopyridoindole (BPI) derivatives strongly stabilize triple helices which contain a stretch of four or more T-A x T base triplets [47]. This is reflected in a stabilization of the triple-helical structure which is not seen with ethidium bromide. It was shown earlier that the junction between triplex and duplex structures constitutes a strong binding site for intercalators such as acridine or ellipticine derivatives [48,49]. This property was exploited to design oligonucleotide-intercalator conjugateswhich exhibited a much stronger binding to polypurinepolypyrimidine sequences than the unsubstituted oligonucleotide [50,51]. The highest affinity was obtained when the intercalator was attached to the 5’-end of the third strand (CT) oligonucleotide. We have now synthesized oligonucleotide-BP1 conjugates which exhibit a much higher stabilization than the oligonucleotide-acridine (ellipticine) conjugates.The BP1ring can intercalate within the triplehelical region rather than at the duplex-triplex junction, provided a stretch of T.A x T base triplets is present at the end of the triple helix (B Faucon, unpublished data). Minor-groove-binding ligands such as netropsin bind to triple-helical poly(dT).poly(dA) x poly(dT) without displacing the major-groove-bound third strand at low temperatures [52]. However, binding thermally destabilizes the triplex w duplex + single strand equilibrium while thermally stabilizing the duplex c) single strand equillbrium. A covalent adduct of duocarmycin A with N3 of a speciiic adenine in the minor groove of an intramolecular triple helix has been recently characterized by NMR spectroscopy at low pH [53]. This reaction destabilizes the triple helix as the pH is raised, with a drop of the apparent pK of the cytosines involved in CG x C + triplets by 1.8pH units.

349

350

Nucleic acids Triple-helix formation oligonucleotides with

involving non-natural

bases

The importance of hydrophobic interactions was previously demonstrated by the increase in triple-helix stability observed when cytosines are replaced by 5methylcytosines [ 54,551. Thermodynamic studies have suggestedthat the additional stability that results from methykition at position C5 of cytosine is entropic in’origin [54]. The methyl groups of thymine and 5-methylcytosine form a helical ‘spine’ of hydrophobic groups within the major groove of the triple-helical structure (Fig. 3), which results in a change of hydration of the complex. Oligonucleotides containing the C5 propyne analog of 2’-deoxyuridine together with 5-methyl-2deoxycytidine form more stable triple-helical complexes than oligomers containing thymine and 5-methylcytosine [5G]. In contrast, replacemeht of 5-methylcytosineby its C5 propyne analog decreasesstability as a result of a decrease in pK, of the heterocycle. Both propyne derivatives stabilize the double-helical structure formed with a complementary RNA sequence. These results again emphasize the interplay of hydrophobic interactions and protonation requirements in the stabilization of triplehelical structures involving the (CT) motif. The stability of oligonucleotide-directed triple helices at polypurinepolypyrimidine sequenceswould be strengthened under physiological conditions if: cytosine could be replaced in the (CT) motif by heterocycles that do not require protonation to form two hydrogen bonds with guanine in CG base pairs; and isomorphous base triplets could be designed in both parallel and antiparallel triplexes without requirement for protonation. In addition, it remains a challenge to chemists to design

nucleotide analogs that can recognize the four base pairs (TA CG, AT, GC) from the major-groove side of the double helix instead of two (TA and CG) in polypurinepolypyrimidine sequences. Some examples of base replacement are given in Fig. 1. Deoxycytidine can be replaced by 2’-O-methyl-pseudoisocytidine ($1 [ 57,581.This base does not require protonation to form two hydrogen bonds with guanine in a C.G base pair (Fig. 1). The two base triplets T.A x T and C.G x J, are isomorphous in the Hoogsteen configuration (Fig. 2). Cytosine can also be replaced by goxo-adenine (M) [59] or its Nb-methyl derivative [6O]. Both form two hydrogen bonds with guanine in a C.G base pair (Fig. 1). These base anafogueswere introduced in place of cytosine in oligonucleotides designed to bind in a parallel orientation with respect to the polypurine sequence of double-helical DNA The C.G X M base triplet can adopt two configurations (Hoogsteen- and reverse Hoogsteenlike). One of them is isomorphous to the T*AX T base triplet in the Hoogsteen configuration (Fig. 2). The parallel orientation of the third strand is consistent with the observation that Soxo-adenosine adopts a v conformation around the glycosidic bond. Another deoxyribonucleoside, I-(2-deoxy-P-o-ribofranosyl)-3-methyl-5amino-lH-pyrazolo[4,3,d]-pyrimidine-7-one (Pl, Fig. l), was also designed to form two hydrogen bonds with guanine of a C.G Watson-Crick base pair [61]. The C.G x Pl triplet is nearly isomorphous to the T.A x T base triplet in Hoogsteen configuration (Fig. 2). AU the above derivatives (pseudo-isocytosine, M and Pl) when incorporated in place of cytosines in pyrimidine oligonucleotides extend the stability of triple-helical complexes to higher pH. They are particularly useful when the target DNA contains a stretch of guanines because

Fig. 3. Stereoview of an energy-minimized triple helix where the third-strand oligodeoxynucleotide contains alternating thymine and S-methylcytosine fmC) bases. The two base triplets T.A x T and C.G x mC+ in the Hoogsteen configuration are isomorphous (Fig. 2). The third strand is oriented parallel to the polypurine strand with anti glycosidic conformation. This figure highlights the helical ‘spine’ of hydrophobic methyl groups (dots represent van der Waals volumes) located in the wide groove separating the third strand and the polypurine strand of the Watson-Crick double helix.

Oligonucleotidedirected

the stability of triplexes containing contiguous C.G x C + triplets decreasesmarkedly when the pH is raised. Oligonucleotides containing 7-diaza-2’-deoxyxanthosine (X; see Fig. 1) and guanine bind polypurine sequences with higher aBnity than similar oligonucleotides containing thymine and guanine [62]. The orientation of the third strand is antiparallel to the polypurine sequence. No comparison was made with oligonucleotides containing adenine and guanine. It should be noted that CG x G and T.A x X triplets are isomorphous in the reverse Hoogsteen conIiguration, which leads to the observed antiparallel orientation if nucleosides adopt an anti conformation. The same report also described the decreasedcapacity of oligonucleotides containing 7diaza-guanine instead of guanine to form triple helices under physiological conditions [62].7-Diaza-guanine was used to decreasethe propensity of oligonucleotides containing stretchesof guanine to self-associatevia formation of guanine quartets. Oligonucleotides were recently synthesized with 2aminopurine (AI’; Fig. 1) to recognize T.A base pairs instead of thymine or adenine [ 631. The two base triplets T.A x AP and C.G x G are isomorphous in the Hoogsteen coniiguration (Fig. 2). Molecular modeling predicts that a parallel orientation should be favored with all nucleotides in the anticonformation (Fig. 4). The nonnatural deoxyribonucleoside 1-(2-deoxy-P-Dribofuranosyl)-4-(3-benzamidophenyl)imidazole (D3; Fig. 1) was incorporated into pyrimidine oligonucleotides and shown to bind purinepyrimidine base pairs with an aEinity higher than that with which it binds to pyrimidinepurine base pairs [64]. The stability of base triplets decreases in the order A.T x D3 ss GCxD3 >

triple-helix

formation

Sun

and Helene

T.A x D3 > CG x D3. Oligonucleotides containing D3 are able to recognize polypurine sequenceswhich are inter-. rupted by thymine and/or cytosine. D3 does not however discriminate between A.T and GC base pairs, and the stability of T.A x D3 and C.G x D3 base triplets depends on their interactions with nearest neighbor triplets. These results neverthelessrepresent an attempt to design non-natural nucleotides that may provide a more general solution to sequence-specilic recognition of double-helical DNA from the major groove. It should be pointed out that even though hydrogen-bonding interactions are important for the recognition of Watson-Crick base pairs, the available results demonstrate the importance of other interactions such as stacking with neighboring base triplets and other van der Waalscontacts, together with the isomorphism of base triplets to avoid distortions of the third-strand backbone.

Oligonucleotides

with

modified

backbones

Changes in backbone chemistry can have dramatic effects on triple-helix formation and stability. We have‘ described above that replacing deoxyribonucleotides in the third strand by ribonucleotides strongly enhances triple-helix stability (C Escude et al, unpublished data) [16-181. Figure 5 shows that an 11-mer (C,U)-containing oligoribonucleotide exhibits a shift in melting temperature (AT,) of - 17 “C at pH 6 when compared with the corresponding (CT) oligodeoxyribonucleotide [ 181. The AT,.,,is further increased by 4 “C when 2’-OH is replaced by 2’-O-methyl; however, this effect is sequencedependent as 2’-O-methyl substitution in the (G,U) mo-

Fig. 4. Stereoview of energy-minimized triple helix where the third strand contains alternating 2-aminopurine (API and guanine in a parallel orientation with respect to the polypurine Watson-Crick strand, with anti glycosidic conformation of the nucleotides. The two base triplets T.A X AP and CC x C are isomorphous in the Hoogsteen configuration (see Fig. 21, which leads to a regular backbone conformation of the third strand, as observed also in a canonical triple helix made of T.A x T and CC x C+ Hoogsteen base triplets (see Fig. 3).

351

352

Nucleic acids

tif in reverseHoogsteen coni&uration inhibits triple-helix formation (RH Durland, abstract 3-2, NAh4A Conference, Can&n, January 1993).

s' d(m) 5' r(S) 5' 2'O-t42-r (UvuCCvCCUtu)

E * $

0.6

; it 2

0.6

3' 3’ 3’

11X ll[x3 11tx*

'

2

Fig. 5. Stability of triple helices made with a third strand containing deoxyribose, ribose or its 2’-O-methyl derivative. The target sequence is boxed in the double-helical target. The melting curveswere recorded at pH6.0 in the presence of 10 mM sodium cacodylate, IOOmM NaCI, 1 mM spermine and 1 mM EDTA. Reprinted with permission [18].

Several factors are certainly playing an important role in determining the stability of these different triple-helical complexes. The conformation of the single-stranded third strand is expected to be different for DNA, RNA and 2’-0-methylribose backbones. The spatial disposition of 2’substituents with respect to the two strands of the WatsonCrick duplex depends on whether the third strand is ‘parallel or antiparallel with respect to the polypurine sequence. In the parallel (C,U> motif, the 2’-O-methyl groups lill the narrow groove between the third strand and the polypurine strand, allowing favorable van der Waalsinteractions with the backbone of the polypurine strand, whereas in the antiparallel (G,U) motif, the 2’-O-methylgroups do not havean appropriate location to enhance the interstrand interactions. These observations clearly open a new field of investigation aimed at enhancing the contribution to triplehelix stability of hydrophobic interactions between base and/or sugarsubstituents.It should be noted that replacing deoxyribonucleotides in one or both strands of the

double-helical target by ribonucleotides has dramatic effects on triplex stability. To describe these complexes, we will use the samenomenclature as that used throughout this review for base triplets, i.e. R*DX R means that the double helix is composed of a polypyrimidine RNA (R) and a polypmine DNA (D) and the third strand is an RNA Two of these combinations (R.Rx D and D.Rx D) were detected by neither Roberts and Crothers [I71 nor us (C Escude et al, unpublished data). The relative stabilities seem to depend however on base composition and sequence and on experimental conditions. For the target sequence s’GGAGAGGAGGGA3’ used by Roberts and Crothers [17], the order of decreasing stabilities was found to be R*Dx R > D.D x R >R.RxR >R.DxD >D.DxD, D.RxR For the sequence s’AAAGGAGGAGA3’,the order at pH 6 was D.DxR >R.DxR >D.DxD >R.RxR,D.RxR,R.DxD (C Escude et al, unpublished data). In a recent study at pH7 and 25 ‘C, using the aflinity cleavagemethod, the order of decreasing stability was found to be D.D X D, D*DxR, R.DxR, R.DxD >D.RxR, R.RxR >=-R.RxD, D.Rx D (PB Cervan, abstract, NAMA Conference, Cancurt, January 1993, p49). These results have clear implications for the recognition of double-helical structures in RNAas well as RNA-DNAhybrids that may arise under some circumstances(e.g. during transcription). They also suggest that triple-helix formation on a single-stranded polypurine RNA sequence may be strongly enhanced by changing the sugars in the two oligopyrimidine sequences (see below). The carbocyclic analog of 5-methyl-2’-deoxycytidine, in which substitution of the furanosyl oxygen with a methy lene generates a cyclopentane ring, was recently introduced into third-strand oligonucleotides and shown to increasethe stability of triple-helix formation directed by pyrimidine oligonucleotides [651. In contrast, the carbocyclic analog of thymidine decreased the stability of the triple-helical complex. These results were explained by an increase in the pK, of cytosine (which extends the stability range toward higher pH values) and an unfavorable (Cl’-exo) pucker of the cyclopentane ring. Oligonucleotides synthesized with the unnatural u anomers of nucleotide units have been shown to bind double-helical DNA to form triple helices with only slightly lower stability than corresponding P-anomeric natural oligomers [66,67]. An oligo- [a] -deoxynucleotide containing only thymines binds in a paraliel orientation with respect to the polypurine target, as does the l3 oligomer. Yet (C,T) [a] -oligomers bind in the reverseorientation. Becauseof the anomeric inversion, the parallel and antiparallel orientations of a-oligomers correspond to reverseHoogsteen and Hoogsteen con@urations, respectively.The change in orientation observed when cytosines are introduced in the third strand reflects the gain in stability of the triple-helical complex as a result of the isomorphism of the T.A x T and C.G x C+ base triplets in the Hoogsteen configuration. Even though the reverse Hoogsteen configuration would be slightly more stable for each of the triplets, the absence of isomorphism of the two triplets in this configuration leads to unfavorable distortions of the third-strand backbone.

Oligonucleotide-directed

Oligodeoxynucleotides bearing a neutral backbone have also been tested for their ability to form triple helices. Fomracetal, S-thioformacetal, methylphosphonate (MP) and (methoxyethyl)phosphoramidate (MEA) linkageswere introduced at four 5methyl CpT steps in a 14mer (C,T) oligonucleotide [ 681. The fonnacetal-containing oligomer binds equally well as the phosphodiesteronly oligonucleodde whereas the MP oligomer was bound more weakly and the MEA and S-thioformacetal oligomers gave only very weak interactions. Pyrimidine oligonucleotides containing an MP backbone were shown to require lower pH than the phosphodiester oligomer to form a triple-helix with a polypurine sequence [69]. Oligomers containing a polyamide backbone (PNA) have been synthesized recently [70,71]. They bind strongly to complementary single-stranded DNA sequences and are able to bind double-helical DNA by strand displacement. The studies reported so far havedealt only with pyrimidine oligomers, which form 2:l complexes with two PNAs bound to single-stranded targetsaswell as to DNA in the strand-displacement complex. Further studies are required to determine whether strand displacement can be observed at sequencesother than polypurinepolypyrimidine sequences and whether triple-helix formation is an intermediate in the process of strand displacement.Recentinvestigations have shown that nucleic acid processing enzymes (reverse transcriptase and RNA polymerase) are arrested at sites where a 2:l complex is formed on the template strand [72].

Oligonucleotide-directed on a single-stranded

triple-helix

triple-helix

formation

Kinetics of oligonucleotide-directed formation

Sun and H&he

triple-helix

The binding of oligonucleotides to the major groove of double-helical DNA is slow compared with double-helix formation. This was first analyzed by probing triple-helix formation by inhibition of restriction enzyme cleavage [80]. More recently, a complete description of kinetic parameters was provided by a study of the hysteresis observed upon dissociation and subsequent reassociation of a (CT) oligonucleotide on a double-helical polypurinepolypyrimidine target followed by uv absorbance spectroscopy at pH6.8 [34]. The activation energy for association is negative, with association rate constants which are several orders of magnitude slower than those measured for double-helix formation. The association rate constant increases with salt concentration (NaCl or MgC12)but is independent of the presence of a mismatch in the center of the triple-helical complex. In contrast, the dissociation rate constant is salt-independent but strongly increases when a mismatch exists in the center of the triple helix. The greatest effect is seen when a T.A X C mismatch is formed instead of a canonical T.A x T triplet. The results have been interpreted according to a nucleation-zipping model where three to five base triplets are involved in the nucleation step. When oligonucleotide clamps are used to form a triple helix on a single-stranded template (see paragraph above), the association of the Hoogsteen part is strongly accelerated because of the restricted diifusion arising from covalent linkage to the Watson-Crick part.

formation

target

A single-stranded polypurine sequence can be recognized by a complementary oligopyrimidine to form a Watson-Crick double helix, which in turn can be recognized by a second oligopyrimidine via Hoogsteen hydrogen bonding. The two pyrimidine oligonucleotides can be linked together by a tetra- or pentanucleotide [731, by a synthetic hexaethylene giycol linker [74] or a terephtalamide derivative [75]. The two oligopyrimidines can also be linked at both ends to form circular structures [76-781. Most of the hydrogen-bonding possibilities of purine basesin the single strand are used to recognize the target: four hydrogen bonds are formed with adenine and five with guanine [74]. This leads to a high discrimination of mutant sequencesas a result of the disruption of both Watson-Crick and Hoogsteen hydrogen bonds 175-781. The oligonucleotide segment involved in Watson-Crick hydrogen bonding can be made a little longer than the Hoogsteen part, thereby creating a duplex-triplex junction. An intercalating agent can be attached to the 5’-end of the Hoogsteen part in order to take advantageof the additional stabilization provided by intercalation at the duplex-triplex junction [74]. Such an oligonucleotide clamp produces a strong arrest of nucleic acid processing enzymes [791.

Concluding

remarks

This review has not addressed the formation of threestranded structures mediated by recombinases such as Escbeticbia coli RecAprotein. These structures involve a third strand which has polarity identical and sequence to that of one of the Watson-Crick strands. These triplestranded complexes remain stable when RecAprotein is removed provided that the length of shared homology is at least 26 base pairs [Sl]. But the structure of these complexes has not been elucidated yet. Oligonucleotide-directed triple-helix formation has led to very interesting and novel applications in different areas of research,including artiiicial control of gene expression [5,82], site-specific cleavageof chromosomal DNA [6,7], site-specific modification of DNA [82], and diagnostic procedures [83]. (The reader is referred to recent reviews in this area [ 5-7,821.) In the near future, new developments are expected in the design of highly sequencespeciiic triple-helix-forming oligonucleotides with higher affiity for short target sites in double-helical DNA. It still remains a challenge to chemists to design the appropriate molecules that could recognize all four base pairs within the major groove of the double helix with enough discrimination to select a unique base-pair sequence in the genome of human cells.

353

354

Nucleic acids

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J-S Sun and C Helene, Laboratoire de Biophysique, INSERMU.201 CNRSURA 481, Musium National d’Histoire NaturelIe, 43 rue Cutler, 75231 Paris Cede%05, France.