Structure and Applications of Intermolecular DNA Triplexes

Structure and Applications of Intermolecular DNA Triplexes JAY E. GEE,*t*§

DONALD M. MILLERt

ABSTRACT: Current DNA binding drugs are not sequence specific. Triplex-forming oligonucleotides will bind targeted duplex DNA sites in a sequence-specific manner. A new class of DNA binding molecules based on triple-helical DNA formation promises a sequence-specific method of targeting discrete regions of DNA. DNA modifying molecules linked to third strands have been shown to modify only regions of DNA to which they were targeted. Current research will increase the understanding of triplex DNA structure and will lead to improved DNA binding drugs. KEY INDEXING TERMS: Chemical probing; Chemotherapy; DNA binding drugs; Intermolecular DNA triplex. [Am J Med Sci 304(6):366-372.]

I

nterest in intermolecular triplex DNA has increased in recent years because of its possible use as a tool for molecular biology and as a basis for chemotherapy agents.l -3 The use of triplex-forming oligonucleotides promises to provide a site-specific method of targeting discrete sequences of DNA to a degree not achievable by current DNA binding molecules.2-6 Two major types of DNA triplexes have been described-intramolecular and intermolecular. For both types, a third strand associates with a duplex tract that is purine-rich in one strand and thus pyrimidine-rich in its complement. 'l'he third strand may be either purine-rich or pyrimidine-rich. In an intermolecular triplex, a separate third strand associates with a target duplex DNA (Figure lA).2,4,7-9 In an intramolecular triplex, the third strand is a portion of one of the strands of the duplex that has folded back to associate

with the purine-pyrimidine tract. Intramolecular triplexes are associated with either supercoiled plasmids containing mirror repeats of purine-pyrimidine tracts10-13 or single DNA strands that can fold back on themselves.I3-17 History

Intermolecular triplexes were first observed in RNA polymers. Felsenfeld and Rich found that a poly-rA RNA strand associated with two poly-rU strands to form a triplex structure.IS Research by Riley and coworkers showed that a poly-dA strand would first associate with one poly-dT complement to form a duplex. Under high-salt conditions, a third poly-dT strand then would associate with the duplex to form a triple-helical structure.19 Triplexes were presumed to be stabilized by Hoogsteen-type hydrogen bonds (Figure IB) between the bases in the third strand and bases in the poly-purine strand of the duplex.1,7,lO When a poly-G:poly-C tract formed a triplex DNA structure, a poly-G third strand or a poly-C third strand could be used. When a poly-C third strand was used, acidic conditions were needed to protonate the N3 position of the cytosine.7,10,ll,20 When a poly-G third strand was used, a triplex could be formed at a physiologic pH, but Mg++ ions were required.8,ll,21 Cations such as Mg++, spermine, and spermidine are presumed to enhance triplex formation by shielding the negatively charged phosphodiester backbone of DNA.3,22 For Hoogsteen type hydrogen bonding, bases in the third strand bond with the purine bases in the target duplex. In general, the best triplets when a polypyrimidine third strand is used are T:A_TI0,23 and C:GC+.7,l0,23 When a polypurine third strand is used, the best triplets are T:A-A 23 and C:G_G.ll,21,23 Structure

From the Departments of *Internal Medicine and tBiochemistry, and the :j:Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, and §Birmingham VA Medical Center, Birmingham. This work was supported by grants {rom NIH (CA 42664, CA 42337), Leukemia Society of America, New York, New York, and the VA Medical Research Service, Washington, D.C. Correspondence: Donald M. Miller, 520 WTI, University ofAlabama at Birmingham, BHFB 288, UAB Station, Birmingham, AL 35294.

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Third strands in triplexes can be pyrimidine-rich or purine-rich. The bulk of the research has focused on the structure ofpolypyrimidine third strands. The major methods used to probe this unique DNA structure have been x-ray crystallography,24,25 nuclear magnetic resonance (NMR),27-29 ultraviolet absorption spectroscopy,27,28,30-32 circular dichroism,28,31,33 enzymatic probing,34 and chemical probing.4-6,9,21,35-41 December 1992 Volume 304 Number 6

Gee and Miller

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X-Ray Crystallography

Amott and Selsing analyzed x-ray diffraction data on fibers of a poly-dT:poly-dA:poly-dT triplex and fibers of a poly-dA:poly-dT duplex. They found that the A:T duplex maintained the expected B family structure. However, the T:A-T triplex yielded an A type DNA conformation. This suggested that the binding of a third strand induces a B to A conformational change. The A family of DNA helices are characterized by a deep major groove, which they suggested could easily accommodate the third strand. They also found that for their poly-Y:poly-R:poly-Y triplex, the poly-Y strand of the duplex is anti-parallel to the poly-R strand, as expected, but that the poly-Y third strand is parallel to the poly-R strand.24 Less x-ray data is available for poly-R:poly-R:polyY triplexes. Campos and Subirana's crystallography data show that the C:G-G triplex is stabilized by Mg++ ions.25 This supports work by Kohwi and Kohwi-Shigematsu,11 Cooney and coworkers,s and Beale and Dervan.21 However, further work is needed to yield fine structure details. Nuclear Magnetic Resonance

NMR studies of intermolecular triplexes have verified the unique Hoogsteen-type hydrogen bond that was presumed to stabilize these structures.lO In a T:A-T triplet, the hydrogen at the N3 of the thymines has been shown to be engaged in Watson-Crick hydrogen bonding and Hoogsteen hydrogen bonding.26-29 In a C:G-C+ triplet, the N3 of the cytosine has been shown to be protonated at low pH and involved in Hoogsteentype hydrogen bonding.26,28,29 Radhakrishnan and coworkers have characterized an intramolecular triplex that has yielded data applicable to intermolecular triplexes. They used a triplex that contains predominantly C:G-G base triplets with THE AMERICAN JOURNAL OF THE MEDICAl. SCIENCES

a few T:A-T triplets. Their data indicated that the guanine bases of the third strand maintained an anti torsion angle about the glycosidic bond. The N -7 proton of the guanine base in the duplex strand also was shown to be involved in Hoogsteen-type hydrogen bonding to the guanines in the third strand.42 Chemical Probes

Chemical probes have been used extensively to study DNA structure. By studying the reactivity pattern of DNA that has been subjected to chemical modification, the accessibility or lack of accessibility of a given chemical probe can be determined. This yields structural information about a given section ofDNA.5,9.lo.41 Francois and coworkers used the minor groove binder (phenanthrolenehCu chelate, which cleaves DNA in the presence of reducing agents. Using a polypyrimidine third strand, they found that triplex formation protected that target site from cleavage. They also found greater cleavage at the triplex-duplex junction 3' of the third strand, although there was no perturbation at the junction 5' of the third strand.5 Diethylpyrocarbonate (DEPC) reacts with the N-7 position of purines, whereas dimethylsulfate (DMS) reacts predominantly at the N-7 position of guanines.10 Collier and coworkers probed a homopyrimidine third strand using DEPC and DMS. They found that triplex formation protected the purine-rich strand of the duplex from chemical attack. This was attributed to the involvement of the N-7 of the purines in Hoogsteentype hydrogen bonding, which would make them inaccessible to the probes.41 Osmium tetroxide OS04 forms an osmate ester with the C-5, C-6 double bond of pyrimidines.10 Hartman and coworkers probed a triplex-forming sequence from the human papillomavirus type 11 with DMS, DEPC, and OS04' They found the triplex formation protected 367

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the underlying duplex from chemical attack. They also found there was a greater reactivity at bases adjacent to triplex-duplex junction. with a greater reactivity found at the site 3' of the homopyrimidine third strand. They proposed that the triplex is in an A type of conformation, while the flanking duplex is in the B type of conformation. This would induce a bend in the DNA. making the bases at the junctions more accessible to chemical modification.9 Pyrimidine dimers result from irradiation of DNA by ultraviolet light. After irradiated DNA is treated with piperidine. fragments that indicate the site of damage can be isolated. Lyamichev and coworkers showed that after addition of a triplex-forming oligonucleotide and irradiation with ultraviolet light. the target site is protected from damage. This ''photofootprinting" technique promises to be useful in probing DNA triplexes.43 Unked Chemical Probes

Oligonucleotides linked to chemical probes have been used extensively to study triplex structure. A variety of probes that can act as nucleases have been attached site specifically to oligonucleotides capable of triplex formation. Upon triplex formation, the cleaving moiety will site specifically cleave the duplex DNA. depending upon the orientation of the third strand as it lies in the major groove of the duplex targei.4,6,21,34-40 Ethylenediaminetetraacetic acid (EDTA)-Fe (IT) can generate a diffusible hydroxyl radical that can cleave the DNA backbone.1o The EDTA-Fe(IT) moiety has been linked to purine-rich and pyrimidine-rich triplex-forming oligonucleotides. Dervan's group has demonstrated that pyrimidine-rich third strands bind parallel to the purine-rich strand of the duplex.4,35,37 They also have shown that when a purine-rich third strand is used, it is in an anti-parallel orientation with respect to the purine-rich strand of the duplex.21 Hogan's group used an eosin linked to a purine-rich triplex-forming oligonucleotide. Under irradiation from an argon laser, they induced cleavage of the DNA through the production of a singlet oxygen by the eosin moiety. Their work also indicated an anti-parallel orientation of a purine-rich third strand with respect to the purine-rich strand of the duplex.6 Helene and ~oworkers have made triplex-targeted cleavers by using a variety of ligands, such as phenanthroline-copper,39,40 azidoproflavine,44 azidophenacyl.4S and an ellipticine derivative.38 Their work also has confirmed that a polypyrimidine third strand lies in a parallel orientation with respect to the polypurine strand of the duplex.38-40,44,4S Pei and coworkers covalently attached a mutant staphylococcal nuclease to a polypyrimidine triplexforming oligonucleotide. When the nuclease was tethered to the 5' end of the oligonucleotide. the authors observed site-specific cleavage of both strands of the target duplex at the triplex-duplex junction.34

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Intermolecular Triplexes as a Tool in Molecular Biology Site-Specific DNA Cleavage. Restriction endonucle-

ases recognize and cleave at target sites of 4-8 base pairs in size. Because their selectivity may not be sufficient for cleavage oflarge fragments of DNA. such as for genome mapping. using a more discrete cleaving tool may be advantageous. The previously described linked chemical probes that have been shown to cleave DNA site specifically may be used for such purposes.4,21,35,37-40,46 Strobel and Dervan demonstrated site-specific cleavage in a genomic-size target. They targeted a triplex-forming oligonucleotide linked to an EDTA-Fe cleaving moiety to chromosome m of Saccharomyces cerevisiae, which is 340 kbp in size. After the chromosome was treated, bands were detected, indicating that discrete site-specific cleavage had occurred.46 Intermolecular triplex formation has been shown to prevent the binding of proteins such as DNA methylases and restriction endonucleases.47-49 Dervan used an "Achilles heel cleavage"50 technique to mediate sitespecific cleavage in genomic-size targets. In this technique. a triplex is overlapped onto a sequence that is recognized by a methylase and a restriction endonuclease. The third strand oligonucleotide is targeted to a sufficiently long sequence, so that not all the recognition sites of the methylase and the restriction endonuclease will be involved in triplex formation. After triplex is allowed to form, DNA methylase is added. Triplex formation prevents the methylase from methylating the DNA in the underlying duplex. After the third strand oligonucleotide and methylase are removed, the restriction endonuclease is added. The methylated sites of the DNA are not cleaved. However, the sites that were protected from methylation by triplex formation are accessible for cleavage. This technique allows discrete targeting and cleavage of DNA.51,52 TherapeutiC Uses of Intermolecular Triplexes. Current DNA binding drugs are not truly sequence specific.3 Because purine/pyrimidine-rich stretches occur relatively often in eukaryotic genomes,10,53 the use of sequence-specific binding drugs based on triplex-forming oligonucleotides promises a level of DNA specificity that will revolutionize chemotherapy. The characterization of genes that play important roles in human disease has provided excellent targets for transcriptional modulation. Triplex targeting of DNA binding drugs may allow novel therapeutic agents to be developed for illnesses as diverse as cancer and AIDS.l,3,8,33,37 Transcriptional Repression. Morgan and Wells found that DNA involved in triplex formation with RNA third strands were not effective templates for transcription by Escherichia coli RNA polymerase.54 More recently, Duval-Valentin and coworkers showed that a triplex-forming oligonucleotide targeted downstream from a transcriptional start site also represses tranDecember 1992 Volume 304 Number 6

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scription.55 However, the bulk of transcriptional inhibition studies have used oligonucleotides targeted upstream of the transcriptional start site by inhibiting the binding of proteins such as transcription factors.6,S,33,47,56-59 Using purine-rich third strands, Hogan's group has successfully formed in vitro triplex in the G-C-rich promoter regions of the human c-myc,S epidermal growth factor receptor gene, and the mouse insulin receptor gene.6 The triplex-forming site in the human c-myc promoter is at the binding site of several cellular factors. 6 Triplex formation has been shown to repress in vitro transcription of the c-myc gene, presumably by blocking protein binding.s Sp1 is a transcription factor that increases transcription levels upon binding to its site in gene promoters.so Using a mutated murine metallothionein promoter that contained an Sp1 transcription factor binding site, Maher and coworkers showed that intermolecular triplex formation prevented the binding of the Sp1 protein.47 In more recent work, they showed that the formation of triplex would repress in vitro transcription by blocking Sp1 binding. They also found that triplex formation alone upstream of the transcription start site would repress transcription. It was proposed that the repression in transcription was not strictly due to prevention of transcription factor binding. Their data indicated that triplex formation induced a conformational change that prevented the formation of initiation complexes required for transcription.56 Using a pyrimidine-rich third strand linked to an intercalator, Grigoriev and coworkers showed that triplex formation prevented the binding of the NF kappa B protein to its site on the interleukin-2 receptor alpharegulatory (IT..2R alpha) sequence in vitro. Using an artificial reporter construct placed inside HSB2 cells, a human tumor T cell line, they also showed that introduction of the triplex-forming oligonucleotide reduced transcription levels in vivo.57 Studies done on wild type genes in vivo have given promising results. Orson and coworkers also targeted the kappa B site on the IT..2R alpha gene. They synthesized a purine-rich triplex-forming oligonucleotide and tested its uptake by peripheral blood mononuclear cells. The oligonucleotides isolated from nuclei after treatment showed resistance to degradation up to 21 hours. They also found that addition of the triplexforming oligonucleotide suppressed transcription of the IT..2R alpha gene but did not suppress the IT..2R beta gene nor the c-myc gene.59 Postel and coworkers used a purine-rich triplexforming oligonucleotide targeted to the c-myc promoter in HeLa cells. They showed that the oligonucleotide is not only taken up by the cells, but its target site in the nucleus resists DNase I treatment, presumably after triplex formation. A control oligonucleotide had no effect on resistance to DNase L The mRNA levels from the c-myc gene were found to be reduced in the presence THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

of the triplex-forming oligonucleotide, whereas the control oligonucleotide had no effect.58 McShan and coworkers targeted oligonucleotides that contained an amine group on the 3' terminus to the transcriptional start site and Sp1 binding sites of the human immunodeficiency virus 1 (HIV-1). They found that in vitro transcription with a HeLa cell extract was inhibited by triplex formation. Healthy MT4 cells will reaggregate after dispersal, whereas ones infected with HIV lose this ability because of cytopathic effects.61 They found that a nontriplex-forming oligonucleotide had no effect on cytopathology, whereas treatment with a triplex-forming oligonucleotide allowed reaggregation. In infected U937 cells, they found that treatment with a triplex-forming oligonucleotide resulted in profound inhibition of the full length viral transcript, but that control oligonucleotide had no effect. They also found that viral titers dropped up to fourfold when cells were treated with triplex-forming oligonucleotides, and that the viability of infected cells was substantially improved by treatment with the triplex-forming oligonucleotides.33 Strategies for Improving Effectiveness Base Composition. When C-rich third strand is used

to form triplex over a G-C-rich tract, acidic conditions are required to protonate the N-3 position of the third strand cytosine. This hydrogen is required for Hoogsteen-type hydrogen bonding.lo,n The use of 5-methylcytosines permits triplex formation at a neutral pH by providing the required hydrogen.47,62 Other "unnatural" residues, such as inosine23 or deoxyribonucleosides,37,63 also may be used. Krawczyk and coworkers found that ~-methyl-8oxo-2'-deoxyadenosine (M) can be substituted for 5methylcytosine. Third strands that contained M were shown to be pH independent in triplex formation. They also had a higher affinity for triplex formation than third strands that contained 5-methylcytosine.64 Studies with Mismatched Triplex-Forming Oligonucleotides. Polypurine-polypyrimidine tracts will not

always be available as target sites. An understanding of what bases are allowed in the third strand over a given base pair will result in the design of higher affinity oligonucleotides.9,21,23,35,37,65,66 In a pyrimidine-rich third strand, Griffin and Dervan found that a guanine will stabilize triplex formation over a T:A base pair where the T is in the purine-rich strand of the target duplex. They proposed that the G in the third strand formed a hydrogen bond with the T in the duplex to form an A:T-G triplet.35 NMR data support a novel hydrogen bond between the third strand G and the T of the duplex.67 Studies by Yoon and coworkers confirmed the A:T-G triplet and found evidence for a G:C-T triplet using gel mobility shift and high-pressure liquid chromatography analysis.66 Studies of nearest neighbor effects on triplex formation by a polypyrimidine third strand have been

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Intermolecular DNA Triplexes

done. The C:G-C+ triplet was shown to be more affected by flanking sequences than was the T:A-T triplet. The A:T-G triplet also was shown to be stabilized by being flanked by two T:A-T triplets.sa Computer studies have indicated that the introduction of an interruption in a polypurine-polypyrimidine tract is detrimental to triplex formation. 65 Roberts and Crothers demonstrated that a third strand will discriminate against a duplex that contains just one mismatch. They have based a DNA isolation technique on this discrimination, which they call "stringency clamping."68 Linkers. Home and Dervan successfully formed a triplex at a site where the polypurine-polypyrimidine tract switches strands. They used a target duplex with a 10 bp tract of purines adjacent to a 10 bp tract of pyrimidines. For a third strand to form triplex, it was necessary for the third strand to "crossover" to maintain the Hoogsteen hydrogen bonds with the purines in the duplex at this site. This was accomplished by using a bidirectional polypyrimidine oligonucleotide that contained a 1,2dideoxy-D-ribose that linked. The triplex-forming tracts in the oligonucleotide were maintained at the proper distance and orientation for triplex formation.36 Ono and coworkers also have used third strands with linkers that form triplexes at purine-pyrimidine tracts that switch strands. Their third strands contain linker groups consisting of phosphates and 1,3-propanediols that allow polarity changes in the sugar-phosphate backbone.69 Home and Dervan tested a third strand containing an abasic site over various interruptions in a polypurine-polypyrimidine tract. They found that their abasic site-substituted third strands had lower affinities than some of those that contained standard bases over the interruptions. They attributed this loss in affinity to loss of base stacking interactions and hydrogen bonds.37 Backbone Modifications. Modifications to the DNA phosphodiester backbone of oligonucleotides have been used to increase nuclease resistance.2 Oligonucleotides containing alpha nucleosides32.44.70.71 or phosphorothioates72 have been shown to be triplex formers. In naturally occurring nucleic acids, the nucleosides are in the beta configuration. Anomers synthesized with the nucleosides in the alpha configuration have been shown to resist nucleases. 7o Polypyrimidine alpha oligonucleotides have been shown to form triplexes. Unlike natural polypyrimidine beta oligonucleotides that bind parallel to the polypurine strand of the duplex, the alpha type can bind in an anti-parallel or a parallel orientation, depending upon sequence.32,44.71 Latimer and coworkers synthesized a variety of polynucleotides containing phosphorothioates. The nuclease resistance of the polymers tended to be base composition dependent. Duplexes and triplexes were shown to be formed by these polymers, but further work is needed to determine the usefulness of phosphoro370

thioates at increasing the effectiveness of third strand oligonucleotides.72 Conjugates. As noted before, various chemical moieties can be covalently linked to oligonucleotides. Various conjugates with oligonucleotides have been made to increase triplex binding affinity or resistance to cellular nucleases. The use of such conjugates will improve the effectiveness of triplex-forming oligonucleotides.2,3,32,33,39.40.41.57.73.74 Conjugates made with acridine and polypyrimidine triplex-forming oligonucleotides have been studied extensively. Acridine alone intercalates randomly. However, when it's tethered to a triplex-forming oligonucleotide, site-specific intercalation is found. The triplex gains affinity while the intercalator gains specificity.30,32,39,57.73 Acridine conjugates also have been shown to be readily taken up by cells.2 Using polypyrimidine triplex-forming oligonucleotides with and without a linked acridine, Sun and coworkers showed that the conjugate dissociated from the duplex at a higher temperature than the third strand alone, which indicated a greater thermal stability. Using fluorescence spectroscopy, they also determined that the acridine intercalated at the junction of the triplex and the duplex.30 Birg and coworkers found that a triplex-forming acridine-oligonucleotide conjugate inhibited cytopathic effects of SV40 infection of CV1 cells, whereas control conjugates that had no target sites did not affect cell viability. They also found that acridine alone had no effect. The triplex-forming conjugate inhibited viral DNA synthesis, whereas controls had no effect.73 McShan and coworkers showed that triplex-forming oligonucleotides with a 3' amine blocking group targeted to the HIV-1 virus resisted nuclease degradation. MT4 cells also were shown to readily take up this modified oligonucleotide.33 Summary

Triplex formation promises a site-specific method of targeting DNA.I-4 Site-specific cleavage beyond that allowed by standard restriction endonucleases is now possible for molecular biology studies, such as in genomic mapping.4.51.52 Chemotherapy treatments based on the inhibition of expression of specific oncogenes by triplex-forming oligonucleotides may become available soon.2,6.58 Further research on triplex structure and affinity will result in more effective third strand oligonucleotides and derivatives useful in a biologic setting. Triplex-forming compounds are likely to permit the therapeutic modulation of gene expression.2,3.6,21 Acknowledgments

We thank Becky Carroll and Barbara Wilson for help in preparing this manuscript. References 1. Helene C, Toulme J-J: Specific regulation of gene expression by

antisense, sense and antigene nucleic acids. Biochem Biophys Acta 1049:99-125, 1990. December 1992 Volume 304 Number 6

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December 1992 Volume 304 Number 6