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Recent improvements in antigene technology
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Recent improvements in antigene technology Sabrina Buchini and Christian J Leumann DNA triple-helix-based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother-like treatment in comparison to the flourishing field of oligonucleotide-based control of translation (antisense technology). This was mostly due to lack of affinity of triplex-forming oligonucleotides to their DNA target, to sequence restriction constraints imposed by the triple helical recognition motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine–purine inversion sites as well as sugar modifications, for example, the 20 -aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid sugar unit, which greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence-specific genomic DNA manipulation is starting to become a useful tool in biotechnology. Addresses Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland e-mail: [email protected]
Current Opinion in Chemical Biology 2003, 7:717–726 This review comes from a themed section on Biopolymers Edited by Thomas Carell and Peter Seeberger 1367-5931/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2003.10.007
Abbreviations AE-TFO 20 -aminoethoxy TFO PNA polyamide or peptide nucleic acids TFO triplex-forming oligonucleotide
Introduction Selective artificial control of gene expression is a longstanding dream in biotechnology and human therapy. Oilgonucleotides seem perfectly suited for this purpose because of their unique base–base recognition properties. In the antisense approach, short oligonucleotides are designed to bind to specific sequences of an mRNA of interest via Watson–Crick duplex formation, to block gene expression on the level of translation according to various molecular mechanisms. This approach is widely explored not only in the therapeutic area, where approximately 70–80 oligonucleotides targeted to various diseases are currently in clinical trials, but also as tools to downregulate gene expression in the area of functional www.current-opinion.com
genomics. In the antigene approach (Figure 1), oligonucleotides bind sequence selectively to genomic, doublestranded DNA and interfere with transcription and the DNA processing machinery via triple helix formation. Some intrinsic advantages of the antigene over the antisense principle can be identified. First, there are only two target copies of DNA per diploid cell as compared with the hundreds to thousands of mRNA copies that have to be targeted in the antisense approach. This should dramatically reduce the amount of oligonucleotide needed for activity. Moreover, not only transcriptional activation and deactivation but also gene knockout as well as targeted mutagenesis, targeted recombination and sequence-selective manipulation of genomic DNA can be achieved. Despite the potential benefits for biotechnology in general, the investment of resources has been limited, and therewith progress in this field has been slow. Consequently, there is still no antigene oligonucleotide in clinical trials. Two stable DNA triple-helical motifs are known that differ according to the orientation and base composition of the third strand. In the parallel or pyrimidine binding motif, the homo-pyrimidine triplex-forming oligonucleotide (TFO) is parallel oriented to the duplex purine strand forming H-bonded Hoogsteen Cþ GC and T AT basetriplets. The antiparallel or purine motif is characterized by G GC, A AT and T AT reverse Hoogsteen base-triplets between a purine rich TFO that is antiparallel oriented to the purine strand of the DNA duplex (Figure 2). In both motifs, the third strand lies in the DNA major groove, and requires a target DNA homopurine–homopyrimidine sequence tract, ideally of 15–30 nucleotides. The potential advantages in targeting genomic DNA by oligonucleotides are counterbalanced by several restrictions common to all oligonucleotide-based approaches (e.g. biostability, cell permeability and non-specific binding of the oligonucleotides or analogues used). Further, the triplex approach shows specific restrictions. Some of them are motif-dependent while others are of general nature. For the parallel motif, the severest restriction is the pHsensitivity of the Cþ GC base triplet, which is intrinsically unstable under physiological conditions due to the low pKa of the third strand cytosines. Triplex stability in the antiparallel motif is markedly sequence-dependent due to alternative structure formation (e.g. G tetrads), of the corresponding GA or GT rich TFOs under physiological conditions (especially Kþ concentration). Finally, there are common problems in both motifs. The most prominent ones are related to target accessibility, low Current Opinion in Chemical Biology 2003, 7:717–726
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Figure 1
Nucleus Protein 1
mRNA 2
Major groove
3 + DNA target
Triple helix
Cytoplasm TFO 1
2
3
Modulation of gene expression
Site-specific mutagenesis
Modification of genomic DNA
Transcription initiation and inactivation
Directed mutagenesis Homologous recombination Replication inhibition
DNA cleavage or crosslinking at selected gene loci Current Opinion in Chemical Biology
Basic antigene concepts and corresponding applications. Gene expression can be upregulated or downregulated on the level of transcription via selective triple-helix formation, preferably at promoter sites. Selective modification of the genome includes site-specific mutagenesis via triplex delivered mutagens (e.g. psoralen), or homologous recombination through triplex delivered donor DNA via DNA repair. Further applications arise from triplex-targeted chemical modification of the gene (e.g. cleavage, site-specific cross-linking or alkylation).
overall affinity of TFOs to their target, resulting in poor competition with DNA binding proteins, and the restriction of the known binding motifs to homopurine/ homopyrimidine DNA sequence tracts. The occurrence and function of triple helical structures in vivo [1], the use of TFOs as modulators of gene expression [2] and as gene modifiers [3], and the more chemical and molecular recognition aspects of nucleobase modifications [4] have all been comprehensively reviewed elsewhere. In our opinion, this review highlights the most relevant advances in the past two years on the development of oligonucleotide analogues with improved binding and sequence recognition properties as well as in gene targeting.
Increasing triplex stability There are a few basic concepts in improving the binding of TFOs to duplex targets that have been successfully applied in the past few years to increase triplex stability. First, the introduction of positively charged amino functions on the sugar and/or base unit, designed to make additional binding contacts between phosphodiester residues of the target Watson–Crick duplex besides the Current Opinion in Chemical Biology 2003, 7:717–726
base–base interactions (dual recognition concept). Second, the use of conformationally adapted or constrained TFOs. Finally, the use of TFOs with a non-charged backbone. Most recent progress has focused on the parallel binding motif of TFOs. Figure 3 displays the molecular structures of some modifications discussed in more detail below.
Dual recognition The best explored and most promising parallel motif TFOs are those containing an aminoethoxy side-chain at C(20 ) of the ribonucleoside units, developed by Cuenoud and collegues [5] (Figure 3). Under physiological conditions, the amino function of this side-chain is protonated and leads to an increase in thermal triplex stability of ca. 3.58C per modification at pH 7.0. Fully modified 20 -aminoethoxy TFOs (AE-TFOs) can lead to triplexes that are more stable than the target duplex alone. The gain in thermodynamic stability (DG) of ca. 0.5 kcal/mol for each modified unit is mostly due to a 1000-fold enhanced association rate compared with that of a DNA–TFO. This is an important feature considering that association kinetics for triplex formation are substantially lower as compared with duplex formation. The stabilizing effect was shown by NMR to arise from www.current-opinion.com
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Figure 2
(a) Parallel motif
N O
N H
H N H
H N
H
N
O N N
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H
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Molecular structures of the canonical Hoogsteen and reverse Hoogsteen base triplets in the known parallel and antiparallel triple helical binding motifs.
specific hydrogen bonds of the protonated amino function to the pro-R non-bridging phosphate oxygen of the purine strand of the underlying duplex target [6]. The 20 -amino side-chain occurs in a gaucheþ conformation and shows astonishingly low conformational mobility in the triplex [7]. Variations in the length of the side chain did not further improve binding efficiency. AE-TFOs have recently been shown to vastly broaden in vitro and in vivo applications of antigene oligonucleotides (see below). Another point of attachment of alkylamino chains pointing towards the same spatial direction as in AE-TFOs is the position C(40 ) of the deoxyribose unit. Matsuda and co-workers recently synthesized the corresponding 40 amino-TFOs and found some thermal triplex stabilization for the ethylamino side chain; however, only by less than 18C per modification [8]. Shortening or lengthening www.current-opinion.com
the tether between C(40 ) and the amino function consistently reduced affinity relative to the two-carbon linker. There is no doubt, however, that the efficiency of this system does not match that of the AE-TFOs. Related to the same concept, our group has embarked on the evaluation of the properties of pyrrolidino-pseudonucleosides [9]. According to modelling based on X-ray data available for parallel motif triplexes, the furanose ring oxygen is close to a pro-R non-bridging phosphate oxygen of the purine duplex strand. Thus, replacement of this oxygen by nitrogen was expected to bring about additional stability in triplex formation. As a tribute to chemical stability, the linkage between base and amino sugar had to be changed into a C-glycosidic linkage, as in the pseudonucleosides. Experiments with TFOs containing the base uracil or N-1-methyl uracil for recognizing Current Opinion in Chemical Biology 2003, 7:717–726
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Figure 3
Conformationally constrained or uncharged
Dual (base and phosphate) recognition O O
O
Py
O O –O P O O
O
Py
+H N 3
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PyrrolidinoTFOs
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n O O –O P O O DNA n=1, LNA (BNA) n=2, ENA
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ψPy Current Opinion in Chemical Biology
Molecular structures of TFOs with potential dual recognition properties, with conformationally constrained sugar analogues and with uncharged backbone structures.
adenine in the parallel binding motif, however, showed triplex destabilization [10], whereas those containing the base isocyctosine increased triplex stability at neutral pH by ca. 28C in Tm per modification [11]. The reason for the differential stabilization/destabilization of the pyrrolidino pseudonucleosides is unclear.
Variation of TFO conformation, and sugar backbone modified TFOs Generally, RNA-TFOs form more stable triplexes than do DNA-TFOs in the pyrimidine binding motif. Various NMR studies on DNA-triplexes were in favor of structures in which not all deoxyribose units of a TFO adopt the same sugar pucker. Thus, the overall helical conformation of DNA triplexes is somewhere between A and B [12]. A recent study with 20 -deoxy- and 20 -O-methyl-RNA TFOs showed marked sequence-dependent differences in stability that cannot easily be rationalized with known rules in conformational analysis [13]. Thus, extrapolation of triplex stabilities from determined sequences to any sequence is difficult. Within this context, we developed a combinatorial method to determine conformational effects of TFOs on triplex stability [14]. The method is outlined in Figure 4. In short, we prepared a 12-mer oligonucleotide library of TFOs via the ‘split and combine’ technique in which any except the 30 -terminal position is occupied by either a ribo- or a deoxyribonucleoside while the bases are Current Opinion in Chemical Biology 2003, 7:717–726
kept invariant. This enables conformational variation within a given base recognition frame, due to the intrinsic preferences of the deoxyribo- (20 -endo) and ribo- (30 endo) nucleosides. The library was then screened for stable triplex binders by fractionated affinity chromatography on an agarose immobilized DNA target in a temperature gradient. The resulting fractions were deconvoluted by mass spectrometry (nature and number of respective nucleoside units in the TFOs) and partial alkaline hydrolysis followed by gel electrophoresis (sequential position of ribonucleosides). This information was then used to build focused libraries for concept validation and isolation of individuals. With this method, the complete conformational space of a chimeric TFO containing ribound deoxyribonucleotides towards its DNA target could be screened. Two main pieces of information resulted from this analysis: first, there exists no ‘magic combination’ of a chimeric TFO with enhanced binding relative to the allRNA-TFO; and second, there is less sequence/stability variation for Cþ-GC than for T(U)-AT triplet formation. With slight variations of this combinatorial assay, sequence/stability effects of TFOs containing non-natural backbone modifications can also be addressed, which is certainly of interest as conformational variations in the corresponding TFOs can be higher in these cases. Another interesting approach involves the use of TFOs built from conformationally constrained nucleoside units. As with duplex formation, less entropy loss upon triplex www.current-opinion.com
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Figure 4 Ribonucleosides
Deoxyribonucleosides
O
O
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Base O
O
O OH
O
3′-endo 2′-endo DNA target: 5′- AAAAGAGGAGGG TTTTCTCCTCCC-3′ TFO Library:
5′-JJJJKJKKJKKC-
J = dT,rU K = rC, dC
Library size: 211 = 2048
TFO library +
Hybridize DNA target Affinity chromatography selection (temp. gradient)
Solid phase
Focused library preparation
Definition of binding features
Deconvolution (ESI-MS base digest) Current Opinion in Chemical Biology
Schematic representation of the combinatorial approach developed for establishing a structure/stability profile of TFOs with conformational variation in the backbone. Within a given base recognition frame imposed by the target duplex, an oligonucleotide library with 2048 individual oligonucleotides was constructed, containing either a ribo- rU/rC or a deoxyribo- (dT/dC) sugar unit at each position in the chain by the split and combine technique. For convenience, deoxycytidine was chosen as the 30 -terminus. This library was then loaded on the agarose-supported DNA target and fractionated via affinity chromatography in a temperature gradient. The fractions were then deconvoluted by mass spectrometry combined with alkaline digestion to determine the preferred composition and location of ribonucleosides within the sequences. This information was then used to produce focused libraries for a subsequent round of selection.
formation, and thus higher triplex stability is expected. Given the preference of RNA- over DNA-TFOs in the parallel binding motif, oligonucleotide analogues that are constrained in a 30 -endo (RNA-like) conformation should be advantageous. Imanishi and co-workers investigated triplex formation with the conformationally constrained RNA analogue LNA in the pyrimidine motif and found substantial increases in thermal and thermodynamic stability of corresponding triplexes at neutral pH [15] (Figure 3). (The abbreviation BNA is synonymous for LNA and was coined by the Imanishi group. We use here consistently the better known abbreviation LNA.) There exists an optimal number of LNA-residues within a DNA-TFO that exert a stabilizing effect. Interestingly, fully modified LNA-TFOs do not form triplexes with DNA targets at all. A kinetic analysis of triplex formation with LNA revealed that most of the observed increase in the binding constant at neutral pH arises from a decreased dissociation rate [16]. Data also exist on triplex binding www.current-opinion.com
with the LNA-analogue ENA, containing a 20 ,40 -ethylene bridge [17]. ENA is able to stabilize triplex formation at neutral pH roughly to the same extent as LNA. Notably, fully modified ENA-TFOs do bind to their DNA target while fully modified LNA-TFOs do not. This phenomenon is not completely understood. Given the properties of LNA-TFOs and AE-TFOs, it is tempting to propose a combination of conformational constriction (reducing the rate of dissociation) with a properly positioned alkylamino function as in AE-TFOs (increasing the rate of association) to unite the best properties of both and produce even stronger binding TFOs (see also Update). Representatives of the class of neutral backbone oligonucleotide analogues with interesting antigene properties are morpholino-DNA and the polyamide or peptide nucleic acids (PNAs). Dimeric bis-PNA pyrimidine Current Opinion in Chemical Biology 2003, 7:717–726
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sequences bind to DNA duplexes via strand invasion (Ploop formation), thus offering the possibility for sitespecific modulation of gene expression or modification of the gene. Because the properties of PNA have been extensively reviewed elsewhere, we do not include them here systematically and refer the interested reader to two recent papers [18,19]. The morpholino oligonucleotides (Figure 3) bind to target DNA in the parallel but not in the antiparallel motif and are nuclease resistant. Binding in the parallel motif requires low pH and takes place in the absence of Mg2þ or Kþ ions (salt free conditions). A 25-mer morpholino oligonucleotide targeted to the HER-2/neu promoter region in the presence of 140 mM Kþ with and without 10 mM Mg2þ at pH 5.0 shows ca. twofold increased binding as compared with an unmodified DNA-TFO [20]. Thus, it is not stability that excels in this system. The advantages are more related to cellular uptake and distribution due to the non-charged backbone.
formation was assayed by Tm analysis as well as by a nuclease protection assay in mixtures of TFOs competing for the DNA target. The anthraquinone unit A (Figure 5) was found to give highest affinity to the TA site when positioned in the center of the TFO. It is, however, not specific, presumably because of unspecific intercalation. Although no hit or lead was discovered, the combinatorial rationale behind the approach is certainly of interest.
Expanding the sequence recognition range
Deoxynucleosides with acceptor or double acceptor bases of the pyridone (P) [26] or pyrimidone (4HT) [27] type have recently been investigated as units to target CG inversion sites in parallel TFOs (Figure 5). They are essentially C(4) carbonyl deletion mutants of thymine, which disables their binding to an AT base pair while maintaining the binding profile to CG sites. Both bases were shown to be highly selective for CG inversion sites but both have compromised affinity when compared with a canonical base-triple. Extension of the aromatic surface while maintaining the double acceptor nature of the base, as in Q (Figure 5) did not solve the affinity problem. In contrast, the selectivity for CG recognition was compromised by almost isoenergetic recognition of a GC basepair [28].
An obvious drawback in antigene technology is the restriction of TFO binding to homopurine/homopyrimidine DNA target sequences. Although such sequences are statistically over-represented in the genome it would be desirable to have bases at hand that specifically recognize pyrimidine bases or whole pyrimidine/purine Watson– Crick base-pairs. Recognizing pyrimidine bases is hampered by the fact that only one H-bond donor or acceptor site of C and T is available for binding in the major groove. Recognizing T in a AT base-pair is even more complicated because of the presence of the methyl group, which leads to steric interference with the TFO backbone, especially in the parallel binding motif. Alternatively, one can envisage the use of combinations of known binding motifs in one TFO or design new binding motifs from scratch. Research into this direction has been ongoing fot more than 10 years, but a general solution is elusive. The following section highlights a few recent variations on this theme.
Base modifications A series of new base analogues of the thiazolylaniline or -benzimidazol type, recognizing a TA inversion site, have been proposed recently by Fourrey and Benhida [21,22,23]. The design of these bases was inspired by earlier work by the Dervan group on the base D3, which showed substantial affinity but lacked sequence specificity due to non-specific intercalation. Within the parallel motif, the base S (Figure 5) which is the best representative of this class of base analogues so far, was found to bind to a TA inversion site with an affinity that almost matches that of a T-AT canonical base triplet, and with reasonable selectivity. Another approach to find new bases recognizing TA inversion sites was based on a combinatorial synthesis/nuclease protection assay by Richert and co-workers [24]. In this case, a series of different aromatic units were attached to an acyclic ribose surrogate and incorporated into parallel TFOs. Triplex Current Opinion in Chemical Biology 2003, 7:717–726
Approaches in our group to overcome the sequence restriction in the antiparallel binding motif have led to the evaluation of the base 2-aminopurine in TFOs at TA inversion sites. In a study comprising both anomers (a/b) of the N-9 or N-7 regioisomeric deoxyribonucleosides of 20 -aminopurine in DNA TFOs, we found that the N7aisomer preferentially recognizes a TA inversion site with acceptable but not exceptional base-pair selectivity and affinity [25].
An alternative way to resolve the homopurine requirement for triplex formation consists in the design of TFOs with new major groove binding motifs. In this context, we [29] and others [30] have explored with limited success TFOs that are designed to recognize purine bases on either strand of the Watson–Crick duplex by using a structural switch. In these oligonucleotides, an N7/N9 and/or an a/b structural switch on aminopurine, hypoxanthine, thymine or 4-guanidinocyctosine nucleosides were used to recognize consecutive purine bases on either strand of the target DNA. The lack of affinity in such switch-TFOs is probably due to missing intrastrand stacking of the bases at the switch site. An interesting novel approach was recently discussed by Gold and colleagues using 2-aminoquinazoline C-nucleosides [31].
Combinations of base and sugar modifications An obvious task in further expanding the triplex recognition range of TFOs lies in the combination of specific pyrimidine–purine inversion site bases with overall triplex-enhancing backbone modifications. Progress in www.current-opinion.com
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Figure 5
Targeting T-A inversions DNA O
S O
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Current Opinion in Chemical Biology
Molecular structures of recently investigated base analogues for targeting TA or CG inversion sites on otherwise homopurine/homopyrimidine duplex targets.
this field has been very successful and will be highlighted in three examples from the recent literature. Replacing the deoxyribose unit on the 2-pyridone nucleoside by an LNA sugar unit (2-pyridone-LNA-TFOs, Figure 6) led to an increase in Tm of 98C in a corresponding TFO 15-mer binding to a DNA target with a CG inversion site at neutral pH, without compromising selectivity [26,32]. Up to three CG inversions within a 15mer TFO, adding up to 20% of pyrimidine content within the purine sequence of the target DNA, could be recognized in this way. Additional gain in stability could be realized by replacing standard pyrimidine nucleosides forming canonical base-triples, by corresponding LNA units in the TFO. This is a good example on how sugar and base modification work in symbiosis. Another demonstration of the power of combination is the case of 20 O-aminoethyl nucleosides containing the 5propargylamino-U base. In the context of a TFO, this www.current-opinion.com
nucleoside analogue carries two positive charges at neutral pH. It was shown by DNase I footprinting and thermal denaturation studies on intramolecular parallel motif triplexes that these bisubstituted nucleoside derivatives produce a large increase in triplex stability, much greater than that produced by either of the monosubstituted analogues [33]. Also, propargylguanidino-U bases were recently reported to enhance triplex stability [34]. Although these cases do not contribute to the expansion of the triplex recognition code, it is conceivable that a propargylamino modification of, for example, a pyridone or pyrimidone base might additionally enhance CG recognition in the same way. We have developed the 20 -aminoethyl nucleoside containing the base 4-deoxothymine (4HT) (Figure 6). The aminoethyl modification also adds, in this case, favorably to triplex stability for CG inversion recognition [35].
Other developments It is clear that there are many other ways to increase triplex stability or modulate target recognition of TFOs. Current Opinion in Chemical Biology 2003, 7:717–726
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Figure 6
NH3+ O NH O
N
O
O O O –O P O O
N O
N
O
O
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NH3+
Propargylamino-UAE-TFOs
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Current Opinion in Chemical Biology
Promising TFO candidates combining stability increasing sugar modifications with either stability increasing or recognition range expanding base modifications.
Well explored concepts include the use of TFOs containing triplex-specific intercalators. The extreme variant of target binding is covalent attachment of a TFO to the DNA duplexes (e.g. via psoralen cross linking or cross linking with reactive base analogues). One recent example of the latter kind uses 2-amino-6-vinyl purine deoxyribosides in parallel TFOs as an electrophilic unit that reacts irreversibly with the amino functions of adenine at TA inversion sites [36]. This might be useful for monitoring TA interruptions within homopurine sequence tracts. Another interesting addition to the already large palette of functional TFOs are pyrimidine oligonucleotides that carry an azobenzene unit as a base substitute. By irradiation with light at two specific wavelengths cistrans isomerization can be effected, which enables or inhibits binding of the TFO to its target [37]. This might allow photoregulation of cellular processes based on antigene technology.
Antigene applications As pointed out in the Introduction, there are several cellular functions that can be controlled via the antigene principle. In the past, lack of success in such applications was mostly due to insufficient target affinity. With the new generation of strong binding TFOs, this picture will change. Most advanced at this stage are investigations with psoralene-modified DNA-TFOs, AE-TFOs and PNA-clamp-TFOs. Some examples of the recent literature are compiled below. With a 30 -psoralene conjugated 16 bp DNA-TFO, designed to bind in the antiparallel motif to the third intron of the human intercellular adhesion molecule 1 (ICAM-1) gene, repressor activity was found in vitro, in cell nuclei and in a human keratinocyte cell line (A431). This shows Current Opinion in Chemical Biology 2003, 7:717–726
that the ICAM 1 target sequence in the chromatin context in cell nuclei is available for triplex formation [38]. With a restriction protection assay to physically detect triple helix directed psoralene cross-links at chromosomal sites in mammalian cells, it was shown that an (antiparallel) TFO could bind to a chromosomal target site in human cells even in the absence of transcription [39]. Substantial increases in target site binding of the TFO was observed upon specific induction of transcription. These findings provide evidence that physiological activity at a chromosomal target influences its accessibility to TFOs and suggests that gene targeting may be most effective at highly expressed chromosomal loci. An AE-TFO designed to bind in parallel fashion to the IgE germline proximal promoter overlapping with the binding site of three proteins inhibited binding of all three proteins in a dose-dependent way in vitro. Functionally, this resulted in a selective inhibition of IL-4 induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells [40]. This is a successful application of 20 -aminoethoxy-TFOs towards specific modulation of gene expression via DNA triplex formation. Targeted gene knockout by AE-TFOs was reported by Seidman and collegues [41,42]. Psoralene-linked aminoethoxy-TFOs were designed to target and mutagenise a site in the hamster HPRT gene. Knockout frequencies that are 300-400-fold above background could be observed. Site-specific mutagenesis of a chromosomal gene could also be achieved with AE-TFOs in the parallel binding motif in a cellular assay. It was reported that the targeting efficiency of the TFO was sensitive to the cellcycle status. Targeted mutagenesis showed the greatest activity in the S phase with ca. 20–30% of adduct formation. Ca. 75–80% of these adducts were repaired faithfully, leading to a mutation frequency of ca. 5% [43]. A variation of site-specific mutagenesis was reported in which covalent crosslinking of a TFO to its target was achieved via the reactive base 2-amino-6-vinyl purine of the TFO with a nucleophilic amino function of a neighboring cytosine unit [44]. This opens the way for using other mutagens than psoralene in triplex-targeted mutation experiments. Site-directed inhibition of DNA replication was recently shown with DNA-TFOs and PNA. Triplex-mediated inhibition of DNA replication targeted to the polypurine sequence tract of HIV-1 was observed with both, covalent psoralene adduct forming DNA-TFOs or non-covalent binding bisPNAs [45]. Another appealing application of triplexes in vivo is inducing site-directed recombination with donor DNA fragments. In a recent report, a bis-PNA clamp was coupled to a 40-nt donor DNA fragment that was homologous www.current-opinion.com
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to an adjacent region in the target gene. The PNA–DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency of at least 60-fold above background [46]. Recombination was found to be dependent on the nucleotide excision repair factor XPA. Thus, it seems that recombination is DNA repair driven and induced by a helical distortion of the DNA target upon bis-PNA– DNA triplex formation. This may be a potentially useful strategy to achieve targeted correction of defective genes.
This is a comprehensive review with 462 references, covering all relevant nucleobase modifications and their effect on triplex stability up to the end of 2001. 5.
Cuenoud B, Casset F, Hu¨ sken D, Natt F, Wolf RM, Altmann K-H, Martin P, Moser HE: Dual recognition of double-stranded DNA by 2-aminoethoxy-modified oligonucleotides. Angew Chem Int Ed Engl 1998, 37:1288-1291.
6.
Blommers MJJ, Natt F, Jahnke W, Cuenoud B: Dual recognition of double-stranded DNA by 2(-aminoethoxy-modified oligonucleotides: the solution structure of an intramolecular triplex obtained by NMR spectroscopy. Biochemistry 1998, 37:17714-17725.
7.
Carlomagno T, Blommers MJJ, Meiler J, Cuenoud B, Griesinger C: Determination of aliphatic side-chain conformation using cross-correlated relaxation: application to an extraordinarily stable 2(-aminoethoxy-modified oligonucleotide triplex. J Am Chem Soc 2001, 123:7364-7370.
8.
Atsumi N, Ueno Y, Kanazaki M, Shuto S, Matsuda A: Nucleosides and nucleotides. Part 214: thermal stability of triplexes containing 4(-a-C-aminoalkyl-2(-deoxynucleosides. Bioorg Med Chem 2002, 10:2933-2939.
9.
Ha¨ berli A, Leumann CJ: Synthesis of pyrrolidino C-nucleosides via Heck reaction. Org Lett 2001, 3:489-492.
Conclusions Antigene applications in general have already, and will do more so in the future, greatly benefited from recent advances in increasing triplex stability and modulating base recognition specificities by designed sugar and base modifications. Especially, modifications of TFOs exploiting the parallel binding motif show great promise in this respect. The combination of general triplex enhancing structural features (e.g. the 20 -aminoethoxy modifications or conformationally constrained sugars, such as in LNA) with novel bases for selective pyrimidine-purine inversion site recognition lends hope to the disappearance of the sequence recognition constraints for TFOs in the near future. Thus, the day where antigene oligonucleotides will complement and in some cases even challenge antisense oligonucleotides as biomolecular tools or therapeutics may come soon.
Update At the stage of proofreading of this manuscript, an article appeared describing the synthesis and duplex binding properties of 20 -aminoethyl modified 20 -amino-LNA [47]. This structure perfectly combines the features of conformational rigidity and dual binding and may be an interesting candidate as high affinity TFO. However, triplex binding data are yet missing.
Acknowledgements The authors thank the Swiss National Science foundation for ongoing financial support of our research in the field of molecular recognition of DNA.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Zain R, Sun J-S: Do natural DNA triple-helical structures occur and function in vivo? Cell Mol Life Sci 2003, 60:862-863.
2.
Guntaka RV, Varma BR, Weber KT: Triplex-forming oligonucleotides as modulators of gene expression. Int J Biochem Cell Biol 2003, 35:22-31.
3.
Seidman MM, Glazer PM: The potential for gene repair via triple helix formation. J Clin Invest 2003, 112:487-494.
4.
Robles J, Grandas A, Pedroso E, Luque FJ, Eritja R, Orozco M: Nucleic acid triple helices: stability effects of nucleobase modifications. Curr Org Chem 2002, 6:1333-1368.
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10. Ha¨ berli A, Leumann CJ: DNA binding properties of oligodeoxynucleotides containing pyrrolidino C-nucleosides. Org Lett 2002, 4:3275-3278. 11. Ha¨ berli A, Mayer A, Leumann CJ: Pyrrolidino-DNA. Nucleosides Nucleotides Nucleic Acids 2003, 22:1187-1189. 12. Tarko¨ y M, Phipps AK, Schultze P, Feigon J: Solution structure of an intramolecular DNA triplex linked by hexakis(ethylene glycol) units: d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6TCTCTCTT). Biochemistry 1998, 37:5810-5819. 13. Cassidy RA, Puri N, Miller PS: Effect of DNA target sequence on triplex formation by oligo-2(-deoxy- and 2(-Omethylribonucleotides. Nucleic Acids Res 2003, 31:4099-4108. 14. Bernal-Me´ ndez E, Leumann CJ: Conformational diversity versus nucleic acid triplex stability, a combinatorial study. J Biol Chem 2001, 276:35320-35327. We find this to be a potentially very useful combinatorial method to rapidly screen sequence effects on triplex (or duplex) binding. The advantage over selex and related methods is that there is no enzymatic amplification step necessary, thus allowing also the screening of sugar-modified nucleotide units. 15. Obika S, Uneda T, Sugimoto T, Nanbu D, Minami T, Doi T, Imanishi T: 2(-O,4(-C-Methylene bridged nucleic acid (2(,4(-BNA): Synthesis and triplex forming properties. Bioorg Med Chem 2001, 9:1001-1011. 16. Torigoe H, Hari Y, Sekiguchi M, Obika S, Imanishi T: 2(-O,4(-Cmethylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies. J Biol Chem 2001, 276:2354-2360. 17. Koizumi M, Morita K, Daigo M, Tsutsumi S, Abe K, Obika S, Imanishi T: Triplex formation with 2(-O,4(-C-ethylene-bridged nucleic acids (ENA) having C3(-endo conformation at physiological pH. Nucleic Acids Res 2003, 31:3267-3273. This variant of LNA was shown to have poorer RNA single-strand binding properties compared with LNA. This seems not to be the case in triplex formation where it shows similar Tm enhancements to LNA, but does not suffer from the non-binding of fully modified oligonucleotides to their DNA target. 18. Demidov VV, Frank-Kamenetskii MD: Sequence-specific targeting of duplex DNA by peptide nucleic acids via triplex strand invasion. Methods 2001, 23:108-122. 19. Nielsen PE: Targeting double stranded DNA with peptide nucleic acids (PNA). Curr Med Chem 2001, 8:545-550. 20. Basye J, Trent JO, Gao D, Ebbinghaus SW: Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif. Nucleic Acids Res 2001, 29:4873-4880. Current Opinion in Chemical Biology 2003, 7:717–726
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21. Guianvarc’h D, Benhida R, Fourrey J-L, Maurisse R, Sun J-S: Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine.pyrimidine interruption. Chem Commun (Camb) 2001:1814-1815. This paper describes one of the best nucleobase analogues for TA inversion site recognition in DNA-TFOs, in terms of affinity. 22. Guianvarc’h D, Fourrey J-L, Maurisse R, Sun J-S, Benhida R: Design of artificial nucleobases for the recognition of the AT inversion by triple-helix forming oligonucleotides: a structurestability relationship study and neighbour base effect. Bioorg Med Chem 2003, 11:2751-2759.
been investigated so far. Thus, the rigorous proof of principle has yet to come. 34. Roig V, Asseline U: Oligo-2(-deoxyribonucleotides containing uracil modified at the 5-position with linkers ending with guanidinium groups. J Am Chem Soc 2003, 125:4416-4417. 35. Buchini S, Leumann CJ: Dual recognition of a pyrimidine-purine inversion site: synthesis and binding properties of triplex forming oligonucleotides containing 2(-aminoethoxy-5methyl-1H-pyrimidin-2-one ribonucleosides. Tetrahedron Lett 2003, 44:5065-5068. 36. Nagatsugi F, Matsuyama Y, Maeda M, Sasaki S: Selective crosslinking to the adenine of the TA interrupting site within the triple-helix. Bioorg Med Chem Lett 2002, 12:487-489.
23. Guianvarc’h D, Fourrey J-L, Maurisse R, Sun J-S, Benhida R: Synthesis, incorporation into triplex forming oligonucleotide, and binding properties of a novel 2(-deoxy-C-nucleoside featuring a 6-(thiazolyl-5)benzimidazole nucleobase. Org Lett 2003, 4:4209-4212.
37. Liang X, Asanuma H, Komiyama M: Photoregulation of DNA triplex formation by azobenzene. J Am Chem Soc 2002, 124:1877-1883.
24. Mokhir AA, Connors WH, Richert C: Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices. Nucleic Acids Res 2001, 29:3674-3684.
38. Besch R, Giovannangeli C, Kammerbauer C, Degitz K: Specific inhibition of ICAM-1 expression mediated by gene targeting with triplex-forming oligonucleotides. J Biol Chem 2002, 277:32473-32479.
25. Parel SP, Leumann CJ: Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides. Nucleic Acids Res 2001, 29:2260-2267.
39. Macris MA, Glazer PM: Transcription depedence of chromosomal gene targeting by triplex-forming oligonucleotides. J Biol Chem 2003, 278:3357-3362.
26. Obika S, Hari Y, Sekiguchi M, Imanishi T: A 2(,4(-bridged nucleic acid containing 2-pyridone as a nucleobase: efficient recognition of a C-G interruption by triplex formation with a pyrimidine motif. Angew Chem Int Ed Engl 2001, 40:2079-2081. 27. Pre´ vot-Halter I, Leumann C: Selective recognition of a C-G base-pair in the parallel DNA triple-helical binding motif. Bioorg Med Chem Lett 1999, 9:2657-2660. 28. Pre´ vot I, Leumann CJ: Evaluation of novel third strand bases for the recognition of a C-G base-pair in the parallel DNA triplehelical binding motif. Helv Chim Acta 2002, 85:502-515. 29. Parel SP, Marfurt J, Leumann CJ: DNA triple-helix formation at pyrimidine-purine inversion sites. Nucleosides Nucleotides Nucleic Acids 2001, 20:411-417. 30. Doronina SO, Behr J-P: Synthesis of 4-guanidinopyrimidine nucleosides for triple helix-mediated guanine and cytosine recognition. Tetrahedron Lett 1998, 39:547-550. 31. Li JS, Fan YH, Zhang Y, Marky LA, Gold B: Design of triple helix forming C-glycoside molecules. J Am Chem Soc 2003, 125:2084-2093. 32. Obika S, Hari Y, Sekiguchi M, Imanishi T: Stable oligonucleotide directed triplex formation at target sites with CG interruptions: strong sequence-specific recognition by 2(,4(-bridged nucleic-acid-containing 2-pyridones under physiological conditions. Chemistry 2002, 8:4796-4802. This article describes probably the best CG inversion binder known so far. The combination of conformationally restricted sugar analogues with novel bases that selectively recognize pyrimidine bases is a very promising strategy to alleviate the sequence restriction problem in DNA triplex formation. 33. Sollogoub M, Darby RA, Cuenoud B, Brown T, Fox KR: Stable DNA triple helix formation using oligonucleotides containing 2(-aminoethoxy-5-propargylamino-U. Biochemistry 2002, 41:7224-7231. An interesting way to improve triplex stability that may be applied also to non-standard bases. Unfortunately, only monomolecular triplexes have
Current Opinion in Chemical Biology 2003, 7:717–726
40. Stu¨ tz AM, Hoeck J, Natt F, Cuenoud B, Woisetschla¨ ger M: Inhibition of interleukin-4- and CD40-induced IgE germline gene promoter activity by 2(-aminoethoxy-modified triplexforming oligonucleotides. J Biol Chem 2001, 276:11759-11765. 41. Puri N, Majumdar A, Cuenoud B, Natt F, Martin P, Boyd A, Miller PS, Seidman MM: Targeted gene knockout by 2(-O-aminoethyl modified triplex forming oligonucleotides. J Biol Chem 2001, 276:28991-28998. 42. Puri N, Majumdar A, Cuenoud B, Natt F, Martin P, Boyd A, Miller PS, Seidman MM: Minimum number of 2(-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides. Biochemistry 2002, 41:7716-7724. This paper contains important information as to the design of AE-TFOs related to knockout activity. It also contains a side by side comparison of the functionality of AE-TFOs with standard 20 -OM-TFOs. 43. Majumdar A, Puri N, Cuenoud B, Natt F, Martin P, Khorlin A, Dyatkina N, George AJ, Miller PS, Seidman MM: Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide. J Biol Chem 2003, 278:11072-11077. 44. Nagatsugi F, Sasaki S, Miller PS, Seidman MM: Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog. Nucleic Acids Res 2003, 31:e31. 45. Diviacco S, Rapozzi V, Xodo LE, He´ le`ne C, Quadrifoglio F, Giovannangeli C: Site-directed inhibition of DNA replication by triple helix formation. FASEB J 2001, 15:2660-2668. 46. Rogers FA, Vasquez KM, Egholm M, Glazer PM: Site-directed recombination via bifunctional PNA-DNA conjugates. Proc Natl Acad Sci USA 2002, 99:16695-16700. Achieving correction of defective genes via triplex induced site directed recombination with donor DNA is a very appealing goal as it is complementary to gene therapy. There is still a long way to go though. 47. Sorensen MD, Petersen M, Wengel J: Functionalized LNA (locked nucleic acid): high-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated, 2(-amino-LNA monomers. Chem Commun 2003:2130-2132.
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Recent improvements in antigene technology Sabrina Buchini and Christian J Leumann DNA triple-helix-based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother-like treatment in comparison to the flourishing field of oligonucleotide-based control of translation (antisense technology). This was mostly due to lack of affinity of triplex-forming oligonucleotides to their DNA target, to sequence restriction constraints imposed by the triple helical recognition motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine–purine inversion sites as well as sugar modifications, for example, the 20 -aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid sugar unit, which greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence-specific genomic DNA manipulation is starting to become a useful tool in biotechnology. Addresses Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland e-mail: [email protected]
Current Opinion in Chemical Biology 2003, 7:717–726 This review comes from a themed section on Biopolymers Edited by Thomas Carell and Peter Seeberger 1367-5931/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2003.10.007
Abbreviations AE-TFO 20 -aminoethoxy TFO PNA polyamide or peptide nucleic acids TFO triplex-forming oligonucleotide
Introduction Selective artificial control of gene expression is a longstanding dream in biotechnology and human therapy. Oilgonucleotides seem perfectly suited for this purpose because of their unique base–base recognition properties. In the antisense approach, short oligonucleotides are designed to bind to specific sequences of an mRNA of interest via Watson–Crick duplex formation, to block gene expression on the level of translation according to various molecular mechanisms. This approach is widely explored not only in the therapeutic area, where approximately 70–80 oligonucleotides targeted to various diseases are currently in clinical trials, but also as tools to downregulate gene expression in the area of functional www.current-opinion.com
genomics. In the antigene approach (Figure 1), oligonucleotides bind sequence selectively to genomic, doublestranded DNA and interfere with transcription and the DNA processing machinery via triple helix formation. Some intrinsic advantages of the antigene over the antisense principle can be identified. First, there are only two target copies of DNA per diploid cell as compared with the hundreds to thousands of mRNA copies that have to be targeted in the antisense approach. This should dramatically reduce the amount of oligonucleotide needed for activity. Moreover, not only transcriptional activation and deactivation but also gene knockout as well as targeted mutagenesis, targeted recombination and sequence-selective manipulation of genomic DNA can be achieved. Despite the potential benefits for biotechnology in general, the investment of resources has been limited, and therewith progress in this field has been slow. Consequently, there is still no antigene oligonucleotide in clinical trials. Two stable DNA triple-helical motifs are known that differ according to the orientation and base composition of the third strand. In the parallel or pyrimidine binding motif, the homo-pyrimidine triplex-forming oligonucleotide (TFO) is parallel oriented to the duplex purine strand forming H-bonded Hoogsteen Cþ GC and T AT basetriplets. The antiparallel or purine motif is characterized by G GC, A AT and T AT reverse Hoogsteen base-triplets between a purine rich TFO that is antiparallel oriented to the purine strand of the DNA duplex (Figure 2). In both motifs, the third strand lies in the DNA major groove, and requires a target DNA homopurine–homopyrimidine sequence tract, ideally of 15–30 nucleotides. The potential advantages in targeting genomic DNA by oligonucleotides are counterbalanced by several restrictions common to all oligonucleotide-based approaches (e.g. biostability, cell permeability and non-specific binding of the oligonucleotides or analogues used). Further, the triplex approach shows specific restrictions. Some of them are motif-dependent while others are of general nature. For the parallel motif, the severest restriction is the pHsensitivity of the Cþ GC base triplet, which is intrinsically unstable under physiological conditions due to the low pKa of the third strand cytosines. Triplex stability in the antiparallel motif is markedly sequence-dependent due to alternative structure formation (e.g. G tetrads), of the corresponding GA or GT rich TFOs under physiological conditions (especially Kþ concentration). Finally, there are common problems in both motifs. The most prominent ones are related to target accessibility, low Current Opinion in Chemical Biology 2003, 7:717–726
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Figure 1
Nucleus Protein 1
mRNA 2
Major groove
3 + DNA target
Triple helix
Cytoplasm TFO 1
2
3
Modulation of gene expression
Site-specific mutagenesis
Modification of genomic DNA
Transcription initiation and inactivation
Directed mutagenesis Homologous recombination Replication inhibition
DNA cleavage or crosslinking at selected gene loci Current Opinion in Chemical Biology
Basic antigene concepts and corresponding applications. Gene expression can be upregulated or downregulated on the level of transcription via selective triple-helix formation, preferably at promoter sites. Selective modification of the genome includes site-specific mutagenesis via triplex delivered mutagens (e.g. psoralen), or homologous recombination through triplex delivered donor DNA via DNA repair. Further applications arise from triplex-targeted chemical modification of the gene (e.g. cleavage, site-specific cross-linking or alkylation).
overall affinity of TFOs to their target, resulting in poor competition with DNA binding proteins, and the restriction of the known binding motifs to homopurine/ homopyrimidine DNA sequence tracts. The occurrence and function of triple helical structures in vivo [1], the use of TFOs as modulators of gene expression [2] and as gene modifiers [3], and the more chemical and molecular recognition aspects of nucleobase modifications [4] have all been comprehensively reviewed elsewhere. In our opinion, this review highlights the most relevant advances in the past two years on the development of oligonucleotide analogues with improved binding and sequence recognition properties as well as in gene targeting.
Increasing triplex stability There are a few basic concepts in improving the binding of TFOs to duplex targets that have been successfully applied in the past few years to increase triplex stability. First, the introduction of positively charged amino functions on the sugar and/or base unit, designed to make additional binding contacts between phosphodiester residues of the target Watson–Crick duplex besides the Current Opinion in Chemical Biology 2003, 7:717–726
base–base interactions (dual recognition concept). Second, the use of conformationally adapted or constrained TFOs. Finally, the use of TFOs with a non-charged backbone. Most recent progress has focused on the parallel binding motif of TFOs. Figure 3 displays the molecular structures of some modifications discussed in more detail below.
Dual recognition The best explored and most promising parallel motif TFOs are those containing an aminoethoxy side-chain at C(20 ) of the ribonucleoside units, developed by Cuenoud and collegues [5] (Figure 3). Under physiological conditions, the amino function of this side-chain is protonated and leads to an increase in thermal triplex stability of ca. 3.58C per modification at pH 7.0. Fully modified 20 -aminoethoxy TFOs (AE-TFOs) can lead to triplexes that are more stable than the target duplex alone. The gain in thermodynamic stability (DG) of ca. 0.5 kcal/mol for each modified unit is mostly due to a 1000-fold enhanced association rate compared with that of a DNA–TFO. This is an important feature considering that association kinetics for triplex formation are substantially lower as compared with duplex formation. The stabilizing effect was shown by NMR to arise from www.current-opinion.com
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Figure 2
(a) Parallel motif
N O
N H
H N H
H N
H
N
O N N
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H
N N
H N
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T-AT Current Opinion in Chemical Biology
Molecular structures of the canonical Hoogsteen and reverse Hoogsteen base triplets in the known parallel and antiparallel triple helical binding motifs.
specific hydrogen bonds of the protonated amino function to the pro-R non-bridging phosphate oxygen of the purine strand of the underlying duplex target [6]. The 20 -amino side-chain occurs in a gaucheþ conformation and shows astonishingly low conformational mobility in the triplex [7]. Variations in the length of the side chain did not further improve binding efficiency. AE-TFOs have recently been shown to vastly broaden in vitro and in vivo applications of antigene oligonucleotides (see below). Another point of attachment of alkylamino chains pointing towards the same spatial direction as in AE-TFOs is the position C(40 ) of the deoxyribose unit. Matsuda and co-workers recently synthesized the corresponding 40 amino-TFOs and found some thermal triplex stabilization for the ethylamino side chain; however, only by less than 18C per modification [8]. Shortening or lengthening www.current-opinion.com
the tether between C(40 ) and the amino function consistently reduced affinity relative to the two-carbon linker. There is no doubt, however, that the efficiency of this system does not match that of the AE-TFOs. Related to the same concept, our group has embarked on the evaluation of the properties of pyrrolidino-pseudonucleosides [9]. According to modelling based on X-ray data available for parallel motif triplexes, the furanose ring oxygen is close to a pro-R non-bridging phosphate oxygen of the purine duplex strand. Thus, replacement of this oxygen by nitrogen was expected to bring about additional stability in triplex formation. As a tribute to chemical stability, the linkage between base and amino sugar had to be changed into a C-glycosidic linkage, as in the pseudonucleosides. Experiments with TFOs containing the base uracil or N-1-methyl uracil for recognizing Current Opinion in Chemical Biology 2003, 7:717–726
720 Biopolymers
Figure 3
Conformationally constrained or uncharged
Dual (base and phosphate) recognition O O
O
Py
O O –O P O O
O
Py
+H N 3
4′-AminoTFOs
PyrrolidinoTFOs
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NH
(H3C)
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N+ N
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n O O –O P O O DNA n=1, LNA (BNA) n=2, ENA
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N N P O O MorpholinoTFOs NH2
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H H ψPy N+
O –O P O O
n O –O P O O NH3+
2′-AminoethoxyTFOs
O
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NH O
ψPy Current Opinion in Chemical Biology
Molecular structures of TFOs with potential dual recognition properties, with conformationally constrained sugar analogues and with uncharged backbone structures.
adenine in the parallel binding motif, however, showed triplex destabilization [10], whereas those containing the base isocyctosine increased triplex stability at neutral pH by ca. 28C in Tm per modification [11]. The reason for the differential stabilization/destabilization of the pyrrolidino pseudonucleosides is unclear.
Variation of TFO conformation, and sugar backbone modified TFOs Generally, RNA-TFOs form more stable triplexes than do DNA-TFOs in the pyrimidine binding motif. Various NMR studies on DNA-triplexes were in favor of structures in which not all deoxyribose units of a TFO adopt the same sugar pucker. Thus, the overall helical conformation of DNA triplexes is somewhere between A and B [12]. A recent study with 20 -deoxy- and 20 -O-methyl-RNA TFOs showed marked sequence-dependent differences in stability that cannot easily be rationalized with known rules in conformational analysis [13]. Thus, extrapolation of triplex stabilities from determined sequences to any sequence is difficult. Within this context, we developed a combinatorial method to determine conformational effects of TFOs on triplex stability [14]. The method is outlined in Figure 4. In short, we prepared a 12-mer oligonucleotide library of TFOs via the ‘split and combine’ technique in which any except the 30 -terminal position is occupied by either a ribo- or a deoxyribonucleoside while the bases are Current Opinion in Chemical Biology 2003, 7:717–726
kept invariant. This enables conformational variation within a given base recognition frame, due to the intrinsic preferences of the deoxyribo- (20 -endo) and ribo- (30 endo) nucleosides. The library was then screened for stable triplex binders by fractionated affinity chromatography on an agarose immobilized DNA target in a temperature gradient. The resulting fractions were deconvoluted by mass spectrometry (nature and number of respective nucleoside units in the TFOs) and partial alkaline hydrolysis followed by gel electrophoresis (sequential position of ribonucleosides). This information was then used to build focused libraries for concept validation and isolation of individuals. With this method, the complete conformational space of a chimeric TFO containing ribound deoxyribonucleotides towards its DNA target could be screened. Two main pieces of information resulted from this analysis: first, there exists no ‘magic combination’ of a chimeric TFO with enhanced binding relative to the allRNA-TFO; and second, there is less sequence/stability variation for Cþ-GC than for T(U)-AT triplet formation. With slight variations of this combinatorial assay, sequence/stability effects of TFOs containing non-natural backbone modifications can also be addressed, which is certainly of interest as conformational variations in the corresponding TFOs can be higher in these cases. Another interesting approach involves the use of TFOs built from conformationally constrained nucleoside units. As with duplex formation, less entropy loss upon triplex www.current-opinion.com
Recent improvements in antigene technology Buchini and Leumann 721
Figure 4 Ribonucleosides
Deoxyribonucleosides
O
O
Base
Base O
O
O OH
O
3′-endo 2′-endo DNA target: 5′- AAAAGAGGAGGG TTTTCTCCTCCC-3′ TFO Library:
5′-JJJJKJKKJKKC-
J = dT,rU K = rC, dC
Library size: 211 = 2048
TFO library +
Hybridize DNA target Affinity chromatography selection (temp. gradient)
Solid phase
Focused library preparation
Definition of binding features
Deconvolution (ESI-MS base digest) Current Opinion in Chemical Biology
Schematic representation of the combinatorial approach developed for establishing a structure/stability profile of TFOs with conformational variation in the backbone. Within a given base recognition frame imposed by the target duplex, an oligonucleotide library with 2048 individual oligonucleotides was constructed, containing either a ribo- rU/rC or a deoxyribo- (dT/dC) sugar unit at each position in the chain by the split and combine technique. For convenience, deoxycytidine was chosen as the 30 -terminus. This library was then loaded on the agarose-supported DNA target and fractionated via affinity chromatography in a temperature gradient. The fractions were then deconvoluted by mass spectrometry combined with alkaline digestion to determine the preferred composition and location of ribonucleosides within the sequences. This information was then used to produce focused libraries for a subsequent round of selection.
formation, and thus higher triplex stability is expected. Given the preference of RNA- over DNA-TFOs in the parallel binding motif, oligonucleotide analogues that are constrained in a 30 -endo (RNA-like) conformation should be advantageous. Imanishi and co-workers investigated triplex formation with the conformationally constrained RNA analogue LNA in the pyrimidine motif and found substantial increases in thermal and thermodynamic stability of corresponding triplexes at neutral pH [15] (Figure 3). (The abbreviation BNA is synonymous for LNA and was coined by the Imanishi group. We use here consistently the better known abbreviation LNA.) There exists an optimal number of LNA-residues within a DNA-TFO that exert a stabilizing effect. Interestingly, fully modified LNA-TFOs do not form triplexes with DNA targets at all. A kinetic analysis of triplex formation with LNA revealed that most of the observed increase in the binding constant at neutral pH arises from a decreased dissociation rate [16]. Data also exist on triplex binding www.current-opinion.com
with the LNA-analogue ENA, containing a 20 ,40 -ethylene bridge [17]. ENA is able to stabilize triplex formation at neutral pH roughly to the same extent as LNA. Notably, fully modified ENA-TFOs do bind to their DNA target while fully modified LNA-TFOs do not. This phenomenon is not completely understood. Given the properties of LNA-TFOs and AE-TFOs, it is tempting to propose a combination of conformational constriction (reducing the rate of dissociation) with a properly positioned alkylamino function as in AE-TFOs (increasing the rate of association) to unite the best properties of both and produce even stronger binding TFOs (see also Update). Representatives of the class of neutral backbone oligonucleotide analogues with interesting antigene properties are morpholino-DNA and the polyamide or peptide nucleic acids (PNAs). Dimeric bis-PNA pyrimidine Current Opinion in Chemical Biology 2003, 7:717–726
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sequences bind to DNA duplexes via strand invasion (Ploop formation), thus offering the possibility for sitespecific modulation of gene expression or modification of the gene. Because the properties of PNA have been extensively reviewed elsewhere, we do not include them here systematically and refer the interested reader to two recent papers [18,19]. The morpholino oligonucleotides (Figure 3) bind to target DNA in the parallel but not in the antiparallel motif and are nuclease resistant. Binding in the parallel motif requires low pH and takes place in the absence of Mg2þ or Kþ ions (salt free conditions). A 25-mer morpholino oligonucleotide targeted to the HER-2/neu promoter region in the presence of 140 mM Kþ with and without 10 mM Mg2þ at pH 5.0 shows ca. twofold increased binding as compared with an unmodified DNA-TFO [20]. Thus, it is not stability that excels in this system. The advantages are more related to cellular uptake and distribution due to the non-charged backbone.
formation was assayed by Tm analysis as well as by a nuclease protection assay in mixtures of TFOs competing for the DNA target. The anthraquinone unit A (Figure 5) was found to give highest affinity to the TA site when positioned in the center of the TFO. It is, however, not specific, presumably because of unspecific intercalation. Although no hit or lead was discovered, the combinatorial rationale behind the approach is certainly of interest.
Expanding the sequence recognition range
Deoxynucleosides with acceptor or double acceptor bases of the pyridone (P) [26] or pyrimidone (4HT) [27] type have recently been investigated as units to target CG inversion sites in parallel TFOs (Figure 5). They are essentially C(4) carbonyl deletion mutants of thymine, which disables their binding to an AT base pair while maintaining the binding profile to CG sites. Both bases were shown to be highly selective for CG inversion sites but both have compromised affinity when compared with a canonical base-triple. Extension of the aromatic surface while maintaining the double acceptor nature of the base, as in Q (Figure 5) did not solve the affinity problem. In contrast, the selectivity for CG recognition was compromised by almost isoenergetic recognition of a GC basepair [28].
An obvious drawback in antigene technology is the restriction of TFO binding to homopurine/homopyrimidine DNA target sequences. Although such sequences are statistically over-represented in the genome it would be desirable to have bases at hand that specifically recognize pyrimidine bases or whole pyrimidine/purine Watson– Crick base-pairs. Recognizing pyrimidine bases is hampered by the fact that only one H-bond donor or acceptor site of C and T is available for binding in the major groove. Recognizing T in a AT base-pair is even more complicated because of the presence of the methyl group, which leads to steric interference with the TFO backbone, especially in the parallel binding motif. Alternatively, one can envisage the use of combinations of known binding motifs in one TFO or design new binding motifs from scratch. Research into this direction has been ongoing fot more than 10 years, but a general solution is elusive. The following section highlights a few recent variations on this theme.
Base modifications A series of new base analogues of the thiazolylaniline or -benzimidazol type, recognizing a TA inversion site, have been proposed recently by Fourrey and Benhida [21,22,23]. The design of these bases was inspired by earlier work by the Dervan group on the base D3, which showed substantial affinity but lacked sequence specificity due to non-specific intercalation. Within the parallel motif, the base S (Figure 5) which is the best representative of this class of base analogues so far, was found to bind to a TA inversion site with an affinity that almost matches that of a T-AT canonical base triplet, and with reasonable selectivity. Another approach to find new bases recognizing TA inversion sites was based on a combinatorial synthesis/nuclease protection assay by Richert and co-workers [24]. In this case, a series of different aromatic units were attached to an acyclic ribose surrogate and incorporated into parallel TFOs. Triplex Current Opinion in Chemical Biology 2003, 7:717–726
Approaches in our group to overcome the sequence restriction in the antiparallel binding motif have led to the evaluation of the base 2-aminopurine in TFOs at TA inversion sites. In a study comprising both anomers (a/b) of the N-9 or N-7 regioisomeric deoxyribonucleosides of 20 -aminopurine in DNA TFOs, we found that the N7aisomer preferentially recognizes a TA inversion site with acceptable but not exceptional base-pair selectivity and affinity [25].
An alternative way to resolve the homopurine requirement for triplex formation consists in the design of TFOs with new major groove binding motifs. In this context, we [29] and others [30] have explored with limited success TFOs that are designed to recognize purine bases on either strand of the Watson–Crick duplex by using a structural switch. In these oligonucleotides, an N7/N9 and/or an a/b structural switch on aminopurine, hypoxanthine, thymine or 4-guanidinocyctosine nucleosides were used to recognize consecutive purine bases on either strand of the target DNA. The lack of affinity in such switch-TFOs is probably due to missing intrastrand stacking of the bases at the switch site. An interesting novel approach was recently discussed by Gold and colleagues using 2-aminoquinazoline C-nucleosides [31].
Combinations of base and sugar modifications An obvious task in further expanding the triplex recognition range of TFOs lies in the combination of specific pyrimidine–purine inversion site bases with overall triplex-enhancing backbone modifications. Progress in www.current-opinion.com
Recent improvements in antigene technology Buchini and Leumann 723
Figure 5
Targeting T-A inversions DNA O
S O
DNA O
N N H
O
N Ac H
O N O HN
O DNA
O O DNA A
S N
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dR N
N
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O
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Antiparallel motif Targeting C-G inversions N N dR P
O
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N
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dR Q
Parallel motif
Current Opinion in Chemical Biology
Molecular structures of recently investigated base analogues for targeting TA or CG inversion sites on otherwise homopurine/homopyrimidine duplex targets.
this field has been very successful and will be highlighted in three examples from the recent literature. Replacing the deoxyribose unit on the 2-pyridone nucleoside by an LNA sugar unit (2-pyridone-LNA-TFOs, Figure 6) led to an increase in Tm of 98C in a corresponding TFO 15-mer binding to a DNA target with a CG inversion site at neutral pH, without compromising selectivity [26,32]. Up to three CG inversions within a 15mer TFO, adding up to 20% of pyrimidine content within the purine sequence of the target DNA, could be recognized in this way. Additional gain in stability could be realized by replacing standard pyrimidine nucleosides forming canonical base-triples, by corresponding LNA units in the TFO. This is a good example on how sugar and base modification work in symbiosis. Another demonstration of the power of combination is the case of 20 O-aminoethyl nucleosides containing the 5propargylamino-U base. In the context of a TFO, this www.current-opinion.com
nucleoside analogue carries two positive charges at neutral pH. It was shown by DNase I footprinting and thermal denaturation studies on intramolecular parallel motif triplexes that these bisubstituted nucleoside derivatives produce a large increase in triplex stability, much greater than that produced by either of the monosubstituted analogues [33]. Also, propargylguanidino-U bases were recently reported to enhance triplex stability [34]. Although these cases do not contribute to the expansion of the triplex recognition code, it is conceivable that a propargylamino modification of, for example, a pyridone or pyrimidone base might additionally enhance CG recognition in the same way. We have developed the 20 -aminoethyl nucleoside containing the base 4-deoxothymine (4HT) (Figure 6). The aminoethyl modification also adds, in this case, favorably to triplex stability for CG inversion recognition [35].
Other developments It is clear that there are many other ways to increase triplex stability or modulate target recognition of TFOs. Current Opinion in Chemical Biology 2003, 7:717–726
724 Biopolymers
Figure 6
NH3+ O NH O
N
O
O O O –O P O O
N O
N
O
O
O
NH3+
Propargylamino-UAE-TFOs
O O –O P O O
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4HT-AE-TFOs
N
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O O –O P O O 2-Pyridone-LNATFOs
Current Opinion in Chemical Biology
Promising TFO candidates combining stability increasing sugar modifications with either stability increasing or recognition range expanding base modifications.
Well explored concepts include the use of TFOs containing triplex-specific intercalators. The extreme variant of target binding is covalent attachment of a TFO to the DNA duplexes (e.g. via psoralen cross linking or cross linking with reactive base analogues). One recent example of the latter kind uses 2-amino-6-vinyl purine deoxyribosides in parallel TFOs as an electrophilic unit that reacts irreversibly with the amino functions of adenine at TA inversion sites [36]. This might be useful for monitoring TA interruptions within homopurine sequence tracts. Another interesting addition to the already large palette of functional TFOs are pyrimidine oligonucleotides that carry an azobenzene unit as a base substitute. By irradiation with light at two specific wavelengths cistrans isomerization can be effected, which enables or inhibits binding of the TFO to its target [37]. This might allow photoregulation of cellular processes based on antigene technology.
Antigene applications As pointed out in the Introduction, there are several cellular functions that can be controlled via the antigene principle. In the past, lack of success in such applications was mostly due to insufficient target affinity. With the new generation of strong binding TFOs, this picture will change. Most advanced at this stage are investigations with psoralene-modified DNA-TFOs, AE-TFOs and PNA-clamp-TFOs. Some examples of the recent literature are compiled below. With a 30 -psoralene conjugated 16 bp DNA-TFO, designed to bind in the antiparallel motif to the third intron of the human intercellular adhesion molecule 1 (ICAM-1) gene, repressor activity was found in vitro, in cell nuclei and in a human keratinocyte cell line (A431). This shows Current Opinion in Chemical Biology 2003, 7:717–726
that the ICAM 1 target sequence in the chromatin context in cell nuclei is available for triplex formation [38]. With a restriction protection assay to physically detect triple helix directed psoralene cross-links at chromosomal sites in mammalian cells, it was shown that an (antiparallel) TFO could bind to a chromosomal target site in human cells even in the absence of transcription [39]. Substantial increases in target site binding of the TFO was observed upon specific induction of transcription. These findings provide evidence that physiological activity at a chromosomal target influences its accessibility to TFOs and suggests that gene targeting may be most effective at highly expressed chromosomal loci. An AE-TFO designed to bind in parallel fashion to the IgE germline proximal promoter overlapping with the binding site of three proteins inhibited binding of all three proteins in a dose-dependent way in vitro. Functionally, this resulted in a selective inhibition of IL-4 induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells [40]. This is a successful application of 20 -aminoethoxy-TFOs towards specific modulation of gene expression via DNA triplex formation. Targeted gene knockout by AE-TFOs was reported by Seidman and collegues [41,42]. Psoralene-linked aminoethoxy-TFOs were designed to target and mutagenise a site in the hamster HPRT gene. Knockout frequencies that are 300-400-fold above background could be observed. Site-specific mutagenesis of a chromosomal gene could also be achieved with AE-TFOs in the parallel binding motif in a cellular assay. It was reported that the targeting efficiency of the TFO was sensitive to the cellcycle status. Targeted mutagenesis showed the greatest activity in the S phase with ca. 20–30% of adduct formation. Ca. 75–80% of these adducts were repaired faithfully, leading to a mutation frequency of ca. 5% [43]. A variation of site-specific mutagenesis was reported in which covalent crosslinking of a TFO to its target was achieved via the reactive base 2-amino-6-vinyl purine of the TFO with a nucleophilic amino function of a neighboring cytosine unit [44]. This opens the way for using other mutagens than psoralene in triplex-targeted mutation experiments. Site-directed inhibition of DNA replication was recently shown with DNA-TFOs and PNA. Triplex-mediated inhibition of DNA replication targeted to the polypurine sequence tract of HIV-1 was observed with both, covalent psoralene adduct forming DNA-TFOs or non-covalent binding bisPNAs [45]. Another appealing application of triplexes in vivo is inducing site-directed recombination with donor DNA fragments. In a recent report, a bis-PNA clamp was coupled to a 40-nt donor DNA fragment that was homologous www.current-opinion.com
Recent improvements in antigene technology Buchini and Leumann 725
to an adjacent region in the target gene. The PNA–DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency of at least 60-fold above background [46]. Recombination was found to be dependent on the nucleotide excision repair factor XPA. Thus, it seems that recombination is DNA repair driven and induced by a helical distortion of the DNA target upon bis-PNA– DNA triplex formation. This may be a potentially useful strategy to achieve targeted correction of defective genes.
This is a comprehensive review with 462 references, covering all relevant nucleobase modifications and their effect on triplex stability up to the end of 2001. 5.
Cuenoud B, Casset F, Hu¨ sken D, Natt F, Wolf RM, Altmann K-H, Martin P, Moser HE: Dual recognition of double-stranded DNA by 2-aminoethoxy-modified oligonucleotides. Angew Chem Int Ed Engl 1998, 37:1288-1291.
6.
Blommers MJJ, Natt F, Jahnke W, Cuenoud B: Dual recognition of double-stranded DNA by 2(-aminoethoxy-modified oligonucleotides: the solution structure of an intramolecular triplex obtained by NMR spectroscopy. Biochemistry 1998, 37:17714-17725.
7.
Carlomagno T, Blommers MJJ, Meiler J, Cuenoud B, Griesinger C: Determination of aliphatic side-chain conformation using cross-correlated relaxation: application to an extraordinarily stable 2(-aminoethoxy-modified oligonucleotide triplex. J Am Chem Soc 2001, 123:7364-7370.
8.
Atsumi N, Ueno Y, Kanazaki M, Shuto S, Matsuda A: Nucleosides and nucleotides. Part 214: thermal stability of triplexes containing 4(-a-C-aminoalkyl-2(-deoxynucleosides. Bioorg Med Chem 2002, 10:2933-2939.
9.
Ha¨ berli A, Leumann CJ: Synthesis of pyrrolidino C-nucleosides via Heck reaction. Org Lett 2001, 3:489-492.
Conclusions Antigene applications in general have already, and will do more so in the future, greatly benefited from recent advances in increasing triplex stability and modulating base recognition specificities by designed sugar and base modifications. Especially, modifications of TFOs exploiting the parallel binding motif show great promise in this respect. The combination of general triplex enhancing structural features (e.g. the 20 -aminoethoxy modifications or conformationally constrained sugars, such as in LNA) with novel bases for selective pyrimidine-purine inversion site recognition lends hope to the disappearance of the sequence recognition constraints for TFOs in the near future. Thus, the day where antigene oligonucleotides will complement and in some cases even challenge antisense oligonucleotides as biomolecular tools or therapeutics may come soon.
Update At the stage of proofreading of this manuscript, an article appeared describing the synthesis and duplex binding properties of 20 -aminoethyl modified 20 -amino-LNA [47]. This structure perfectly combines the features of conformational rigidity and dual binding and may be an interesting candidate as high affinity TFO. However, triplex binding data are yet missing.
Acknowledgements The authors thank the Swiss National Science foundation for ongoing financial support of our research in the field of molecular recognition of DNA.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Zain R, Sun J-S: Do natural DNA triple-helical structures occur and function in vivo? Cell Mol Life Sci 2003, 60:862-863.
2.
Guntaka RV, Varma BR, Weber KT: Triplex-forming oligonucleotides as modulators of gene expression. Int J Biochem Cell Biol 2003, 35:22-31.
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Seidman MM, Glazer PM: The potential for gene repair via triple helix formation. J Clin Invest 2003, 112:487-494.
4.
Robles J, Grandas A, Pedroso E, Luque FJ, Eritja R, Orozco M: Nucleic acid triple helices: stability effects of nucleobase modifications. Curr Org Chem 2002, 6:1333-1368.
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10. Ha¨ berli A, Leumann CJ: DNA binding properties of oligodeoxynucleotides containing pyrrolidino C-nucleosides. Org Lett 2002, 4:3275-3278. 11. Ha¨ berli A, Mayer A, Leumann CJ: Pyrrolidino-DNA. Nucleosides Nucleotides Nucleic Acids 2003, 22:1187-1189. 12. Tarko¨ y M, Phipps AK, Schultze P, Feigon J: Solution structure of an intramolecular DNA triplex linked by hexakis(ethylene glycol) units: d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6TCTCTCTT). Biochemistry 1998, 37:5810-5819. 13. Cassidy RA, Puri N, Miller PS: Effect of DNA target sequence on triplex formation by oligo-2(-deoxy- and 2(-Omethylribonucleotides. Nucleic Acids Res 2003, 31:4099-4108. 14. Bernal-Me´ ndez E, Leumann CJ: Conformational diversity versus nucleic acid triplex stability, a combinatorial study. J Biol Chem 2001, 276:35320-35327. We find this to be a potentially very useful combinatorial method to rapidly screen sequence effects on triplex (or duplex) binding. The advantage over selex and related methods is that there is no enzymatic amplification step necessary, thus allowing also the screening of sugar-modified nucleotide units. 15. Obika S, Uneda T, Sugimoto T, Nanbu D, Minami T, Doi T, Imanishi T: 2(-O,4(-C-Methylene bridged nucleic acid (2(,4(-BNA): Synthesis and triplex forming properties. Bioorg Med Chem 2001, 9:1001-1011. 16. Torigoe H, Hari Y, Sekiguchi M, Obika S, Imanishi T: 2(-O,4(-Cmethylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies. J Biol Chem 2001, 276:2354-2360. 17. Koizumi M, Morita K, Daigo M, Tsutsumi S, Abe K, Obika S, Imanishi T: Triplex formation with 2(-O,4(-C-ethylene-bridged nucleic acids (ENA) having C3(-endo conformation at physiological pH. Nucleic Acids Res 2003, 31:3267-3273. This variant of LNA was shown to have poorer RNA single-strand binding properties compared with LNA. This seems not to be the case in triplex formation where it shows similar Tm enhancements to LNA, but does not suffer from the non-binding of fully modified oligonucleotides to their DNA target. 18. Demidov VV, Frank-Kamenetskii MD: Sequence-specific targeting of duplex DNA by peptide nucleic acids via triplex strand invasion. Methods 2001, 23:108-122. 19. Nielsen PE: Targeting double stranded DNA with peptide nucleic acids (PNA). Curr Med Chem 2001, 8:545-550. 20. Basye J, Trent JO, Gao D, Ebbinghaus SW: Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif. Nucleic Acids Res 2001, 29:4873-4880. Current Opinion in Chemical Biology 2003, 7:717–726
726 Biopolymers
21. Guianvarc’h D, Benhida R, Fourrey J-L, Maurisse R, Sun J-S: Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine.pyrimidine interruption. Chem Commun (Camb) 2001:1814-1815. This paper describes one of the best nucleobase analogues for TA inversion site recognition in DNA-TFOs, in terms of affinity. 22. Guianvarc’h D, Fourrey J-L, Maurisse R, Sun J-S, Benhida R: Design of artificial nucleobases for the recognition of the AT inversion by triple-helix forming oligonucleotides: a structurestability relationship study and neighbour base effect. Bioorg Med Chem 2003, 11:2751-2759.
been investigated so far. Thus, the rigorous proof of principle has yet to come. 34. Roig V, Asseline U: Oligo-2(-deoxyribonucleotides containing uracil modified at the 5-position with linkers ending with guanidinium groups. J Am Chem Soc 2003, 125:4416-4417. 35. Buchini S, Leumann CJ: Dual recognition of a pyrimidine-purine inversion site: synthesis and binding properties of triplex forming oligonucleotides containing 2(-aminoethoxy-5methyl-1H-pyrimidin-2-one ribonucleosides. Tetrahedron Lett 2003, 44:5065-5068. 36. Nagatsugi F, Matsuyama Y, Maeda M, Sasaki S: Selective crosslinking to the adenine of the TA interrupting site within the triple-helix. Bioorg Med Chem Lett 2002, 12:487-489.
23. Guianvarc’h D, Fourrey J-L, Maurisse R, Sun J-S, Benhida R: Synthesis, incorporation into triplex forming oligonucleotide, and binding properties of a novel 2(-deoxy-C-nucleoside featuring a 6-(thiazolyl-5)benzimidazole nucleobase. Org Lett 2003, 4:4209-4212.
37. Liang X, Asanuma H, Komiyama M: Photoregulation of DNA triplex formation by azobenzene. J Am Chem Soc 2002, 124:1877-1883.
24. Mokhir AA, Connors WH, Richert C: Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices. Nucleic Acids Res 2001, 29:3674-3684.
38. Besch R, Giovannangeli C, Kammerbauer C, Degitz K: Specific inhibition of ICAM-1 expression mediated by gene targeting with triplex-forming oligonucleotides. J Biol Chem 2002, 277:32473-32479.
25. Parel SP, Leumann CJ: Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides. Nucleic Acids Res 2001, 29:2260-2267.
39. Macris MA, Glazer PM: Transcription depedence of chromosomal gene targeting by triplex-forming oligonucleotides. J Biol Chem 2003, 278:3357-3362.
26. Obika S, Hari Y, Sekiguchi M, Imanishi T: A 2(,4(-bridged nucleic acid containing 2-pyridone as a nucleobase: efficient recognition of a C-G interruption by triplex formation with a pyrimidine motif. Angew Chem Int Ed Engl 2001, 40:2079-2081. 27. Pre´ vot-Halter I, Leumann C: Selective recognition of a C-G base-pair in the parallel DNA triple-helical binding motif. Bioorg Med Chem Lett 1999, 9:2657-2660. 28. Pre´ vot I, Leumann CJ: Evaluation of novel third strand bases for the recognition of a C-G base-pair in the parallel DNA triplehelical binding motif. Helv Chim Acta 2002, 85:502-515. 29. Parel SP, Marfurt J, Leumann CJ: DNA triple-helix formation at pyrimidine-purine inversion sites. Nucleosides Nucleotides Nucleic Acids 2001, 20:411-417. 30. Doronina SO, Behr J-P: Synthesis of 4-guanidinopyrimidine nucleosides for triple helix-mediated guanine and cytosine recognition. Tetrahedron Lett 1998, 39:547-550. 31. Li JS, Fan YH, Zhang Y, Marky LA, Gold B: Design of triple helix forming C-glycoside molecules. J Am Chem Soc 2003, 125:2084-2093. 32. Obika S, Hari Y, Sekiguchi M, Imanishi T: Stable oligonucleotide directed triplex formation at target sites with CG interruptions: strong sequence-specific recognition by 2(,4(-bridged nucleic-acid-containing 2-pyridones under physiological conditions. Chemistry 2002, 8:4796-4802. This article describes probably the best CG inversion binder known so far. The combination of conformationally restricted sugar analogues with novel bases that selectively recognize pyrimidine bases is a very promising strategy to alleviate the sequence restriction problem in DNA triplex formation. 33. Sollogoub M, Darby RA, Cuenoud B, Brown T, Fox KR: Stable DNA triple helix formation using oligonucleotides containing 2(-aminoethoxy-5-propargylamino-U. Biochemistry 2002, 41:7224-7231. An interesting way to improve triplex stability that may be applied also to non-standard bases. Unfortunately, only monomolecular triplexes have
Current Opinion in Chemical Biology 2003, 7:717–726
40. Stu¨ tz AM, Hoeck J, Natt F, Cuenoud B, Woisetschla¨ ger M: Inhibition of interleukin-4- and CD40-induced IgE germline gene promoter activity by 2(-aminoethoxy-modified triplexforming oligonucleotides. J Biol Chem 2001, 276:11759-11765. 41. Puri N, Majumdar A, Cuenoud B, Natt F, Martin P, Boyd A, Miller PS, Seidman MM: Targeted gene knockout by 2(-O-aminoethyl modified triplex forming oligonucleotides. J Biol Chem 2001, 276:28991-28998. 42. Puri N, Majumdar A, Cuenoud B, Natt F, Martin P, Boyd A, Miller PS, Seidman MM: Minimum number of 2(-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides. Biochemistry 2002, 41:7716-7724. This paper contains important information as to the design of AE-TFOs related to knockout activity. It also contains a side by side comparison of the functionality of AE-TFOs with standard 20 -OM-TFOs. 43. Majumdar A, Puri N, Cuenoud B, Natt F, Martin P, Khorlin A, Dyatkina N, George AJ, Miller PS, Seidman MM: Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide. J Biol Chem 2003, 278:11072-11077. 44. Nagatsugi F, Sasaki S, Miller PS, Seidman MM: Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog. Nucleic Acids Res 2003, 31:e31. 45. Diviacco S, Rapozzi V, Xodo LE, He´ le`ne C, Quadrifoglio F, Giovannangeli C: Site-directed inhibition of DNA replication by triple helix formation. FASEB J 2001, 15:2660-2668. 46. Rogers FA, Vasquez KM, Egholm M, Glazer PM: Site-directed recombination via bifunctional PNA-DNA conjugates. Proc Natl Acad Sci USA 2002, 99:16695-16700. Achieving correction of defective genes via triplex induced site directed recombination with donor DNA is a very appealing goal as it is complementary to gene therapy. There is still a long way to go though. 47. Sorensen MD, Petersen M, Wengel J: Functionalized LNA (locked nucleic acid): high-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated, 2(-amino-LNA monomers. Chem Commun 2003:2130-2132.
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