Triple Helix-forming Oligonucleotides Research Articles

Exploring the Cellular Activity of Camptothecin-Triple-Helix-Forming Oligonucleotide Conjugates

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

Developing a programmed restriction endonuclease for highly specific DNA cleavage

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4–8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(γ-maleimidobutryloxy) succinimide ester to a TFO (5′-NH2-[CH2]6 or 12-MPMPMPMPMPPPPPPT-3′, with M being 5-methyl-2′-deoxycytidine and P being 5-[1-propynyl]-2′-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO (‘addressed’ site: 5′-TTTTTTTCTCTCTCTCN∼10CAGCTG-3′), leaving ‘unaddressed’ PvuII sites intact. The preference for cleavage of an ‘addressed’ compared to an ‘unaddressed’ site is >1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.

The Development of Bioactive Triple Helix‐Forming Oligonucleotides

We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.

An extra dimension in nucleic acid sequence recognition

Watson-Crick base pairing is a natural molecular recognition process that has been exploited in molecular biology and universally adopted in many fields. An additional mode of nucleic acid sequence recognition that could be used in combination with normal base pairing would add an exta dimension to nucleic acid interactions and open up many new applications. In principle the triplex approach could provide this if developed to recognize any DNA sequence. To this end modified nucleosides have been incorporated into triple-helix-forming oligonucleotides (TFOs) and used to recognize mixed sequence DNA with high selectivity and affinity at neutral pH. Continuing developments are directed towards improving TFO affinity at high pH and increasing triplex association kinetics. A number of applications of triplexes are currently being explored.

Triplex targeted genomic crosslinks enter separable deletion and base substitution pathways.

We have synthesized triple helix forming oligonucleotides (TFOs) that target a psoralen (pso) interstrand crosslink to a specific chromosomal site in mammalian cells. Mutagenesis of the targeted crosslinks results in base substitutions and deletions. Identification of the gene products involved in mutation formation is important for developing practical applications of pso-TFOs, and may be informative about the metabolism of other interstrand crosslinks. We have studied mutagenesis of a pso-TFO genomic crosslink in repair proficient and deficient cells. Deficiencies in non homologous end joining and mismatch repair do not influence mutation patterns. In contrast, the frequency of base substitutions is dependent on the activity of ERCC1/XPF and polymerase ζ, but independent of other nucleotide excision repair (NER) or transcription coupled repair (TCR) genes. In NER/TCR deficient cells the frequency of deletions rises, indicating that in wild-type cells NER/TCR functions divert pso-TFO crosslinks from processes that result in deletions. We conclude that targeted pso-TFO crosslinks can enter genetically distinct mutational routes that resolve to base substitutions or deletions.

Sequence-dependent cleavage of HBV DNA by combining with triple helix-forming oligodeoxyribonucleotides modified with manganese porphyrin in vitro

To observe the ability of triple helix-forming oligonucleotides (TFO) modified with manganese porphyrin to combine with and cleave HBV DNA fractions. The ends of TFO were modified with manganese porphyrin and acridine; At 37 degrees C and pH 7.4 condition in vitro, TFO modified with manganese porphyrin and acridine were bound with 32P labeled HBV DNA fragments, the affinity and specificity binding to target sequence were tested by electrophoretic mobility shift and DNase 1 footprinting assays, the ability to cleave HBV DNA fractions was observed with cleavage experiments. TFO modified with manganese porphyrin and acridine could bind to target sequence in a sequence-dependent manner with Kd values of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target sequence in the region forming triple DNA. In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target HBV-DNA in sequence-dependent manner.

Triple Helix-Forming Oligonucleotides Conjugated to New Inhibitors of Topoisomerase II: Synthesis and Binding Properties

Triplex-forming oligonucleotides (TFOs) are among the most specific DNA ligands and represent an important tool for specific regulation of gene expression. TFOs have also been used to target DNA-modifying molecules to obtain irreversible modifications on a specific site of the genome. A number of molecules have been recognized to target topoisomerase II and stabilize double-stranded cleavage mediated by this enzyme thus determining permanent DNA damage. Among these poisons, etoposide (VP16), a 4'-demethylepipodophyllotoxin derivative, is widely used in cancer chemotherapy. In the aim to design DNA site-specific molecules, three analogues of VP16 (1, 2, and 3), recently described (Duca et al. J. Med. Chem. 2005, 48, 596-603), were attached to TFOs, together with a fourth one, of which the synthesis is reported here. Two different oligonucleotides, differing by the length (a 16-mer and a 20-mer), and two different linker arms between the oligonucleotide and the drug were used. The coupling reaction between the drug and the TFO was further improved. For the first time, we also report the synthesis of TFO conjugates bearing two molecules of inhibitor linked to the same oligonucleotide end. In total, 16 new conjugates were synthesized and evaluated for their ability to form triple helices. The loss in triplex stability due to the conjugation of the TFO to compounds that do not interact with DNA is compensated by the presence of the ethylene glycol linker arm. This stabilization effect is more pronounced at the 3' end than at the 5' end. All conjugates form a stable triplex selectively on the DNA target at 37 degrees C and pH 7.2.

369. Targeted Gene Modification Via Triple Helix-Forming Oligonucleotides

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. The resulting triplexes have been shown to constitute a DNA structure that can provoke binding by the damage recognition factors, XPA and RPA, and to stimulate DNA repair via the nucleotide excision repair (NER) pathway. Based on these observations, experiments were undertaken to determine the extent to which triplex structures can stimulate recombination events within genomic DNA in a site-directed manner. Studies in which TFOs were targeted to episomal SV40 genomes in COS cells suggested that intermolecular triplexes could stimulate recombination between tandemly repeated reporter gene sequences in a pathway dependent on XPA and other NER factors. Experiments in mouse Ltk-cells containing a dual TK gene substrate demonstrated that intrachromosomal gene conversion could be provoked by high affinity triplex formation, at frequencies as high as 1-2%, if the TFOs were delivered into the cells by microinjection. Recent work in both COS cells using an episomal target and in human cell free extracts using plasmid substrates has further demonstrated that triplex formation can promote intermolecular recombination. TFOs were shown to induce recombination between a target gene and short DNA fragment, in cases where the fragment is either covalently linked to or is completely separate from the TFO. In the extracts, immunodepletion and complementation with purified proteins demonstrated a requirement for XPA and for the human recombinase, HsRad51, in the reaction. In recent work, the ability of TFOs to stimulate recombination and thereby mediate gene repair in a dose-dependent manner at a chromosomal locus in mammalian cells has been established. Overall, this work raises the possibility that DNA binding ligands such as TFOs and related molecules such as a peptide nucleic acids (PNAs) may be useful in strategies designed to mediate targeted genome modification and genetic therapy by stimulating recombination in site-specific manner.

Improved synthesis of daunomycin conjugates with triplex-forming oligonucleotides. The polypurine tract of HIV-1 as a target

Triple helix-forming oligonucleotides (TFOs) are promising agents for the control of gene expression, as they can selectively bind to a chosen oligopyrimidine·oligopurine region of a gene of interest thus interfering with its expression. The stability of the triplex formed by the TFO and the duplex is often too poor for successful applications of TFOs in vivo and the conjugation of a DNA intercalating moiety to the TFO is a common way to enhance the TFO affinity for its target. In a previous work, we investigated the properties of daunomycin conjugated TFO (dauno-TFO) and found that this class of compounds showed a higher degree of affinity than native oligonucleotides for an oligopyrimidine·oligopurine duplex target and that the presence of the amino sugar increases such stability. Here, we report a significantly improved synthetic procedure for the preparation of the conjugates, based on the protection of the daunosamine moiety by N-trifluoroacetylation. This protecting group is removed as a final step from the conjugation product by mild basic hydrolysis to give the desired dauno-TFO. Compared to the previous synthetic procedure, the improvement is important. The synthesis is now more reproducible and no side products are formed. Moreover, the thus protected daunomycin derivative is very stable, up to at least one year. Two dauno-TFOs, differing by the length of the oligonucleotide moiety, were prepared to target the polypurine tract (PPT) of HIV-1. Triplex formation by these compounds with model duplexes was studied by UV spectroscopy, thermal gradient gel electrophoresis (TGGE) and gel electrophoretic mobility shift. The experimental results demonstrate that dauno-TFOs bind to the PPT of HIV-1 more strongly than the unconjugated TFOs.

Activation of Camptothecin Derivatives by Conjugation to Triple Helix-Forming Oligonucleotides

Topoisomerase I (topo I) is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy. Camptothecins (CPTs) reversibly trap topo I in covalent complex with DNA but exhibit limited sequence preference. The utilization of conjugates such as triplex-forming oligonucleotides (TFOs) to target a medicinal agent (like CPT) to a specific genetic sequence and orientation within the DNA has been accomplished successfully. In this study, different attachment points of the TFO to CPT (including positions 7, 9, 10, and 12) were investigated and our findings confirmed and extended previous conclusions. Interestingly, the conjugates induced specific DNA cleavage by topo I at the triplex site even when poorly active or inactive CPT derivatives were used. This suggests that the positioning of the drug in the cleavage complex by the sequence-specific DNA ligand is able to stabilize the ternary complex, even when important interactions between topo I and CPT are disrupted. Finally, certain TFO-CPT conjugates were able to induce sequence-specific DNA cleavage with the topo I mutants R364H and N722S that are resistant to camptothecin. The TFO-CPT conjugates are thus valuable tools to study the interactions involved in the formation of the ternary complex and also to enlarge the family of compounds that poison topo I. The fact that an inactive CPT analogue can act as a topo I poison when appropriately coupled to a TFO provides a new perspective at the level of drug design.

Amino-functionalized DNA: the properties of C5-amino-alkyl substituted 2'-deoxyuridines and their application in DNA triplex formation

The incorporation of C5-amino-modified 2′-deoxyuridine analogues into DNA have found application in nucleic acid labelling, the stabilization of nucleic acid structures, functionalization of nucleic acid aptamers and catalysts, and the investigation of sequence-specific DNA bending. In this study, we describe the physicochemical properties of four different C5-amino-modified 2′-deoxyuridines in which the amino group is tethered to the base via a 3-carbon alkyl, Z- or E-alkenyl or alkynyl linker. Conformational parameters of the nucleosides and their pKa values were deduced using 1H NMR. All of them display the expected anti-conformation of the nucleoside with 2′-endo sugar puckers for the deoxyribose ring. A preference for the cisoid conformation for the Z-alkenyl analogue is found, while the E-alkenyl analogue exists exclusively as its transoid conformation. The pKa values range from 10.0 for the analogue with an aliphatic propyl linker to 8.5 for the propargylamino analogue. The analogues have been used for the synthesis of triple-helix forming oligonucleotides (TFOs) in which they replace thymidine in the natural sequence. Oligonucleotides containing the propargylamino analogue display the highest stability especially at low pH, while those containing analogues with propyl and especially Z-alkenyl linkers are destabilized to a great extent. TFOs containing the analogue with the E-alkenyl linker have stability similar to the unmodified structures. The chemical synthesis of TFOs containing the analogue, 5-(3-hydroxyprop-1-ynyl)-2′-deoxyuridine that possesses a neutral but polar side chain show a remarkable stability, which is higher than that of all TFOs containing the alkylamino or alkenylamino analogues and only slightly lower than that of TFOs containing the propargylamino analogue. Both the hydroxyl and propargylamino substitutions impart enhanced triple-helix stability relative to the analogous sequences containing C5-propynyl-2′-deoxyuridine. Furthermore, a similar dependence of stability on pH is found between TFOs containing the hydroxypropynyl modifications and those containing the propargylamino side chains. This suggests that the major factor responsible for stabilizing such triple helices is due to the presence of the alkyne with an attached electronegative group.

Interference with MCP-1 gene expression by vector generated triple helix-forming RNA oligonucleotides

Triple helix-forming oligonucleotides (TFOs) that specifically bind to double-stranded DNA sequences can be rationally designed, while intracellular delivery of single stranded RNA TFOs has not yet been studied in detail. In this report, we demonstrate gene and sequence-specific inhibition of MCP-1 gene expression due to interference of intracellular-generated single-stranded RNA (CU-TFO) with an overlapping SP-1/AP-1 target. Binding of synthetic 19-nucleotide (19-nt) CU-TFO to the MCP-1 promoter duplex was verified by triplex blotting. Furthermore, we confirmed binding of a 1.1-kb fusion transcript containing the 19-nt pyrimidine CU sequence to a plasmid-encoded MCP-1 promoter target duplex at pH 7.0. In tumour necrosis factor-alpha-stimulated HEK cells, CU-TFOs inhibited MCP-1 protein release by 76 +/- 10.2% compared to intracellular-generated control oligonucleotides. Interleukin-8 as a control target gene was not affected by CU-TFO, confirming both highly specific and effective chemokine gene repression by transfectable TFO-shuttle vectors.

Postsynthetic functionalization of triple helix-forming oligonucleotides.

The design of molecules that recognize specific sequence on the deoxyribonucleic acid (DNA) double helix would provide interesting tools to interfere with DNA information processing at an early stage of gene expression. This chapter describes in detail the protocol of conjugation between terminally phosphorylated oligonucleotides and chemically or biologically active ligands possessing electrophilic or nucleophilic functional groups. The synthetic procedure includes chemical activation of oligonucleotide terminal phosphate and introduction in this way of a nucleophilic or electrophilic group (such as amino or carboxyl groups) into oligonucleotide terminus using aliphatic amino group of a ligand or a linker. The attachment of a topoisomerase inhibitor camptothecin to a triple helix-forming oligonucleotide is taken as an example of such synthesis. The described method has general interest because any functional ligand containing a primary or secondary amino group or aliphatic carboxyl group could be attached to the terminal phosphate of an oligonucleotide in a similar way.

Position- and orientation-specific enhancement of topoisomerase I cleavage complexes by triplex DNA structures.

Topoisomerase I (Top1) activities are sensitive to various endogenous base modifications, and anticancer drugs including the natural alkaloid camptothecin. Here, we show that triple helix-forming oligonucleotides (TFOs) can enhance Top1-mediated DNA cleavage by affecting either or both the nicking and the closing activities of Top1 depending on the position and the orientation of the triplex DNA structure relative to the Top1 site. TFO binding 1 bp downstream from the Top1 site enhances cleavage by inhibiting religation and to a lesser extent DNA nicking. In contrast, TFO binding 4 bp downstream from the Top1 site enhances DNA nicking especially when the 3' end of the TFO is proximal to the Top1 site. However, when the orientation of the triplex is inverted, with its 5' terminus 4 bp downstream from the Top1 site, religation is also inhibited. These position- and orientation-dependent effects of triplex structures on the Top1-mediated DNA cleavage and religation are discussed in the context of molecular modeling and effects of TFO on DNA twist and mobility at the duplex/triplex junction.

Sequence and pH effects of LNA-containing triple helix-forming oligonucleotides: physical chemistry, biochemistry, and modeling studies.

Triple helix-forming oligonucleotides (TFOs) have been demonstrated to be capable of interfering with gene expression and modifying genomic DNA in a sequence-specific manner. Partial incorporation of 2'-O,4'-C-methylene linked locked nucleic acid (LNA) residues in TFOs has been shown to enhance significantly triple helix formation, whereas the full-length LNA TFO failed to form a stable triplex. This work is aimed at understanding the triple helix-forming properties of LNA-containing TFOs and at optimally designing their sequences. Both DNA thermal melting, gel retardation, and restriction enzyme experiments as well as modeling studies by molecular mechanics were carried out to investigate the base composition/sequence and pH-dependence effects of LNA-containing TFOs, as well as their structural features underlying triple helix formation. Alternating LNA substitution every 2-3 nucleotides in TFOs is mandatory, whereas the use of thymine LNA residues should be favored under neutral pH conditions. A rule for designing optimal LNA-containing TFOs is proposed. In addition, alternative LNA and 2'-O-methyl residues in TFOs do not significantly improve triple helix formation.

Importance of clustered 2'-O-(2-aminoethyl) residues for the gene targeting activity of triple helix-forming oligonucleotides.

We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in living mammalian cells. We have described psoralen-linked TFOs with 2'-O-methyl and 2'-O-(2-aminoethyl) (2'-AE) substitutions that are active in a gene knockout assay in cultured cells. The assay is based on mutagenesis by psoralen, a photoactive DNA cross-linker. Previous work showed that TFOs with three or four 2'-AE residues were disproportionately more active than those with one or two substitutions. Here we demonstrate that for optimal bioactivity the 2'-AE residues must be clustered rather than dispersed. We have further characterized bioactive and inactive TFOs in an effort to identify biochemical and biophysical correlates of biological activity. While thermal stability is a standard monitor of TFO biophysical activity, we find that T(m) values do not distinguish bioactive and inactive TFOs. In contrast, measurements of TFO association rates appear to correlate well with bioactivity, in that triplex formation occurs disproportionately faster with the TFOs containing three or four 2'-AE residues. We asked if extending the incubation time prior to photoactivation would enhance the bioactivity of a TFO with a slow on rate relative to the TFO with a faster association rate. However, there was no change in bioactivity differential. These results are compatible with a model in which TFO binding in vivo is followed by relatively rapid elution by cellular functions, similar to that described for transcription factors. Under these circumstances, TFOs with faster on rates would be favored because they would be more likely to be in triplexes at the time of photoactivation.

Chemical tools for targeted mutagenesis of DNA based on triple helix formation.

The development of methods for targeted mutagenesis shows promise as an alternative form of gene therapy. Triple helix-forming oligonucleotides (TFOs) provide an attractive strategy for inducing mutations. Especially, alkylation of nucleobases with functionalized TFOs would have potential for site-directed mutation. Several studies have demonstrated that treatment of mammalian cells with TFOs can be exploited to introduce desired sequence changes and point mutations. This review summarizes targeted mutagenesis using reactive TFOs, including studies with photo reactive psolaren derivatives as well as a new reactive derivative recently developed by our group.

Gene targeting by triple helix-forming oligonucleotides.

Effective gene targeting reagents would have widespread utility for genomic manipulation including transgenic cell and animal construction and for gene therapy. They would also be useful in basic research as probes of chromatin structure, and as tools for studying the repair and mutagenesis of targeted DNA damage. We are developing triple helix-forming oligonucleotides (TFOs) for gene targeting in living mammalian cells. Challenges to TFO bioactivity include the impediments to the biochemistry of triplex formation presented by the physiological environment and the charge repulsion between the duplex and the third strand. In addition, there are biological constraints to target access imposed by mammalian chromatin structure. Here we describe the oligonucleotide modification format that appears to support biological activity of TFOs. In addition we show that manipulation of the cell biology, specifically the cell cycle, has a dramatic influence on TFO bioactivity.

Enhancement and Inhibition by 2′-O-Hydroxyethyl Residues of Gene Targeting Mediated by Triple Helix Forming Oligonucleotides

Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2′-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2′-O-hydroxyethyl (2′-HE) substitutions, in combination with other 2′ modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2′-HE residues enhance the bioactivity of TFOs containing 2′-O-methyl (2′-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2′-O-aminoethyl (2′-AE) residues.

Synthesis of novel TFOs bearing an abasic portion and their triplex forming ability with a DNA duplex containing a pyrimidine-gapped polypurine strand.

Novel bi-functional anthraquinone phosphoramidite reagent was prepared from anthraquinolylethylenediamine and 2,2-bis(hydroxymethyl)propionic acid. The amidite reagent was incorporated into the middle position of oligoDNA consisting of pyrimidine residues, exclusively. The resulted oligoDNA was examined as the triple helix forming oligonucleotide (TFO) targeting a homopurine tract of a double helical DNA possessing a single pyrimidine-gap sequence.