Towards mixed sequence recognition by triple helix formation

Journal of the American Chemical Society, 2005

Stable DNA triple helical structures are normally restricted to homopurine sequences. We have described a system of four heterocyclic bases (TRIPsides) that when incorporated into oligomers (oligoTRIPs) can recognize and bind in the major groove to any native sequence of DNA [Li et al., J. Am. Chem. Soc. 2003]. To date, we have reported on triple forming oligomers composed of two of these TRIPsides, i.e., antiTA and antiGC, and their ability to form intramolecular triplexes at mixed purine/pyrimidine sequences. In the present study, we describe the synthesis and characterization of the antiCG TRIPside and its use in conjunction with antiTA and antiGC to form sequence specific intra-and/or inter-molecular triplex structures at mixed purine/pyrimidine sequences that require as many as four major groove crossovers.

Tetrahedron, 1999

Protected forms of 1,2,3-propanetriol and cis, cis-l,3,5-cyclohexanetriol were incorporated onto solid supports which were exploited in the solid phase synthesis of 3'-3' linked oligodeoxyribonucleotides (ODNs), involving only nucleoside 3'-phosphoramidites as building blocks. UV thermal denaturation analysis showed the ability of ODNs with this inversion of polarity motif to cooperatively hybridize with duplexes of the type 5'-(Pu)m(PY)n-3' in an alternate strand recognition approach.

ChemBioChem, 2004

BMC biochemistry, 2002

Background: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.

Biochemistry, 1993

Triple-helical structures involving the interaction of an oligonucleotide third strand with a duplex nucleic acid sequence have recently gained attention as a therapeutic strategy in the "antigene" approach [cf. Eur. J. Cancer 27, 146614711. This method utilizes the triple helix formed from the cellular duplex and an added third strand to directly regulate the activity of a selected gene. The limited stability of nucleic acid triple-helical interactions, particularly if the third strand has backbone modifications such as methylphosphonate or phosphorothioate substitutions, is a limiting condition for the use of this approach. We have designed and synthesized compounds, on the basis of the following three criteria, that we feel should provide selective interactions and significant stabilization of triplexes: appropriate aromatic surface area for stacking with triplex bases in an intercalation complex, positive charge, and limited torsional freedom in the aromatic system to match the propeller twist of the triple-base interactions in the triplex. A series of quinoline derivatives with an alkylamine side chain at the 4-position and with different aryl substituents at the 2-position has been synthesized as our first compounds, A 2-naphthyl derivative provides significant and selective stabilization of the triplex. In a 0.2 M NaCl buffer, the naphthyl derivative increased the Tm for the triplex (triplex to duplex and third strand transition) by approximately 30 O C more than the T, increase for the duplex (duplex to single strands transition). Spectral changes and energy-transfer results indicate that the naphthyl compound and related derivatives bind to the triplex by intercalation. Molecular modeling results indicate very good stacking of the naphthylquinoline ring system with the bases at a triplex intercalation site, but the results also indicate that the ring system is too large to stack optimally with base pairs at a duplex intercalation site.

Nucleic Acids Research, 1987

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [a]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[a]-thymidylate : a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[a]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[a]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in a parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [a]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.

Molekuliarnaia biologiia

The possibility is discussed of stabilizing a DNA triple helix by covalent conjugation to the third strand (through its terminal phosphate) of ligands that have affinity to double and triple helices. Two types of stabilizers are considered: minor groove binders based on oligopyrroles, and triplex-specific intercalators. As a target, a synthetic 29-mer duplex containing a natural polypurine sequence of the human immunodeficiency provirus was employed. The stabilization with minor groove binders requires several conditions to be respected: a sufficiently long linker capable of reaching the minor groove from the major groove, a specific doublestranded structure of the oligopyrrole fragment, and its in-phase fitness to the target sequence. The best stabilizers of a triplex were novel conjugates in which two parallel molecules containing six pyrrole units each are linked to the same 5'-phosphate of a 16-mer triplex-forming oligonucleotide. The stabilizing properties of these derivatives were comparable to those of benzoindoloquinoline (BIQ) intercalators attached to the terminal phosphate of triple-helix forming oligonucleotides.

Nucleic Acids Research, 1996

Triple helices containing C + •G•C triplets are destabilised at physiological pH due to the requirement for base protonation of 2′-deoxycytidine (dC), which has a pK a of 4.3. The C nucleoside 2-amino-5-(2′-deoxy-β-Dribofuranosyl)pyridine (β-AP) is structurally analogous to dC but is considerably more basic, with a pK a of 5.93. We have synthesised 5′-psoralen linked oligodeoxyribonucleotides (ODNs) containing thymidine (dT) and either β-AP or its α-anomer (α-AP) and have assessed their ability to form triplexes with a double-stranded target derived from standard deoxynucleotides (i.e. β-anomers). Third strand ODNs derived from dT and β-AP were found to have considerably higher binding affinities for the target than the corresponding ODNs derived from dT and either dC or 5-methyl-2′deoxycytidine (5-Me-dC). ODNs containing dT and α-AP also showed enhanced triplex formation with the duplex target and, in addition are more stable in serumcontaining medium than standard oligopyrimidinederived ODNs or ODNs derived from dT and β-AP. Molecular modelling studies showed that an α-anomeric AP nucleotide can be accommodated within an otherwise β-anomeric triplex with only minor perturbation of the triplex structure. Molecular dynamics (MD) simulations on triplexes containing either the αor β-anomer of (N1-protonated) AP showed that in both cases the base retained two standard hydrogen bonds to its associated guanine when the 'A-type' model of the triplex was used as the start-point for the simulation, but that bifurcated hydrogen bonds resulted when the alternative 'B-type' triplex model was used. The lack of a differential stability between α-APand β-AP-containing triplexes at pH >7, predicted from the behaviour of the B-type models, suggests that the A-type models are more appropriate.

Journal of Organic Chemistry, 1997

ODNs containing a 3′-3′ phosphodiester junction have been conveniently synthesized through a solid phase procedure involving only 3′-phosphoramidite nucleosides, starting from a modified support linking the first nucleotide through the base. Thermal denaturation studies showed that 16-mers with this inversion of polarity cooperatively bound to 5′-(purine) m (pyrimidine) n -3′-type duplexes by specific recognition of the oligopurine domains alternately on the two DNA strands. The nature of the 3′-3′ junction and nearest neighbors affected the stability of the resulting triplexes.

Chemical Science

Triplex-forming oligonucleotides can target specific DNA sequences by binding in the duplex major groove. Chemical modifications and ligand binding have been explored, for use in a variety of biological applications.

Biochemistry, 1997

Peptide nucleic acid (PNA) is an oligonucleotide mimic in which the backbone of DNA has been replaced by a pseudopeptide. Thymine-rich homopyrimidine PNA oligomers have been found to recognize double-stranded DNA targets by displacement of the pyrimidine DNA strand and forming an internal Watson-Crick-Hoogsteen base-paired PNA(pyr)-DNA(pu)-PNA(pyr) triplex. We here show that cytosine-rich homopyrimidine PNA sequences instead add to double-stranded polynucleotide targets as Hoogsteen strands forming PNA(pyr)-DNA(pu)-DNA(pyr) triplexes. Furthermore, PNA strands with homopurine or alternating thymine-guanine sequences are shown to invade their respective DNA targets by displacing the identical DNA strands of the polynucleotides and forming new PNA-DNA duplexes. These results indicate distinct mechanistic variations as to how PNA interacts with a DNA target depending on choice of nucleobases, which could be of importance for future design of gene-specific diagnostic or therapeutic agents.

Bioorganic & Medicinal Chemistry Letters, 2005

Several substituted quinolin-4-amines and heteroaromatic analogs were synthesized and evaluated for interaction with triplex polydAAE2polydT and duplex polydAAEpolydT by using UV-thermal melting experiments. Excellent triple-helix DNA ligands with high affinity toward TAEAAET triplets and triple/duplex selectivity were designed through a rational approach.

Bioconjugate Chemistry, 1991

Journal of Molecular Recognition, 1994

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz 'H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential dNN(i, i+ 1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an a-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.

Proceedings of the National Academy of Sciences, 1998

DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3,4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteenpaired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and͞or induce triple helix formation under physiological conditions.