Selective formation of stable triplexes including a TA or a CG interrupting site with new bicyclic nucleoside analogues (WNA)

Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules and would become powerful tools for genomic research. As the stabilization of the TFO is partially provided by hydrogen bonds to purine bases, the most stable triplexes form with homopurine/homopyrimidine sequences, and a pyrimidine base in the purine strand of the duplex interrupts triplex formation. If a TFO can recognize sequences including such an interrupting site, the target regions in the genome would be expanded to a greater extent. However, this problem has not been generally solved despite extensive studies. We have previously reported a new base analogue (WNA) constructed of three parts, a benzene ring, a heterocyclic ring, and a bicyclic skeleton to hold these two parts. In this study, we have further investigated modification of WNA systematically and determined two useful WNA analogues, WNA-beta T and WNA-beta C, for selective stabilization of triplexes at a TA and a CG interrupting site, respectively. The triplexes with WNA analogues have exhibited an interesting property in that they are more stable than natural-type triplexes even at low Mg(2+) concentration. From comparison of the results with H-WNA-beta T lacking benzene and those with WNA-H without thymine, it has been suggested that benzene is a major contributor for triplex stability and thymine provides selectivity. Thus, it has been successfully demonstrated that WNA-beta T/TA and WNA-beta C/CG combinations may expand triplex recognition codes in addition to the natural A/AT and G/GC base triplet codes. The results of this study will provide useful information for the design of new WNA analogues to overcome inherent problems for further expansion of triplex recognition codes.     

Recognition of CG inversions in DNA triple helices by methylated 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues

Substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues have been synthesised from 5-alkynyl-uridine derivatives, incorporated into triplex forming oligonucleotides (TFOs) and found to selectively bind CG inversions with enhanced affinity compared to T.     

Selectivity and affinity of DNA triplex forming oligonucleotides containing the nucleoside analogues 2'-O-methyl-5-(3-amino-1-propynyl)uridine and 2'-O-methyl-5-propynyluridine

Triplex forming oligonucleotides (TFOs) containing the nucleoside analogues 2'-O-methyl-5-propynyluridine (1) and 2'-O-methyl-5-(3-amino-1-propynyl)uridine (2) were synthesized. The affinity and selectivity of triplex formation by these TFOs were studied by gel shift analysis, T(m) value measurement, and association rate assays. The results show that the introduction of 1 and 2 into TFOs can improve the stability of the triplexes under physiological conditions. Optimized distribution of 1 or 2 in the TFOs combined with a cluster of contiguous nucleosides with 2'-aminoethoxy sugars resulted in formation of triplexes with further enhanced stability and improved selectivity.     

Electrochemical impedance probing of transcriptional TATA binding protein based on TATA box site-specific binding

Electrochemical impedance probing of TATA binding protein (TBP) based on TATA box site-specific binding was described in this work. A sensitive detection of TBP was developed from TATA box DNA self assembly on the electrode and the impedance changes induced by TBP binding. Electrochemical impedance spectroscopy (EIS) probing of TBP had a sensitivity of 0.8 nM with excellent selectivity. Moreover, the interferences of triplex forming oligonucleotides (TFOs) and anticancer drug daunomycin on TBP binding to TATA box DNA were investigated by EIS. TFOs reduced the stability of TBP binding to TATA box, but daunomycin completely inhibited the TBP binding.     

New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction

New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies.     

DNA duplexes and triplex-forming oligodeoxynucleotides incorporating modified nucleosides forming stable and selective triplexes

We have previously reported DNA triplexes containing the unnatural base triad G-PPI·C3, in which PPI is an indole-fused cytosine derivative incorporated into DNA duplexes and C3 is an abasic site in triplex-forming oligonucleotides (TFOs) introduced by a propylene linker. In this study, we developed a new unnatural base triad A-ψ·C(R1) where ψ and C(R1) are base moieties 2'-deoxypseudouridine and 5-substituted deoxycytidine, respectively. We examined several electron-withdrawing substituents for R1 and found that 5-bromocytosine (C(Br)) could selectively recognize ψ. In addition, we developed a new PPI derivative, PPI(Me), having a methyl group on the indole ring in order to achieve selective triplex formation between DNA duplexes incorporating various Watson-Crick base pairs, such as T-A, C-G, A-ψ, and G-PPI(Me), and TFOs containing T, C, C(Br), and C3. We studied the selective triplex formation between these duplexes and TFOs using UV-melting and gel mobility shift assays.     

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

A sequence of double-stranded DNA (dsDNA) which can be recognized by a triplex-forming oligonucleotide (TFO) is limited to a homopurine-homopyrimidine sequence. To develop novel nucleoside analogues which recognize CG interruption in homopurine-homopyrimidine dsDNA, we synthesized a novel 2'-O,4'-C-methyleneribonucleic acid (2'-O,4'-C-methylene bridged nucleic acid; 2',4'-BNA) that bears the unnatural nucleobases, 2-pyridone (PB) or its 5-methyl congener (mPB); these analogues were introduced into pyrimidine TFOs using a DNA synthesizer. A TFO with a 2'-deoxy-beta-D-ribofuranosyl-2-pyridone (P) or 2',4'-BNA abasic monomer (HB) was also synthesized. The triplex-forming ability of various synthesized 15-mer TFOs and the corresponding homopurine-homopyrimidine dsDNA, which contained a single pyrimidine-purine (PyPu) interruption, was examined in UV melting experiments. It was found that PB and mPB in the TFOs successfully recognized CG interruption under physiological conditions (7 mM sodium phosphate, 140 mM KCl, 5 mM spermine, pH 7.0). Furthermore, triplex formation between the dsDNA target which contained three CG interruptions and the TFO with three PB units was also confirmed. Additional four-point 2',4'-BNA modifications of the TFO containing three PB units significantly enhanced its triplex-forming ability towards the dsDNA and had a Tm value of 43 degrees C under physiological conditions. These results indicate that a critical inherent problem of TFOs, namely, the sequence limitation of the dsDNA target, may be overcome to a large extent and this should promote antigene applications of TFOs in vitro and in vivo.     

Nonpolar isosteres of damaged DNA bases: effective mimicry of mutagenic properties of 8-oxopurines

A substantial fraction of mutations that arise in the cell comes from oxidative damage to DNA bases. Oxidation of purine bases at the 8-position, yielding 8-oxo-G and 8-oxo-A, results in conformational changes (from anti to syn) that cause miscoding during DNA replication. Here we describe the synthesis and biophysical and biochemical properties of low-polarity shape mimics of 8-oxopurines, and we report that these new analogues exhibit remarkable mimicry of the mutagenic properties of the natural damaged bases. A 2-chloro-4-fluoroindole nucleoside (1) was designed as an isosteric analogue of 8-oxo-dG, and a 2-chloro-4-methylbenzimidazole nucleoside (2) as a mimic of 8-oxo-dA. The nucleosides were prepared by reaction of the parent heterocycles with Hoffer's chlorodeoxyribose derivative. Structural studies of the free nucleosides 1 and 2 revealed that both bases are oriented syn, thus mimicking the conformation of the oxopurine nucleosides. Suitably protected phosphoramidite derivatives were prepared for incorporation into synthetic DNAs, to be used as probes of DNA damage responses, and 5'-triphosphate derivatives (3 and 4) were synthesized as analogues of damaged nucleotides in the cellular nucleotide pool. Base pairing studies in 12-mer duplexes showed that 1 and 2 have low affinity for polar pairing partners, consistent with previous nonpolar DNA base analogues. However, both compounds pair with small but significant selectivity for purine partners, consistent with the idea that the syn purine geometry leads to pyrimidine-like shapes. Steady-state kinetics studies of 1 and 2 were carried out with the Klenow fragment of Escherichia coli DNA Pol I (exo-) in single-nucleotide insertions. In the DNA template, the analogues successfully mimicked the mutagenic behavior of oxopurines, with 1 being paired selectively with adenine and 2 pairing selectively with guanine. The compounds showed similar mutagenic behavior as nucleoside triphosphate analogues, being preferentially inserted opposite mutagenic purine partners. The results suggest that much of the mutagenicity of oxopurines arises from their shapes in the syn conformation rather than from electrostatic and hydrogen-bonding effects. The new analogues are expected to be generally useful as mechanistic probes of cellular responses to DNA damage.     

Stability of Hoogsteen‐Type Triplexes – Electrostatic Attraction between Duplex Backbone and Triplex‐Forming Oligonucleotide (TFO) Using an Intercalating Conjugate

Syntheses are described for two novel twisted intercalating nucleic acid (TINA) monomers where the intercalator comprises a benzene ring linked to a naphthalimide moiety via an ethynediyl bridge. The intercalators Y and Z have a 2‐(dimethylamino)ethyl and a methyl residue on the naphthalimide moiety, respectively. When used as triplex‐forming oligonucleotides (TFOs), the novel naphthalimide TINAs show extraordinary high thermal stability in Hoogsteen‐type triplexes and duplexes with high discrimination of mismatch strands. DNA Strands containing the intercalator Y show higher thermal triplex stability than DNA strands containing the intercalator Z . This observation can be explained by the ionic interaction of the protonated dimethylamino group under physiological conditions, targeting the negatively charged phosphate backbone of the duplex. This interaction leads to an extra binding mode between the TFO and the duplex, in agreement with molecular‐modeling studies. We believe that this is the first example of an intercalator linking the TFO to the phosphate backbone of the duplex by an ionic interaction, which is a promising tool to achieve a higher triplex stability.     

Conformationally restricted triplex-forming oligonucleotides (TFOs). Binding properties of α-l-LNA and introduction of the N-glycosylated LNA-guanosine

The method for scaled-up production of α-l-LNA phosphoramidite building blocks containing thymine and 5-methylcytosine nucleobases is described. Binding properties of pyrimidine TFOs modified with α-l-LNA are reported. In contrast to LNA TFOs, the fully modified α-l-LNA forms a stable triplex with a model DNA duplex. Pyrimidine DNA/LNA/α-l-LNA chimeras also efficiently hybridize with a model DNA duplex in the parallel mode. LNA nucleoside containing unnatural N⁷-glycosylated guanine (LNA-⁷G) was synthesized by a convergent method and incorporated into LNA oligonucleotides. The triplex-forming alternating DNA/LNA oligonucleotides containing a single LNA-⁷G modification instead of internal LNA-mC demonstrate improved pH-dependent properties. The single LNA-⁷G modification can also discriminatively reduce competitive binding of TFOs to natural nucleic acids in the antiparallel duplex mode.     

Fluorous base-pairing effects in a DNA polymerase active site

We describe selective "fluorous" effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5'-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene ((F)B) and 4,5,6,7-tetrafluoroindole ((F)I), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo-). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide ((F)I) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene ((F)B). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the (F)B base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson-Crick pairs used in replication.     

Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexyl phosphates as substituents

We have developed new artificial oligonucleotides which distinguish short RNA targets from long ones. The modification of the 5' termini of oligonucleotides by using adenosine derivatives that possess a bulky cyclohexyl phosphate moiety at their base moiety and a phosphate group at the position of their 5'-hydroxyl group maximized their short RNA selectivity. The 2'-O-methyl-RNA (5'-XC(m)A(m)A(m)C(m)C(m)U(m)A(m)C(m)U(m)) having these modifications exhibits ca. 10 °C higher T(m) in the duplexes with the complementary short RNA (3'-GUUGGAUGA-5') than with the long RNA (3'-AUUAUAUGUUGGAUGAUGGUUA-5'). The oligodeoxynucleotides having the same modification exhibited similar selectivity. Such short-RNA selective binding of terminally modified oligonucleotides can be employed to distinguish between mature microRNAs and pre-microRNAs.     

A Click Chemistry Approach to Developing Molecularly Targeted DNA Scissors

Nucleic acid click chemistry was used to prepare a family of chemically modified triplex forming oligonucleotides (TFOs) for application as a new gene-targeted technology. Azide-bearing phenanthrene ligands—designed to promote triplex stability and copper binding—were ‘clicked’ to alkyne-modified parallel TFOs. Using this approach, a library of TFO hybrids was prepared and shown to effectively target purine-rich genetic elements in vitro. Several of the hybrids provide significant stabilisation toward melting in parallel triplexes (>20 °C) and DNA damage can be triggered upon copper binding in the presence of added reductant. Therefore, the TFO and ‘clicked’ ligands work synergistically to provide sequence-selectivity to the copper cutting unit which, in turn, confers high stabilisation to the DNA triplex. To extend the boundaries of this hybrid system further, a click chemistry-based di-copper binding ligand was developed to accommodate designer ancillary ligands such as DPQ and DPPZ. When this ligand was inserted into a TFO, a dramatic improvement in targeted oxidative cleavage is afforded.     

Aminoglycoside-nucleic acid interactions: remarkable stabilization of DNA and RNA triple helices by neomycin

The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E(off) values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.     

Synthesis and DNA duplex recognition of a triplex-forming oligonucleotide with an ureido-substituted 4-phenylimidazole nucleoside

A 4-(3-n-butylureidophenyl)imidazole nucleoside was successfully incorporated into a triplex-forming oligonucleotide (TFO). Binding affinity and base pair selectivity of the TFO containing this non-natural nucleoside were studied with various duplex targets containing all four possible Watson-Crick base pairs opposite the nucleoside analog in the third strand. Triplex thermal stabilities indicate that the synthetic nucleoside acts as a universal base in binding to all four possible Watson-Crick base pairs with moderate affinity but poor selectivity. Based on an analysis of its binding thermodynamics, this can be rationalized by the absence of strong specific interactions and more favorable entropic contributions upon triplex formation.     

Development of bridged nucleic acid analogues for antigene technology

In the last decade, increased efforts have been directed toward the development of oligonucleotide-based technologies for genome analyses, diagnostics, or therapeutics. Among them, an antigene strategy is one promising technology to regulate gene expression in living cells. Stable triplex formation between the triplex-forming oligonucleotide (TFO) and the target double-stranded DNA (dsDNA) is fundamental to the antigene strategy. However, there are two major drawbacks in triplex formation by a natural TFO: low stability of the triplex and limitations of the target DNA sequence. To overcome these problems, we have developed various bridged nucleic acids (BNAs), and found that the 2',4'-BNA modification of oligonucleotides strongly promotes parallel motif triplex formation under physiological conditions. Some nucleobase analogues to extend the target DNA sequence were designed, synthesized, and introduced into the 2',4'-BNA structure. The obtained 2',4'-BNA derivatives with unnatural nucleobases effectively recognized a pyrimidine-purine interruption in the target dsDNA. Some other examples of nucleic acid analogues for stable triplex formation and extension of the target DNA sequence are also summarized.     

Convenient synthesis of nucleoside borane diphosphate analogues: deoxy- and ribonucleoside 5'-P(alpha)-boranodiphosphates

The nucleoside boranophosphates, having one of the nonbridging phosphate oxygens substituted with a borane (BH(3)) group, have shown potential therapeutical applications as aptamers, antisense agents, and antiviral prodrugs. An oxathiaphospholane approach, which does not require exocyclic amine protection of the nucleobase, has been successfully developed to efficiently synthesize 5'-P(alpha)-boranodiphosphates of 2'-deoxythymidine, adenosine, guanosine, and uridine. The approach involves a key intermediate, the borane complex of nucleoside 5'-O-1,3,2-oxathiaphospholane 16, that undergoes a ring-opening reaction catalyzed by 1,4-diazabicyclo[5.4.0]-undec-7-ene to form the protected nucleoside 5'-P(alpha)-boranodiphosphate 18. Treatment of 18 with ammonium hydroxide yielded diastereoisomeric mixtures of nucleoside 5'-P(alpha)-boranodiphosphates 5. This oxathiaphospholane approach ensures the availability of nucleoside 5'-P(alpha)-boranodiphosphate analogues needed for antiviral drug research.     

Modulation of psoralen DNA crosslinking kinetics associated with a triplex-forming oligonucleotide

A triplex-forming oligonucleotide (TFO), HPRT3, conjugated to a psoralen derivative, was designed to target a psoralen reaction site within the HPRT gene. HPRT3 bound with high affinity to a synthetic duplex target sequence. At a uniform UVA radiation dose, the ratio of psoralen monoadducts (MA) to interstrand crosslinks decreased and inverted with increasing TFO concentration. As the TFO concentration increased from 10 nm to 10 microm, the efficiency of psoralen MA formation remained relatively constant but the efficiency of interstrand crosslink formation increased several-fold. Neither shortening the TFO to reduce its dissociation constant nor altering the DNA sequences flanking the TFO binding site altered the concentration dependence of MA and crosslink yields. The psoralen photokinetics associated with 10 nm HPRT3 converted to those associated with 10 microm HPRT3 with the addition of other unrelated TFOs at 10 microm that do not specifically interact with the HPRT3 target sequence. Glycerol at concentrations of 0.5% (vol/vol) or higher also mimicked high TFO concentrations in enhancing crosslink formation. These results demonstrate that while psoralen may be targeted to react at a particular sequence by TFOs, photoreactivity associated with triplex formation is also modulated by sequence-independent factors that may affect the local macromolecular environment.     

yDNA versus xDNA pyrimidine nucleobases: computational evidence for dependence of duplex stability on spacer location

The structural and binding properties of the natural and x- and y-pyrimidines were compared using computational methods. Our calculations show that although the x-pyrimidines favor different orientations about the glycosidic bond compared to the natural pyrimidines, which could have implications for the formation and resulting stability of xDNA duplexes and jeopardize the selectivity of expanded nucleobases, y-pyrimidines have rotational profiles more similar to the natural bases. Increasing the pyrimidine size using a benzene spacer leads to relatively minor changes in the hydrogen-bond strength of isolated Watson-Crick base pairs. However, differences in the anomeric carbon distances in pairs composed of x- or y-pyrimidines suggest yDNA may yield a more optimal expanded structure. By stacking two monomers via their centers of mass, we find that the expanded nucleobases stack much stronger than the natural bases. Additionally, although replacing xT by yT changes the stacking energy by less than 5 kJ mol (-1), replacing xC by yC significantly strengthens complexes with the natural nucleobases (by up to 30%). Calculations on larger duplex models composed of four nucleobases reveal that x- and y-pyrimidines can increase duplex stability of natural helices by strengthening both the intra and interstrand stacking interactions. Furthermore, when the total stability (sum of all hydrogen-bonding and (intrastrand and interstrand) stacking interactions) of the larger models is considered, y-pyrimidines lead to more stable complexes than x-pyrimidines for all but three duplex sequences. Thus, through analysis of a variety of properties, our calculations suggest that the location of the benzene spacer affects the properties of expanded nucleobases and the stability of expanded duplexes, and therefore should be carefully considered when designing future expanded analogues.