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.     

Design, synthesis, and properties of 2',4'-BNA(NC): a bridged nucleic acid analogue

The novel bridged nucleic-acid analogue 2',4'-BNA(NC) (2'-O,4'-C-aminomethylene bridged nucleic acid), containing a six-membered bridged structure with an N-O linkage, was designed and synthesized efficiently, demonstrating a one-pot intramolecular NC bond-forming key reaction to construct a perhydro-1,2-oxazine ring (11 and 12). Three monomers of 2',4'-BNA(NC) (2',4'-BNA(NC)[NH], [NMe], and [NBn]) were synthesized and incorporated into oligonucleotides, and their properties were investigated and compared with those of 2',4'-BNA (LNA)-modified oligonucleotides. Compared to 2',4'-BNA (LNA)-modified oligonucleotides, 2',4'-BNA(NC) congeners were found to possess: (i) equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power, (ii) much better RNA selective binding, (iii) stronger and more sequence selective triplex-forming characters, and (iv) immensely higher nuclease resistance, even higher than the S(p)-phosphorthioate analogue. 2',4'-BNA(NC)-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.     

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.     

N-Methyl substituted 2',4'- BNANC: a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization

Oligonucleotides modified with a novel BNA analogue, 2', 4'-BNA(NC)[N-Me], were synthesized, and in comparison to 2',4'-BNA (LNA), have similarly high RNA affinity, better RNA selectivity and much higher resistance to nuclease degradation, suggesting that the novel BNA analogue may be particularly useful for antisense approaches.     

Synthesis and properties of a bridged nucleic acid with a perhydro-1,2-oxazin-3-one ring

A novel derivative of 2',4'-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with an N-O linkage in the six-membered bridged structure is the unique structural feature of the novel 2',4'-bridged nucleic acid analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl function, which would improve the properties of the modification. The synthesized monomer was incorporated into oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be higher than that of the corresponding natural and 2',4'-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA modified oligonucleotide to 3'-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover, ring-opening of only one modification at the 3'-end of the oligonucleotides was observed, even if two or three HxNA modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in antisense technology.     

High physiological thermal triplex stability optimization of twisted intercalating nucleic acids (TINA)

The structure of the monomer (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol () in twisted intercalating nucleic acids (TINA) was optimized for stabilizing interactions between the intercalator and surrounding nucleobases when used as a triplex forming oligonucleotide (TFO). Enhancement of pi-pi interactions with nucleobases of the TFO was achieved by increasing the aromatic surface using the (R)-1-O-[4-(1-pyrenylethynyl)naphthylmethyl]glycerol monomer (). Bulge insertion of in the middle of a Hoogsteen-type triplex increased the triplex thermal stability, DeltaT(m) = +2.0 degrees C compared with at pH 7.2. Syntheses and thermal denaturation studies of triplexes and duplexes are described for three novel TINA monomers. The influence of pi-pi interactions, link length and the positioning of the ether in the linker in the TINA derivatives are described.     

3'-amino-2',4'-BNA: novel bridged nucleic acids having an N3'-->P5' phosphoramidate linkage

Novel oligonucleotide analogues, containing a 3'-amino-2',4'-BNA unit, were successfully synthesized, and they showed superior duplex and triplex forming ability as well as BNA itself, along with remarkable enzymatic stability.     

Synergistic stabilization of nucleic acid assembly by oligo-N3'-->P5' phosphoramidate modification and additions of comb-type cationic copolymers

Synergic stabilization of DNA triplexes by oligo-N3'-->P5' phosphoramidate (PN) modification and additions of comb-type cationic copolymers was demonstrated. The combination of the copolymer and the PN modification increased triplex K(a) about 4 orders of magnitude. Kinetic analysis revealed that observed stabilization resulted from kinetic complimentarity between increased association rates by the copolymer and decreased dissociation rates by the PN modification of triplex forming oligonucleotides. No countering interference between these stabilizing effects was observed. We propose that kinetic analyses of stabilizing effects permit selection of a rational combination of stabilizing methods for successful synergy in stabilizing complex formation.     

Mutagenesis Mediated by Triple Helix–Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context

Targeted mutagenesis and gene knock-out can be mediated by triple helix-forming oligonucleotides (TFO) linked to mutagenic agents, such as psoralen. However, this strategy is limited by the availability of homopurine/ homopyrimidine stretches at or near the target site because such sequences are required for high-affinity triplex formation. To overcome this limitation, we have tested TFO conjugated to psoralen via linker arms of lengths varying from 2 to 86 bonds, thereby designed to deliver the psoralen at varying distances from the third strand binding site present at the 3'end of the supFG1 mutation reporter gene. Following triplex formation and UVA irradiation, mutations were detected using an SV40-based shuttle vector assay in human cells. The frequency and distribution of mutations depended on the length of the linker arm. Precise targeting was observed only for linker arms of length 2 and 6, which also yielded the highest mutation frequencies (3 and 14%, respectively). Psoralen–TFO with longer tethers yielded mutations at multiple sites, with the maximum distance from the triplex site limited by the linker length but with the distribution within that range influenced by the propensity for psoralen intercalation at A:T base-pair-rich sites. Thus, gene modification can be extended beyond the site of third strand binding but with a decrease in the precision of the targeting.     

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.     

Fluorescently Sensing of DNA Triplex Assembly Using an Isoquinoline Alkaloid as Selector,Stabilizer, Inducer,and Switch‐On Emitter

DNA triplex assembly has attracted a variety of interest in the regulation of genetic expression, drug screening, molecular switches, and sensors. However, these achievements are essentially dependent on the formation and stability of the triplex assembly. Herein, the recognition of DNA triplex assembly with various isoquinoline alkaloids was investigated. We found that natural chelerythrine (CHE) exhibits the highest selectivity in recognizing the triplex structure. The DNA triplex stability is substantially increased upon CHE binding, as opposed to the invariance in the stability of the duplex counterpart. CHE also favors the assembly of the triplex‐forming oligonucleotide (TFO) with its duplex counterpart. The triplex binding switches CHE to a strong fluorescent emitter, which suggests CHE as a useful probe in following triplex assembly. As a unique triplex selector, inducer, and emitter, CHE successfully reports the wide pH‐ and metal‐ion‐dependent tunability of the triplex nanoswitch in a label‐free manner.     

Gold nanoparticles-based colorimetric investigation of triplex formation under weak alkalic pH environment with the aid of Ag+

A novel colorimetric method for investigating triplex formation between oligonucleotide modified Au nanoparticles (AuNPs) under weak alkalic pH environment is developed based upon the specific recognition property of Ag+ with CGC triads. Oligonucleotide 5'-SH-T12-CTTCTTTCCTTTCTTC-3' (oligo-1) is modified on the surface of AuNPs. Upon addition of oligonucleotide 5'-GAAGAAAGGAAAGAAG-3' (oligo-2), triplex formation between oligo-1 modified AuNPs occurred at pH 8.0 with the aid of Ag+, triggers the aggregation of AuNPs, accompany with the solution color change from red to purple. The melting temperature demonstrates a 31 °C increase for the triplex DNA compose of 10 T?A°T triads and 6 C?G°C triads upon addition of Ag+, the disassociation constant (Kd) between Ag+ and C?G°C triads is 3.6 μM. Moreover, triplex formation between AuNPs depending on Ag+ can be used to recognize Ag+ ion with the naked eye, as well as UV-vis absorption spectroscopy.     

Stabilization of parallel triplexes by twisted intercalating nucleic acids (TINAs) incorporating 1,2,3-triazole units and prepared by microwave-accelerated click chemistry

A highly efficient method for postsynthetic modification of unprotected oligonucleotides incorporating internal insertions of (R)-1-O-(4-ethynylbenzyl)glycerol has been developed through the application of click chemistry with water-insoluble pyren-1-yl azide and water-soluble benzyl azide and acceleration by microwave irradiation. The twisted intercalating nucleic acids (TINAs) obtained in these reactions, possessing bulged insertions of (R)-3-O-{4-[1-(pyren-1-yl)-1H-1,2,3-triazol-4-yl]benzyl}glycerol (7), formed parallel triplexes with thermal stabilities of 20.0, 34.0, and 40.0 degrees C at pH 7.2 in the cases of one, two, or three insertions of 7, respectively, separated by three nucleic bases. An oligonucleotide with four insertions of 7--each between three nucleic bases in the sequence--was unable to form complexes with complementary single- or double-stranded DNAs, as a result of self-aggregation of the pyrene moieties. This assumption was supported by the formation of a very strong excimer band at 460 nm in the fluorescence spectra. Molecular modeling of the parallel triplex with bulged insertion of the monomer 7 in the triplex-forming oligonucleotide (TFO) showed that only the pyrene moiety was stacking between the bases of the dsDNA, whereas 1,2,3-triazole did not participate in the triplex stabilization. Thermal denaturation studies of the duplexes and triplexes, as well as the fluorescence properties of TINA-triazole 7, are discussed and compared with previous studies on TINA.     

LNA (locked nucleic acid) and analogs as triplex-forming oligonucleotides

The triplex-forming abilities of some conformationally restricted nucleotide analogs are disclosed and compared herein. 2'-Amino-LNA monomers proved to be less stabilising to triplexes than LNA monomers when incorporated into a triplex-forming third strand. N2'-functionalisation of 2'-amino-LNA monomers with a glycyl unit induced the formation of exceptionally stable triplexes. Nucleotide analogs containing a C2',C3'-oxymethylene linker (E-type furanose conformation) or a C2',C4'-propylene linker (N-type furanose conformation) had no significant effect on triplex stability proving that conformational restriction per se is insufficient to stabilise triplexes.     

Kinetic and thermodynamic analysis of 9‐nitrocamptothecin hydrolysis at physiological pH in the presence and absence of human serum albumin

Kinetic and thermodynamic analysis of the 9‐nitrocamptothecin (9NC) hydrolysis reaction in the presence and absence of human serum albumin (HSA) in phosphate‐buffered saline (PBS) of pH 7.4 was carried out by first derivative absorption spectroscopy. The thermodynamic parameters determined in these studies provided a mechanistic explanation toward the endothermic but yet thermodynamically favorable hydrolysis of 9NC at physiological temperature and pH. In the presence of HSA, the apparent rate constant of 9NC hydrolysis was 3–3.5 times higher than in 9NC solutions alone, whereas the apparent equilibrium constant of 9NC hydrolysis was found to increase at a higher extent in the presence of HSA than in PBS with increasing temperature, reaching almost complete hydrolysis of the 9NC to the 9NC‐carboxylate at 315.15 K. Importantly, the E_a of the 9NC hydrolysis reaction in the presence of HSA was determined to be on average 17 kJ mol^?1 lower than the E_a determined in plain PBS. Moreover, analysis of binding isotherms constructed for the HSA interaction with 9NC, using infinitely cooperative and independent binding models, demonstrated an incredibly higher binding constant for the 9NC‐carboxylate form as compared to the very weak and concentration‐dependent binding for the 9NC‐lactone species at 310.15 K. Taken together, the preferential association of the carboxylate form with HSA and the lower E_a of 9NC hydrolysis in the presence of HSA provide a mechanistic explanation for the shift of lactone–carboxylate equilibria toward the carboxylate product under physiological conditions of pH and ionic strength. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 693–703, 2010     

Stabilization of a Bimolecular Triplex by 3′‐S‐Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single‐stranded nucleic acids are also relevant in third‐strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson–Crick (W–C) base‐paired DNA duplexes and a Hoogsteen base‐paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA‐like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3′‐S‐phosphorothiolate (3′‐SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. ¹H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3′‐SP modifications is more compact. The 3′‐SP modification was previously shown to stabilise G‐quadruplex and i‐motif structures and therefore is now proposed as a generic solution to stabilising multi‐stranded DNA structures.     

Combining the best in triplex recognition: synthesis and nucleic acid binding of a BQQ-neomycin conjugate

Synthesis of a BQQ-neomycin conjugate is reported. The conjugate combines two ligands, one known to intercalate triplexes (BQQ) and another known to bind in the triplex groove (neomycin). The conjugate stabilizes T.A.T, as well as mixed base DNA triplex, better than neomycin, BQQ, or a combination of both. The conjugate selectively stabilizes the triplex (in the presence of physiological salt concentrations), with as little as 4 muM of the ligand leading to a DeltaTm of >60 degrees C. Competition dialysis studies show a clear preference for the drug binding to triplex DNA/RNA over the duplex/single strand structures. Modeling studies suggest a structure of neomycin bound to the larger W-H (Watson-Hoogsteen) groove with BQQ intercalated between the triplex bases.     

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.     

Synthesis and properties of a novel bridged nucleic acid with a P3'' --> N5'' phosphoramidate linkage, 5''-amino-2'',4''-BNA

5'-Amino-2',4'-BNA, a novel analogue of BNA series compounds, was successfully synthesized, and its incorporated oligonucleotides showed potent duplex- and triplex-forming ability and resistance against snake venom phosphodiesterase.     

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.