Wavelength-selective light-triggered strand exchange reaction.

We prepared an oligodeoxynucleotide (ODN) bearing two 4-hydroxy-2-mercaptobenzimidazole nucleobase analogues (SB(NV) and SB(NB)) modified with different photolabile groups. This ODN enabled a light-triggered strand exchange reaction in a wavelength-selective manner.

C5-azobenzene-functionalized locked nucleic acid uridine: isomerization properties, hybridization ability, and enzymatic stability.

Oligonucleotides (ONs) modified with a locked nucleic acid (LNA) are widely used in the fields of therapeutics, diagnosis, and nanotechnology. There have been significant efforts towards developing LNA analogues bearing modified bridges to improve their hybridization ability, nuclease resistance, and pharmacokinetic profiles. Moreover, nucleobase modifications of LNA are useful strategies for the functionalization of ONs. Modifications of the C5-position of pyrimidine nucleobases are particularly interesting because they enable predictable positioning of functional groups in the major groove of the duplex. Here we report the synthesis of C5-azobenzene-functionalized LNA uridine (LNA-U(Az)) and properties of LNA-U(Az)-modified ONs, including isomerization properties, hybridization ability, and enzyme stability. LNA-U(Az) in ON is photo-isomerized effectively and reversibly by irradiation at 365 nm (trans to cis) and 450 nm (cis to trans). LNA-U(Az)-modified ONs show RNA-selective hybridization ability despite the large hydrophobic azobenzene moiety extending into the major groove of the duplex. The enzymatic stability of LNA-U(Az)-modified ONs is higher than that of natural and LNA-modified ONs with or without photo-irradiation. Our results indicate that LNA-U(Az) holds promise for RNA targeting and photo-switchable technologies.

Nucleic acid delivery systems based on poly(galactosyl ureaethyl methacrylate-b-dimethylamino ethyl methacrylate) diblock copolymers.

Elevated low-density-lipoprotein cholesterol (LDL-C) level is a major risk factor leading to cardiovascular diseases. Therefore, since the proprotein convertase subtilisin kexin type 9 (PCSK9) regulates LDL-C receptors, it represents the appropriate target for cholesterol-lowering gene therapy. However, although delivery of antisense oligonucleotides (ODNs) is a promising therapeutic method for the treatment of several diseases, it is fundamental to develop new and efficacious carriers that transport the ODNs into the target cell in a nontoxic manner. This study reports on the synthesis, characterization and in vitro testing of a new liver specific carrier based on linear cationic diblock glycopolymer composed of galactosyl ureaethyl methacrylate (GAMA) and the primary amine-containing dimethylamino ethyl methacrylate (DMAEMA). Delivery experiments proved that the poly(galactosyl ureaethyl methacrylate -b-dimethylamino ethyl methacrylate) diblock copolymer (pGa4D47), internalized in a receptor-mediated manner, exhibited a much faster nuclear transportation than ODNs carried by pDMAEMA homopolymer or glycopolymer bearing glucose moieties.

Promotion of triplex formation by 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification: thermodynamic and kinetic studies.

We analyzed the effect of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with 2',4'-BNA modified TFO was about 20 times larger than that observed with unmodified TFO. The observed increase in the binding constant at neutral pH by the 2',4'-BNA modification resulted from the considerable decrease in the dissociation rate constant.

2'-O,4'-C-Methylene bridged nucleic acid (2',4'-BNA): synthesis and triplex-forming properties.

For development of ideal antisense and antigene molecules, various chemical modifications of oligonucleotides have been studied. However, despite their importance, there is only limited information available on the triplex-forming ability of the conformationally restricted or locked oligonucleotides. We report herein that 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO) significantly enhances the binding affinity towards target dsDNA. On Tm measurements, the triplex with the 2',4'-BNA oligonucleotides were found to be stabilized with deltaTm/modification of +4.3 to +5 degrees C at pH 6.6 compared to the triplexes with the unmodified oligonucleotide. By means of gel-retardation assay, the binding constant of the 2',4'-BNA oligonucleotide at pH 7.0 was at least 300-fold higher than that of the natural oligonucleotide. In addition, the 2',4'-BNA oligonucleotide clearly showed the inhibition of the NF-kappaB transcription factor (p50)-target dsDNA binding by forming a stable triplex at pH 7.0.

Symmetrical cationic triglycerides: an efficient synthesis and application to gene transfer.

Some cationic triglycerides 1Aa-1Cb which have a symmetrical structure were effectively synthesized and formulated into cationic liposomes with the co-lipid dioleoylphosphatidylethanolamine (DOPE) and/or dilauroylphosphatidylcholine (DLPC). The plasmid encoding a luciferase was delivered into CHO cells by using these cationic liposomes. Our symmetrical cationic triglycerides showed high transfection activity when DOPE was used as a co-lipid. Among the symmetrical cationic triglycerides synthesized here, 1Ab and 1Ac, which have an oleoyl group at the 1- and 3-position in the glycerol backbone and also have a relatively long linker connecting the 2-hydroxy group in glycerol with the quaternary ammonium head group, were found to be the most suitable for gene delivery into cells. The transfection activity of the symmetrical cationic triglyceride 1Ab was comparable with that of its asymmetrical congener 6 and several times higher than that of Lipofectin.

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

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.

Inhibition of ICAM-1 gene expression by antisense 2',4'-BNA oligonucleotides.

We have previously reported that 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) had unprecedented binding affinity towards its complementary RNA. We reported herein that the antisense 2',4'-BNA oligonucleotides showed potent inhibitory effect on gene expression of Intercellular Adhesion Molecule-1 (ICAM-1) in living cells. The contribution of RNase H to this antisense effect and adequate stability of 2',4'-BNA oligonucleotides to enzymatic degradation were also demonstrated.

2',4'-BNA bearing unnatural nucleobases: toward the expansion of the target sequence of double-stranded DNA in triplex formation.

It is absolutely necessary for practical application of antigene strategy to create nucleoside analogues recognizing pyrimidine.purine interruption in dsDNA. To develop a nucleoside analogue to interact with T.A interruption, we designed and synthesized a novel 2',4'-BNA monomer (HBB), 2-(2'-O,4'-C-methyleneribofuranosyl)phenol, and it was introduced into a triplex-forming oligonucleotide (TFO). On melting temperature (Tm) measurements, HBB was found to interact with T.A interruption with moderate binding affinity, and unprecedented dU.A base pair recognition ability of HBB was also observed.

Triplex formation involving 2',4'-BNA with 2-pyridone base analogue: efficient and selective recognition of C:G interruption.

We examined the thermodynamic properties of 2',4'-bridged nucleic acid containing 2-pyridone as a nucleobase (PB) to recognize a C interruption in the homopurine strand of the target duplex for pyrimidine motif triplex formation at neutral pH. The triplex formation involving triplex-forming oligonucleotide with PB is highly sequence-selective to specifically recognize C:G target base pair rather than the other G:C, T:A, or A:T base pairs. PB.C:G triad gives significantly larger binding constant than T.C:G triad, which has been known to be the most stable combination in natural base.C:G triad. Our results certainly support the idea that PB could be a key nucleoside to recognize a C interruption in the homopurine strand of the target duplex with high binding affinity and selectivity, and reduce the restriction of target sequences for triplex formation.

[Syntheses and properties of conformationally restrained nucleosides and oligonucleotides analogues].

This review summarizes our efficient syntheses of novel bicyclic nucleoside analogues, 3'-O,4'-C-methyleneribonucleosides (1) (4-BC type nucleoside analogue), 2'-O,4'-C-methyleneribonucleosides (2) (5-BC), 3'-amino-3'-deoxy-3'-N,4'-C-methyleneribonucleosides (3) (aza 4-BC), and 3'-azido- and 3'-amino-3'-deoxy-2'-O,4'-C-methyleneribonucleosides (4, 5) (aza 5-BC). From 1H-NMR and X-ray crystallographic analyses, the 4-BC and aza 4-BC type nucleoside analogues (1, 3) were found to have a S-conformation predominantly, while the conformations of 5-BC and aza 5-BC type nucleoside analogues (2, 4, 5) were exclusively locked in N-form. The 4-BC and 5-BC type nucleoside analogues (1, 2) were effectively introduced into oligonucleotides using a DNA synthesizer. Furthermore, unprecedented hybridizing ability towards complementary RNA and DNA, RNA selectivity, potent triplex forming ability, and sufficient enzymatic stability of these modified oligonucleotides were also confirmed. These results should reveal a promising route to the development of antisense/antigene methodology.

Synthesis and triplex forming ability of conformationally locked oligonucleotides containing unnatural nucleobases: efficient recognition of a C.G interruption.

In order to develop a novel nucleoside analogue which recognizes C.G interruption in homopurine.homopyrimidine DNA, we designed and synthesized a conformationally locked nucleoside analogue, 1-(2-O,4-C-methylene-beta-D-ribofuranosyl)pyridin-2-one (4), and introduced it into a triplex-forming oligonucleotide (TFO). On melting temperature (Tm) measurements, the unprecedented C.G base recognition ability of 4 was observed.

Promotion of triplex formation by a fixed N-form sugar puckering: thermodynamic and kinetic studies.

We analyzed the effect of a fixed N-form sugar puckering of TFO (triplex-forming oligonucleotide) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. Both thermodynamic and kinetic analyses revealed that the binding constant of the pyrimidine motif triplex formation at pH 6.8 with modified TFO containing the fixed N-form sugar puckering was about 20-times larger than that observed with unmodified TFO. Kinetic data also demonstrated that the observed increase in the binding constant at neutral pH by the fixed N-form sugar puckering resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the fixed N-form sugar puckering of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Gene transfer mediated by YKS-220 cationic particles: convenient and efficient gene delivery reagent.

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.

Preparation and properties of 2',5'-linked oligonucleotide analogues containing 3'-O,4'-C-methyleneribonucleosides.

Bicyclic nucleoside analogues, 3'-O,4'-C-methyleneuridine and -5-methyluridine, were successfully incorporated into oligonucleotides via connection with 2',5'-phosphodiester linkage, and hybridization behavior and nuclease stability of the modified oligonucleotides were investigated.

Properties of cationic liposomes composed of cationic lipid YKS-220 having an ester linkage: adequate stability, high transfection efficiency, and low cytotoxicity.

Cationic lipid N-[3-[2-(1,3-dioleoyloxy)propoxy-carbonyl]propyl]-N,N,N-trimethyla mmonium iodide (YKS-220) having a symmetrical and biodegradable structure was employed for the preparation of cationic liposomes with dioleoylphosphatidylethanolamine (DOPE). The stability, transfection activity in several cell lines and cytotoxicity of YKS-220 cationic liposomes were studied. It was found the YKS-220 cationic liposomes were very stable and their transfection activity remained even after storage at 4 degrees C for 12 months. The transfection activity of these liposomes was assayed using CHO, COS, and HepG2 cells and found to be comparable with, or better than, that of other cationic liposomes, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) liposome, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) liposome (Lipofectin), and 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl -1-propanaminium trifluoroacetate (DOSPA) liposome (LipofectAMINE). In addition, the cytotoxicity of YKS-220 cationic liposomes was far lower than that of other cationic liposomes.