Takeshi Imanishi

Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2'-O,4'- C-methyleneribonucleosides

Bicyclic nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneuridine and -cytidine, were incorporated into oligonucleotides, and the binding efficiency of the modified oligonucleotides to the complementary DNA and RNA as well as the CD spectra of the modified DNA-DNA and modified DNA-RNA duplexes were studied.

Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering

2′-O,4′-C-Methyleneuridine and -cytidine, novel bicyclic nucleoside analogs having a typical C3′-endo sugar puckering, were synthesized starting from uridine via a several-step sequence.

Effects on RNA Interference in Gene Expression (RNAi) in Cultured Mammalian Cells of Mismatches and the Introduction of Chemical Modifications at the 3′-Ends of siRNAs

The highly specific posttranscriptional silencing of gene expression induced by double-stranded RNA (dsRNA) is known as RNA interference (RNAi) and has been demonstrated in plants, nematodes, Drosophila, and protozoa, as well as in mammalian cells. The suppression of expression of specific genes by chemically synthesized 21-nucleotide (21-nt) RNA duplexes has been achieved in various lines of mammalian cells, and this technique might prove to be a valuable tool in efforts to analyze biologic functions of genes in mammalian cells. In order to investigate the utility of potential modifications that can be introduced into small interfering RNAs (siRNAs) and also to study their functional anatomy, we synthesized different types of siRNA targeted to mRNA of Jun dimerization protein 2 (JDP2). Our detailed analysis demonstrated that siRNAs with only one mismatch, relative to the target, on the antisense strand had reduced RNAi effect, whereas the corresponding mutation on the sense strand did not interfere with the RNAi. Moreover, one 2-hydroxyethylphosphate (hp) substitution at the 3-end of the antisense strand but not of the sense strand also prevented RNAi, whereas a related modification at the 3-end of either strand, using 2-O,4-C-ethylene thymidine (eT), which is a component of ethylene-bridge nucleic acids (ENA), completely abolished RNAi. These results support the hypothesis that the two strands have different functions in RNAi in cultured mammalian cells and indicate that their chemical modification of siRNAs at the 3-end of the sense strand exclusively is possible, without loss of RNAi activity, depending on the type of modification. Because modification at the 3-end of the antisense strand by hp or eT abolished the RNAi effect, it appears possible that the 3-end is recognized by the RNA-induced silencing complex (RISC).

2′-O,4′-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug

To develop antisense oligonucleotides, novel nucleosides, 2-O,4-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2-O,4-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2-O,4-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.

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.

Triplex-forming enhancement with high sequence selectivity by single 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA) modification

Triplex-forming ability of the oligonucleotides containing one 2′-O,4′-C-methyleneribonucleic acid (2′,4′-BNA) unit was investigated by measurement of the melting temperature (Tm), and the 2′,4′-BNA modification promoted the marked triplex stabilization in a highly sequence-selective manner.

Characterization of plasmid DNA binding and uptake by peritoneal macrophages from class A scavenger receptor knockout mice.

AbstractPurpose. Plasmid DNA (pDNA) has become an important class of macromolecular agent suitable for non-viral gene therapy as well as DNA vaccination. Our recent study has suggested that pDNA is taken up by mouse peritoneal macrophages via a specific mechanism mediated by a receptor similar to the scavenger receptor (SR). This study was designed to further characterize the pDN A uptake by macrophages in order to elucidate the mechanism.nMethods. The binding and uptake of pDNA labeled with 32P or a fluorescent marker were studied in vitro using cultured Chinese hamster ovary (CHO) cells expressing the class A scavenger receptor (SRA) and peritoneal macrophages from SRA-knockout mice.nResults. pDNA binding and uptake by CHO(SRA) cells were minimal and almost identical to that by wild-type CHO cells. Macrophages from the knockout mice showed pronounced pDNA binding and uptake as did the control macrophages. In both types of macrophage, pDNA binding was significantly inhibited by cold pDNA, polyinosinic acid and dextran sulfate but not by polycytidylic acid or Ac-LDL. These results provide direct evidence that SRA is not responsible for the significant binding and subsequent uptake of pDNA by mouse peritoneal macrophages. Further binding experiments revealed that, in addition to polyinosinic acid and dextran sulfate, heparin was a potent inhibitor among a variety of polyanionic compounds such as poly-nucleotides, anionic polysaccharides and modified proteins including Ox-LDL.nConclusions. The present study suggest that pDNA binding and uptake by mouse peritoneal macrophages are mediated by a specific mechanism to some defined polyanions not by scavenger receptors. The finding would be an important basis for further studies to elucidate the mechanism(s) of pDNA uptake by macrophages.

A 2′,4′‐Bridged Nucleic Acid Containing 2‐Pyridone as a Nucleobase: Efficient Recognition of a C⋅G Interruption by Triplex Formation with a Pyrimidine Motif

Significantly enhanced binding affinity to C⋅G base pairs without loss of sequence selectivity is achieved by using a nucleotide containing a 2-pyridone and a 2-O,4-C-methylene-bridged nucleic acid analogue (PB , see picture). The degree of stabilization of the triplex formed enables C⋅G interruptions in a homopurine⋅homopyrimidine double-stranded DNA to be detected.

Synthesis and conformation of 3′,4′-BNA monomers, 3′-O,4′-C-methyleneribonucleosides

Abstract In order to develop novel 2′,5′-linked oligonucleotide analogues aimed for antivirus reagents and antisense/antigene oligonucleotides, novel nucleoside analogues, 3′-O,4′-C-methyleneribonucleosides (3′,4′-BNA monomers) were synthesized via two synthetic routes. The first route starting from uridine utilized a regioselective ring-closure reaction of the 4′-C-(p-toluenesulfonyl)oxymethyluridine derivative. The second route involved a coupling reaction of 1,2,3-tri-O-acetyl-4-C-(p-toluenesulfonyl)oxymethylribofuranose derivative with nucleobases followed by oxetan-ring formation to afford the 3′,4′-BNA monomers bearing all four nucleobases. By means of 1H NMR, X-ray crystallography and computational analysis, the sugar puckering of the 3′,4′-BNA monomers was found to be restricted in S-conformation (C1′-exo–C2′-endo puckering mode).

Triplex formation by an oligonucleotide containing conformationally locked C-nucleoside, 5-(2-O,4-C-methylene-β-d-ribofuranosyl)oxazole

The triplex-forming ability of oligonucleotide analogues containing conformationally locked C-nucleosides, 5-(2-O,4-C-methylene-β-d-ribofuranosyl)oxazole or its 2-phenyl congener, towards a purine sequence of duplex DNA with a single C·G base pair interruption is studied.