Associate
Shuichi Hoshika

Education

BS in Pharmaceutical Sciences. Hokkaido University, Japan (2001)
MS in Pharmaceutical Sciences. Hokkaido University, Japan (2003)
PhD in Pharmaceutical Sciences. Hokkaido University, Japan (2006)

Research summary

My research focuses on the development of rapid and cost-effective sequencing methods based on synthesis of nucleoside derivatives. This would be achieved by multiplex PCR using primers consisting of nucleoside derivatives which form base pairs with natural nucleobases and/or synthetic nucleobases in different hydrogen bonding patterns.

Publications

Incorporation of Multiple Sequential Pseudothymidines by DNA Polymerases and Their Impact on DNA Duplex Structure
Havemann, SA Hoshika, S Hutter, D Benner, SA
Nuc. Nuc. Nuc. acids 27 (3) 261-278 (2008)
<Abstract>

In this article, we focus on the synthesis of aryl C-glycosides via Heck coupling. It is organized based on the type of structures used in the assembly of the C-glycosides (also called C-nucleosides) with the following subsections: pyrimidine C-nucleosides, purine C-nucleosides, and monocyclic, bicyclic, and tetracyclic C-nucleosides. The reagents and conditions used for conducting the Heck coupling reactions are discussed. The subsequent conversion of the Heck products to the corresponding target molecules and the application of the target molecules are also described.

Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4'-thioribonucleosides
Hoshika, S Minakawa, N Shionoya, A Imada, K Ogawa, N Matsuda, A
CHEMBIOCHEM 8 (17) 2133-2138 (2007)
<Abstract>

A detailed study of the modification pattern-RNAi activity relationships by using siRNAs that are modified with 4'-thioribonucleosides has been carried out against photinus luciferase and renilla luciferase genes in cultured mammalian NIH/3T3, HeLa, and MIA PaCa-2 cell lines. When the photinus luciferase gene was targeted, all of the modified siRNAs showed activity equal to, or less than the unmodifed siRNA. In contrast, all modified siRNAs that have a similar modification pattern showed activity equal to or much higher than the unmodified siRNA when tested with the renilla luciferase gene. These results indicated that siRNAs such as RNA33 and RNA53, which each have four residues of the 4'-thioribonucleoside unit on both ends of the sense strand and four residues on the 3'-end of the antisense strand, were the most effective. Accordingly, we succeeded in developing modified siRNAs that have the greatest number of 4'-thioribonucleosides without loss of RNAi activity, and that exhibit potent RNAi activity against two target genes in three different cell lines. Our findings also indicate the significance of target sequences and cell lines when RNAi activity is compared with that of the unmodified siRNA.

RNA interference induced by siRNAs modified with 4 '-thioribonucleosides in cultured mammalian cells
Hoshika, S Minakawa, N Kamiya, H Harashima, H Matsuda, A
FEBS Lett. 579 (14) 3115-3118 (2005)
<Abstract>

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Oligodeoxynucleotides having a loop consisting of 3 '-deoxy-4 '-C-(2-hydroxyethyl)thymidines form stable hairpins
Yamamoto, Y Shuto, S Tamura, Y Kodama, T Hoshika, S Ichikawa, S Ueno, Y Ohtsuka, E Komatsu, Y Matsuda, A
Biochemistry 43 (27) 8690-8699 (2004)
<Abstract>

Components that form stable hairpin loops are highly useful for the development of functional DNA and RNA molecules. We have designed and synthesized a sugar-modified thymidine analogue, 3'-deoxy-4'-C-(2-hydroxyethyl)thymidine (X), as a nucleosidic loop component stabilizing the hairpin structure. The ODNs I-1-4, 5'-d[CGAACG-X-n-CGTTCG]-3' (I-1, n = 1; I-2, n = 2; I-3, n = 3; I-4, n = 4), forming the hairpin loop structures, of which the loop moiety consisted of the analogue X, and also the corresponding unmodified ODNs II-1-4, 5'-d[CGAACG-T-n-CGTTCG]-3' (II-1, n = 1; II-2, n = 2; II-3, n = 3; II-4, n = 4), having a thymidine loop, were synthesized by the phosphoramidite method. The melting temperatures (T m) of the ODNs I-1-4 containing X in the loop moiety at 5 muM were 67.1, 68.1, 73.0, and 69.3 degreesC, respectively, and those of the control natural ODNs II-1-4 were 65.3, 67.0, 69.2, and 68.8 degreesC, respectively. Thus, the ODNs I-1-4 formed a more thermally stable hairpin than the corresponding unmodified ODNs II-1-4 having an equal number of loop residues. The hairpin structures of the modified ODNs I-1-4 and the unmodified ODNs II-1-4 were investigated by CD spectroscopy and molecular mechanics calculations. These results showed that the 4'-branched nucleoside X can stabilize hairpin structures when it is present in the loop moiety, probably due to the flexibility of the one-carbon-elongated 4'-branched structure.

Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3 '-3 '-linked oligodeoxynucleotides composed of asymmetrical sequences
Hoshika, S Ueno, Y Kamiya, H Matsuda, A
Bioorg. Med. Chem. Lett. 14 (12) 3333-3336 (2004)
<Abstract>

In this paper, we describe the synthesis of the 3'-3'-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3'-3'-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine-oligopurine sequences. (C) 2004 Elsevier Ltd. All rights reserved.

Synthesis and physical and physiological properties of 4 '-thioRNA: application to post-modification of RNA aptamer toward NF-kappa B
Hoshika, S Minakawa, N Matsuda, A
Nucl. Acids Res. 32 (13) 3815-3825 (2004)
<Abstract>

We report herein full details of the preparation of 4'-thiouridine, -cytidine, -adenosine and -guanosine phosphoramidites based on our synthetic protocol via the Pummerer reaction. Fully modified 4'-thioRNAs containing four kinds of 4'-thioribonucleoside units were prepared according to the standard RNA synthesis. The T,, values and thermodynamic parameters of a series of duplexes were determined by UV melting and differential scanning calorimetry (DSC) measurements. The resulting overall order of thermal stabilities for the duplexes was 4'-thioRNA:4'-thioRNA >> 4'-thioRNA:RNA > RNA:RNA > RNA:DNA > 4'-thioRNA:DNA. In addition, it was shown that the dominant factor in the stability of the duplexes consisting of 4'-thioRNA was enthalpic in character. The CD spectra of not only 4'-thioRNA:RNA and 4'-thioRNA:4'-thioRNA but also 4'-thioRNA:DNA were all similar to those of duplexes in the A-conformation. The stability of 4'-thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA:RNA nor the 4'-thioRNA: 4'-thioRNA duplexes were digested under the same conditions. The first example of a post-modification of an RNA aptamer by 4'-thioribonucleoside units was demonstrated. Full modification of the aptamer thioRNA3 resulted in complete loss of binding activity. In contrast, modifications at positions other than the binding site were tolerated without loss of binding activity. The post-modified RNA aptamer thioRNA5 was thermally stabilized and resistant toward nuclease digestion. The results presented in this paper will, it is hoped, contribute to the development of 4'-thioRNA as a new generation of artificial RNA.

Nucleosides and nucleotides. 218. Alternate-strand triple-helix formation by the 3 '-3 '-linked oligodeoxynucleotides using a purine motif
Hoshika, S Ueno, Y Matsuda, A
Bioconjugate Chem. 14 (3) 607-613 (2003)
<Abstract>

In this paper, we describe the synthesis of the X-X-linked TFOs that can form the antiparallel triplexes with the duplex DNA target by reverse Hoogsteen hydrogen bonds. Stability of the alternate-strand triplexes between these TFOs and the target DNAs was investigated using the electrophoretic mobility shift assay (EMSA). It was found that the alternate-strand triplexes were significantly stabilized by linking the TFO fragments with the pentaerythritol linker. And, unlike the alternate-strand triplexes composed of the pyrimidine motif, the terminal ammonium ion of the aminobutyl-linker and the intercalator of the TFOs did not contribute to the stability of the alternate-strand triplex comprised of the purine motif. We also tested the ability of the X-X-linked TFOs to inhibit cleavage of the duplex DNA target 17 by the restriction enzyme EcoT14I and found that the 3'-3'-Iinked TFOs 12 and 13 inhibited the cleavage by the enzyme more effectively than the unlinked decamer S. Thus, the TFOs linked with pentaerythritol may be useful as the antigene oligonucleotide to the DNA targets, which have alternating oligopyrimidine-oligopurine sequences.

Nucleosides and nucleotides. 208. alternate-strand triple-helix formation by the 3 '-3 '-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point
Ueno, Y Mikawa, M Hoshika, S Matsuda, A
Bioconjugate Chem. 12 (4) 635-642 (2001)
<Abstract>

The synthesis of 3 ' -3 ' -linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3 ' -3 ' -linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3 ' -3 ' -linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3 ' -3 ' -linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3 ' -3 ' -linked ODN 15 without the intercalator.

Associate
Shuichi Hoshika

Education

Research summary

Publications

Interests

Contact

AssociateShuichi Hoshika

Education

Research summary

Publications

Interests

Contact

Associate
Shuichi Hoshika