Summary of Research Project Results under JSPS FY2001
"Research for the Future Program"

1.Research Institution Tokyo Institute of Technology
2.Research Area Integrated Fields
3.Research Field Production of Novel Useful Substances through Integration of Life Sciences and Chemistry
4.Term of Project FY 1997 〜 FY 2001
5.Project Number 97I00301
6.Title of Project Chemical Approach to Creation of Artificial Nucleic Acids Having New Functions

7.Project Leader
Name Institution,Department Title of Position
Mitsuo, Sekine Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology Professor

8.Core Members

Names Institution,Department Title of Position
You, Kikuchi Toyohashi University of Technology, Faculty of Engineering Professor
Makoto, Komiyama The University of Tokyo, Research Center for Advanced Science and Technology Professor
Akira, Matsuda Hokkaido University, Graduate School of Pharmaceutical Sciences Professor

9.Summary of Research Results

 The aim of this project is to create useful artificial nucleic acid derivatives having a new function which is designed by chemical approaches. During the past five years, the following representative results from this project were obtained. To realize our purpose, we developed new efficient methods for the synthesis of DNA without protection of base residues. As the result, we created a new innovative method called "proton-block method", where the base parts are protected by a simple proton and nearly 100% selective internucleotidic bond formation was achieved at the dimer level. We also found the hitherto most selective method for the internucleotidic bond formation by use of a new activator, i.e., N-hydroxybenzotriazole (HOBT). This reagent enables us to synthesize oligonucleotides without N-phosphorylation. The synthesis of the 5'-terminal 4mer of U1 snRNA having a unique trimethyl-G cap structure was successfully done by use of the solid phase synthesis and used for isolation of 2,2,7-trimethylguanosine-cap binding proteins. Consequently, snurportin 1 was discovered as a TMG-cap carrier to nucleus from cytoplasm. The mechanism of nuclear transport of U1snRNA was now elucidated by this study. We succeeded in synthesize a sterically locked "bent structural motif" of UpU derivative, which was successfully incorporated into RNAs. On the other hand, it was found that oligonucleotides having a sterically fixed linear UpU derivative have considerably higher Tm values than unmodified oligonucleotides. This results suggest that this type of modification could provide a very useful antisense DNA molecules. Incorporation of 2-thiouridine derivatives into RNAs resulted in precise recognition of the complementary base of A without formation of mismatched base pair which has been a serious problem in gene diagnosis. Other innovative research results, the following new nucleic acids were synthesized: N-aminoacyl phosphoramidate derivatives as the most ideal analogs of aminoacyl adenylate, DNA oligomers having 3'-3' terminal linkage capable of bind to DNA duplexes, artificial DNA molecules capable of selective cleavage of target RNAs in specific positions.

10.Key Words

(1)chemical synthesis of DNA、(2)N-acyl DNA、(3)nuclear Transport of U1snRNA
(4)hypermethylated cap structure、(5)bent DNA structure、(6)linear DNA structure
(7)2-thiouridine、(8)gene diagnosis、(9)antisense method