1998 Fiscal Year Final Research Report Summary
Synthesis of Puromycin Tethered RNA and It's Application for Molecular Evolution
| Project/Area Number |
09480139
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
SUGIYAMA Hiroshi Tokyo Medical and Dental University, Institute for Medical and Dental Engineering, Professor, 医用器材研究所, 教授 (50183843)
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| Project Period (FY) |
1997 – 1998
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| Keywords | Puromycin / Protein biosynthesis / mRNA / Molecular evolution |
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| Research Abstract |
A facile method for the synthesis of puromycin-tethered oligonucleotides at the 3'-end is described. The method utilizes a novel CPG support derived from commercially available puromycin. Puromycin-tethered DNA and RNA oligomers were synthesized using a puromycin-tethered CPG support by the usual protocol for automated DNA and RNA synthesis.
Puromycin (Pu) is a member of the 3'-amino-3'-deoxyadenosine family of antibiotics and is known as an extremely powerful inhibitor for protein biosynthesis. Inhibition of protein biosynthesis by Pu is shown to result in the production of a covalent adduct between Pu and the interrupted premature peptide. The cross-linking of an elongated peptide and the 3'-end of mRNA was attempted using Pu-tethered mRNA at the 3'-end in order to accomplish a molecular evolution system of proteins. Thus, we developed a facile synthetic method of Pu-tethered RNA or DNA by preparing a novel Pu-attached CPG support. Using this support, various lengths of Pu-tethered oligonucleotides at the 3'-end up to 40 mer were synthesized. The Pu-attached CPG support was synthesized from commercially available Pu dihydrochloride in four steps. The amino group and 5'-hydroxyl group of Pu were protected by fluorenylmethoxycarbonyl and dimethoxytrityl groups, respectively. A 2'-Hydroxyl group was esterified with succinic anhydride. The resulting product was attached to an LCAA-CPG support under neutral conditions by the recent Fraser method in order to prevent unwanted release of the Fmoc group. The nucleoside attachment for CPG support was found to be 25 j.umole/g by monitoring the released DMTr cation under acidic conditions.
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