Hiroshi Takaku

Solid phase synthesis of oligoribonucleotides by the phosphoramidite approach using 2′-O-1-(2-chloroethoxy)ethyl protection

Abstract The new type protecting group, 1-(2-chloroethoxy)ethyl (Cee) group has been employed for the protection of the 2′-OH groups of ribonucleoside residues in the synthesis of oligoribonucleotides by the phosphoramidite approach en a solid support, using the acid-labile 5′-O-dimethaxytrityl (DMTr) group. This group is completely stable under the acidic conditions required to remove the 5t-terminal protecting groups in oligonucleotide synthesis on a solid support, and yet is easily removable under mild condition of acidic hydrolysis (pH 2.0) for the final unblocking step. The Cee-protected ribonucleoside 3′-phosphoramidite units were evaluated in the synthesis of a series of oligoribonucleotides consisting of the homopolymers of cytidine, the box 9R and 9R sequences of Tetrahymena rRNA, and a leader sequence of phage Qβ-A protein mRNA. A full data for the deprotection and purification of synthetic oligoribonucleotides are also described.

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron.

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3 terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a Bulge-and-Twist (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.