Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of the cleavage reaction and did not affect the amount of Mg2+ bound to the hammerhead RNA. The kinetics and CD spectra for chemically synthesized oligoribonucleotides with a Sp or Rp phosphorothioate diester bond at the cleavage site indicated that 1 mol of Mg2+ binds to the pro-R oxygen of phosphate. The binding constant for Mg2+ was about 10(4) M-1, which represents outer-sphere complexation. The hammerhead ribozyme catalyzes the cleavage reaction via an in-line pathway. This mechanism has been proved for RNA cleavage by RNase A by using a modified oligonucleotide that has an Sp phosphorothionate bond at the cleavage site. From these results, we present the reaction pathway and a model for Mg2+ binding to the hammerhead ribozyme.

No abstract

No abstract

No abstract

Nucleic acidsNucleic acids U 0700 Synthesis and Properties of 2'-O,4'-C-Ethylene-Bridged Nucleic Acids (ENA) asEffective Antisense Oligonucleotides. -(MORITA, K.; TAKAGI, M.; HASEGAWA, C.; KANEKO, M.; TSUTSUMI, S.; SONE, J.; ISHIKAWA, T.; IMANISHI, T.; KOIZUMI*, M.; Bioorg. Med. Chem. 11 (2003) 10, 2211-2226; Explor. Chem. Res. Lab.,

The fidelity of protein biosynthesis rests largely on the correct aminoacylation of transfer RNAs by their cognate aminoacyl‐tRNA synthetases. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl‐tRNA synthetase (GlnRS) provides an excellent system for studying the basis of this highly specific recognition process. Correct aminoacylation depends on the set of nucleotides (identity elements) in tRNA(Gln) responsible for correct interaction with GlnRS. Specific contacts between tRNA(Gln) and GlnRS include the 2‐amino group of guanosines. Therefore, we made a set of tRNA(Gln) variants in which specific guanosines were replaced by inosine using recombinant RNA technology. This resulted in a set of tRNAs that varied by single deletions of the amino group from guanine residues, thus allowing us to test the functional importance of these contacts. In addition, a number of mutants were made by transcription of mutated tRNA genes with base changes at position 10, 16 or 25. In vitro aminoacylation of these mutants showed decreases in the specificity constant (kcat/KM) of up to 300‐fold, with kcat being the parameter most affected. These experiments reveal G10 as a new element of glutamine identity. In addition, the interaction of G2, G3 and G10 with GlnRS via the 2‐amino group is significant for tRNA discrimination. Based on these results, and on earlier data, we propose a complete set of bases as identity elements for tRNA(Gln).

No abstract

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).