Nucleotides -1 to -4 of Hepatitis Delta Ribozyme Substrate Increase the Specificity of Ribozyme Cleavage

Abstract

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.

Examination of the folding pathway of the antigenomic hepatitis delta virus ribozyme reveals key interactions of the L3 loop

“…a 4-thiouridine residue was introduced in position -2. Since a substrate that includes two consecutive pyrimidines in positions -1 and -2 is not efficiently cleaved by HDV ribozyme (Deschênes et al 2000), the cytosine in -1 was replaced by an adenosine. The integrity of the resulting substrate (i.e., Ss 4 U-2) was assessed for cleavage activity in comparison to that of the wild-type substrate (i.e., Swt) under single turnover conditions (Fig.…”

Functional characterization of the SOFAdeltaribozyme

“…The binding domain of dRz (the P1 stem) is composed of one G-U wobble base pair followed by six Watson-Crick base pairs (Perrotta and Been 1991;Nishikawa et al 1999). In addition, the nucleotides (nt) from position À1 to À4 of the substrate-that is, those adjacent to the scissile phosphate, were shown to contribute to the ability of a substrate to be cleaved efficiently (Deschênes et al 2000). Thus, the substrate specificity of dRz cleavage is based on a total of 11 nt, which might be a limiting factor when trying to specifically target an RNA species in a cell (Nielsen 2000).…”

Silencing of SPC2 Expression Using an Engineered δ Ribozyme in the Mouse βTC-3 Endocrine Cell Line

“…The sequence upstream of the cleavage site (i.e. positions Ϫ4 to Ϫ1) was also shown to contribute significantly to the cleavage ability of a substrate (25). This increases the substrate specificity to 11 consecutive nucleotides.…”

“…1B). These criteria were based on the fact that the first pairing of the P1 stem must be a GU wobble bp and that the presence of a guanosine at the cleavage site (position Ϫ1) results in an uncleavable substrate (25). Finally, analysis of the sequences indicated that no other cleavages could occur within the SPC gene family.…”