Comparative Analysis of Antisense RNA, Double-Stranded RNA, and Delta Ribozyme-Mediated Gene Regulation in Toxoplasma gondii

Abstract

RNA tools, namely, antisense RNA, double-stranded RNA (dsRNA), and delta ribozyme, were comparatively analyzed for the development of effective RNA-based gene modulators. The gene encoding uracil phosphoribosyltransferase (UPRT) of Toxoplasma gondii was used as a target and a negative selectable marker. Using plasmid transformation and drug selection assays, we obtained T. gondii transformants resistant to 5-fluoro-2'-deoxyuridine (FDUR), the cytotoxic prodrug and substrate of UPRT, when the plasmids expressing dsRNA and active delta ribozyme were used. No resistant transformants were detected when the plasmids carrying the antisense RNA, the inactive delta ribozyme, or the chloramphenicol acetyltransferase (CAT) genes were used. Parasites generated using the plasmids expressing dsRNA and the delta ribozyme become resistant to FDUR with an LD50 of 50 +/- 5 microM and 25 +/- 8 microM, respectively. These values are approximately 25-fold and 12-fold higher than that of the RH parental parasite strain, indicating that UPRT activity of the transformed parasites was drastically inhibited. Using Northern and Southern blot analysis, we demonstrated that dsRNA and the delta ribozyme interrupt the expression of UPRT. These two RNA tools should, thus, be very useful for the study of gene expression.

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

“…Because the sensitivity of this assay is very high (femtomoles), these results confirm the absence of any active SPC2 in cell line 154. This is in agreement with SPC2-null mice study (29), confirming that the presence of Dyn A-(1-8) is specifically due to SPC2 cleavage activity on its precursor Dyn A- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

“…Thus, the ␦Rz-154 and -394 were placed under the control of the tRNA Val promoter in the expression vectors ptRNA Val -␦Rz-SPC2-154 and -394 and in their catalytically inactive equivalents ptRNA Val -␦RzC47A-SPC2-154 and -394. The ␦RzC47A mutants are inactive versions in which the cytosine in position 47 was mutated for an adenosine (16). These ␦Rz exhibited the same binding but are completely deprived of cleavage activity; therefore, they are ideal controls for antisense effect.…”

Functional characterization of the SOFAdeltaribozyme

“…This hypothesis is well illustrated by the demonstration that dRzs benefit from an outstanding molecular stability in transfected cells (Lévesque et al 2002). Several studies have already successfully used dRz to inhibit gene expression in vivo, confirming its great potential for use as a molecular tool (see Kato et al 2001;Al-Anouti and Ananvoranich 2002;D'Anjou et al 2004;Sheng et al 2004).…”

In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication

“…17,18 The potential of HDV Rzs to cleave in trans was demonstrated in vitro and in vivo targeting various natural RNA species. [19][20][21][22][23] The target recognition mechanism of the HDV Rz is dependent on the formation of the P1 stem that is composed of only 7 base pairs (bp) between the Rz and the substrate (Fig. 1B).…”