Cleavage of an Inaccessible Site by the Maxizyme with Two Independent Binding Arms: An Alternative Approach to the Recruitment of RNA Helicases

Abstract

To overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.

Inhibition of Aminoglycoside 6′- N -Acetyltransferase Type Ib-Mediated Amikacin Resistance by Antisense Oligodeoxynucleotides

“…However, there are bands along the whole region, indicating that ODNs hybridized to nucleotides predicted to be in double-stranded form. It is possible that the predicted structure does not reflect exactly the real structure of the RNA molecule or that the complexity of the spatial structure at this region permits enough breathing at the short double-stranded stems to allow interaction with ODNs and, consequently, cleavage by the RNase H. A case like this has recently been described in which a stem RNA region was digested by a maxizyme due to a binding process that occurred during the breathing of the stem structure (22). The RNase H cleavage products that define region D were identical for all five ODN libraries (Fig.…”

“…1 and 3). An explanation for the detection of fragments cleaved at these predicted doublestranded locations could be that the computer-generated structure is not completely correct or that there is enough breathing at the short double-stranded regions to allow cleavage by RNase H. A stem RNA region has recently been shown to be digested by a single-strand-specific enzyme due to a binding process that occurred during the breathing of the stem structure (22). Furthermore, in all five regions only parts of the loops were identified by RNase H mapping, which may reflect the complexities of the secondary and tertiary structures, which permit only a few nucleotides to be available for interaction with ODNs.…”

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

“…Similar results have been reported by Xu et al ., 1999, in which the expression of human insulin-like growth factor II is not completely inhibited by double hRzs but yields the same efficacy as single hRzs. As an alternative approach, we are currently exploring combining two hRzs as a so-called Maxizyme [ 25 , 29 , 30 , 51 , 52 ] that targets two regions with low tolerance for sequence variation, and may ultimately provide even better suppression of DENV replication in transduced mosquito cells and tissues.…”

External guide sequence technology: a path to development of novel antimicrobial therapeutics

“…B and C). Possible explanations are that either the predicted structure may not reflect the precise structure of the mRNA or the structural complexity of the region permits breathing at the short double‐stranded stems, allowing interaction with the EGS …”