Laser-mediated, site-specific inactivation of RNA transcripts

The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and in vitro characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction.

Magnesium-dependent folding of self-splicing RNA: Exploring the link between cooperativity, thermodynamics, and kinetics

Folding of the Tetrahymena self-splicing RNA into its active conformation involves a set of discrete intermediate states. The Mg2+-dependent equilibrium transition from the intermediates to the native structure is more cooperative than the formation of the intermediates from the unfolded states. We show that the degree of cooperativity is linked to the free energy of each transition and that the rate of the slow transition from the intermediates to the native state decreases exponentially with increasing Mg2+ concentration. Monovalent salts, which stabilize the folded RNA nonspecifically, induce states that fold in less than 30 s after Mg2+ is added to the RNA. A simple model is proposed that predicts the folding kinetics from the Mg2+-dependent change in the relative stabilities of the intermediate and native states.

Inhibition of RNase P RNA cleavage by aminoglycosides

A number of aminoglycosides have been reported to interact and interfere with the function of various RNA molecules. Among these are 16S rRNA, the group I intron, and the hammerhead ribozymes. In this report we show that cleavage by RNase P RNA in the absence as well as in the presence of the RNase P protein is inhibited by several aminoglycosides. Among the ones we tested, neomycin B was found to be the strongest inhibitor with a Ki value in the micromolar range (35 μM). Studies of lead(II)-induced cleavage of RNase P RNA suggested that binding of neomycin B interfered with the binding of divalent metal ions to the RNA. Taken together, our findings suggest that aminoglycosides compete with Mg2+ ions for functionally important divalent metal ion binding sites. Thus, RNase P, which is an essential enzyme, is indeed a potential drug target that can be used to develop new drugs by using various aminoglycosides as lead compounds.