Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology.

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.

Synthesis, incorporation efficiency, and stability of disulfide bridged functional groups at RNA 5'-ends.

Modified guanosine monophosphates have been employed to introduce various functional groups onto RNA 5'-ends. Applications of modified RNA 5'-ends include the generation of functionalized RNA libraries for in vitro selection of catalytic RNAs, the attachment of photoaffinity-tags for mapping RNA-protein interactions or active sites in catalytic RNAs, or the nonradioactive labeling of RNA molecules with fluorescent groups. While in these and in similar applications a stable linkage is desired, in selection experiments for generating novel catalytic RNAs it is often advantageous that a functional group is introduced reversibly. Here we give a quantitative comparison of the different strategies that can be applied to reversibly attach functional groups via disulfide bonds to RNA 5'-ends. We report the preparation of functional groups with disulfide linkages, their incorporation efficiency into an RNA library, and their stability under various conditions.

Two regions of simian virus 40 T antigen determine cooperativity of double-hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding.

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in which this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA structure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (class 4) has remarkably similar properties, although these proteins are phosphorylated on threonine 124, as we show here. By comparing the DNA binding properties of the T124A and class 4 mutant proteins with those of the wild type, we demonstrate that mutant double hexamers bind to viral origin DNA with reduced cooperativity. We report that T124A T-antigen subunits impair the ability of double hexamers containing the wild-type protein to unwind viral origin DNA, suggesting that interactions between hexamers are also required for unwinding. Moreover, the T124A and class 4 mutant T antigens display dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding. We speculate that one surface of the DNA binding domain in each subunit of one hexamer may form a docking site that can interact with each subunit in the other hexamer, either directly with the N-terminal phosphorylated region or with another region that is regulated by phosphorylation.

Oligonucleotide libraries--variatio delectat.

In vitro selection of combinatorial nucleic acid libraries leads to specific target-binding molecules--RNA, single stranded DNA, modified RNA or modified DNA, commonly designated as aptamers--and to novel catalytic nucleic acids. The current state of aptamer and ribozyme technology is such that it establishes itself as a means of obtaining useful tools for molecular biology, diagnostics, molecular medicine and bio-organic chemistry.

A novel ribozyme with ester transferase activity.

BACKGROUND: The 'RNA world' hypothesis proposes that the early history of life on earth consisted of a period in which chemical transformations were catalyzed exclusively by ribozymes. Ribozymes that act as acyl transferases, or catalyze the formation of amide or peptide bonds seem particularly attractive candidates to link the RNA world to the modern protein-nucleic acid world. The central role played by aminoacylated RNAs in today's processes of translating RNA into protein suggests that aminoacyl transfer reactions catalyzed by RNA might have facilitated the development and optimization of the translation apparatus during early evolution. RESULTS: We describe the isolation and characterization of a novel ribozyme that catalyzes the transfer of an amino-acid ester from an aminoacyl donor substrate onto the ribozyme itself. The site of aminoacylation was determined to be at an internal 2'-OH group of a cytosine residue. The aminoacylation depends on the presence of Mg2+ and can be competitively inhibited by the AMP moiety of the aminoacyl donor substrate, suggesting that there is a specific binding pocket for this substrate. The originally selected ribozyme was engineered to act as intermolecular catalyst that transfers the amino acid onto an external 28-residue oligonucleotide. The aminoacylated oligonucleotide was further used to quantify the reverse reaction catalyzed by the ribozyme. CONCLUSIONS: The ribozyme we have isolated is an example of a catalytic RNA with ester transferase activity which uses a substrate that is not templated by Watson-Crick-pairing hydrogen bonds. The reaction catalyzed by the ribozyme expands the scope of RNA catalysis to include acyl transferase activity from an RNA 3' end to an internal 2' position and the reverse. Ribozymes with such activity have been postulated to be evolutionary precursors of ribosomal RNA.