All RNAs were stored at в€’80вЊ in the RNA storage buffer (10 mM MOPS (pH 6.0), 1 mM EDTA). Radiolabeled HC preRNA was also prepared by in vitro transcription in the presence of 32P-О±-UTP, as previously described ( 15). Large-scale transcription of H.c.LSU group II intron precursor RNA (HC preRNA) was carried out as previously described ( 14) using the pLTS101 plasmid linearized with BamHI restriction enzyme. MATERIALS AND METHODS In vitro transcription Implementation of this assay for monitoring splicing and inhibition of the H.c.LSU group II intron provides a major step toward the creation of improved assays for small molecule targeting of self-splicing and pre-mRNA introns. Here we demonstrate that molecular beacons can provide a reliable readout of RNA splicing kinetics and that the resulting methods are sufficiently generalizable and sensitive for application in high-throughput assays for identification of small molecule splicing inhibitors. To our knowledge, however, it has not yet been demonstrated that RNA splicing can be monitored or interrogated using a molecular beacon approach. The fluorophore in the molecular beacon stem-loop is shown in red and the quencher is in black. The part of the molecular beacon complementary to the target sequence is shown in grey. The target sequence is shown as a grey rectangle. Schematic of molecular beacon hybridization to a target sequence. At least one high-throughput screening assay based on molecular beacons has been developed to identify modulators of RNA targets, such as miRNAs ( 13). Molecular beacons have become a powerful chemical biology tool, and they are commonly used to study RNA cellular localization and intronic structure ( 11, 12).

Upon binding a complementary target sequence, however, the beacon will open and hybridize to the target, thereby increasing the distance between the fluorophore and the quencher and allowing fluorescence to serve as a signal for target identification in solution (Figure 2). To achieve the most sensitive detection of nucleic acid sequences, molecular beacons are often designed as stem-loop structures in which the fluorophore and quencher dyes are covalently attached at the termini of an oligonucleotide, in forced proximity with each other.

Molecular beacons are fluorescent DNA-based probes that can detect specific sequences by complementary hybridization ( 10). capsulatum group II intron from the mitochondrial ribosomal large subunit RNA (H.c.LSU) exhibits a relatively slower splicing rate constant relative to other group II introns, rendering it a useful self-splicing model for the development of a small molecule screening assay. capsulatum is a dimorphic fungus responsible for histoplasmosis, which is the most prevalent dimorphic fungal infection in the United States and a significantly underdiagnosed disease globally ( 9). One such intron, found in the ribosomal RNA (rRNA) from the large subunit (LSU) of the mitochondrial ribosome of Histoplasma capsulatumВ ( Supplementary Figure S1), was recently identified by a bioinformatics approach and shown to exhibit self-splicing activity in the presence of catalytic Mg 2+ in vitro ( 8).

While small molecule targeting of RNA is a relatively new field, promising intron drug targets are being continually identified in human pathogens. Thin black line depicts the secondary structure schematic of the intron. Black and grey rectangles represent 5вЂІ- and 3вЂІ-exons, respectively. Schematic of the group II intron self-splicing reaction proceeding via the branching pathway. The results presented herein offer support for a molecular beacon approach to identifying small molecule inhibitors of intron splicing. We demonstrate that the fluorescent assay using molecular beacons can be successfully applied to kinetic characterization of the splicing reaction and determination of inhibition constants for small molecules. We find that a molecular beacon with the loop length of 18 nucleotides selectively recognizes ligated exons formed during self-splicing and exhibits high fluorescent signal upon binding of its target. In this case, we applied it to the autocatalyzed reaction of the H.c.LSU group II intron found in the mitochondria of the pathogenic dimorphic fungus Histoplasma capsulatum. Here, we present the development of a molecular beacon assay for monitoring the accumulation of spliced exons during RNA splicing reactions. Small molecule targeting of self-splicing RNAs like group I and II introns has been limited in part by the lack of a universal high-throughput screening platform for studies of splicing inhibition and kinetics.

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