Fluorescence assays for monitoring RNA-ligand interactions and riboswitch-targeted drug discovery screening.

Riboswitches and other noncoding regulatory RNA are intriguing targets for the development of therapeutic agents. A significant challenge in the drug discovery process, however, is the identification of potent compounds that bind the target RNA specifically and disrupt its function. Essential to this process is an effectively designed cascade of screening assays. A screening cascade for identifying compounds that target the T box riboswitch antiterminator element is described. In the primary assays, moderate to higher throughput screening of compound libraries is achieved by combining the sensitivity of fluorescence techniques with functionally relevant assays. Active compounds are then validated and the binding to target RNA further characterized in secondary assays. The cascade of assays monitor ligand-induced changes in the steady-state fluorescence of an attached dye or internally incorporated 2-aminopurine; the fluorescence anisotropy of an RNA complex; and, the thermal denaturation fluorescence profile of a fluorophore-quencher labeled RNA. While the assays described have been developed for T box riboswitch-targeted drug discovery, the fluorescence methods and screening cascade design principles can be applied to drug discovery efforts targeted toward other medicinally relevant noncoding RNA.

Characterization of a 1,4-disubstituted 1,2,3-triazole binding to T box antiterminator RNA.

The T box riboswitch regulates the transcription of many bacterial genes by structurally responding to cognate non-aminoacylated (uncharged) tRNA. The riboswitch contains multiple conserved RNA elements including a key structural element, the antiterminator, which binds the tRNA acceptor end nucleotides. Previous studies identified a lead 1,4-disubstituted 1,2,3-triazole, GHB-7, that disrupted formation of a tRNA-antiterminator RNA model complex. The affinity and molecular interactions of GHB-7 binding to antiterminator model RNA were characterized as part of a comprehensive T box antiterminator RNA-targeted drug discovery project. In-line probing, UV-monitored thermal denaturation and docking studies all consistently indicated that GHB-7 likely binds to the bulge region of the antiterminator, reduces the flexibility of the bulge nucleotides and, overall, stabilizes the RNA secondary structure. These results begin to elucidate possible mechanisms for ligand-induced inhibition of tRNA binding to T box antiterminator RNA and contribute to the knowledge of how small molecules bind relatively simple RNA structural elements such as bulges.

Characterizing riboswitch function: identification of Mg2+ binding site in T box antiterminator RNA.

T box bacterial genes utilize a riboswitch mechanism to regulate gene expression at the transcriptional level. Complementary base pairing of the 5'-untranslated mRNA with uncharged cognate tRNA stabilizes formation of an antiterminator element and permits complete transcription. In the absence of tRNA, a mutually exclusive RNA terminator element forms and results in transcription termination. This regulatory mechanism requires divalent metal ions at the antitermination event. The structural effects of Mg(2+) binding to antiterminator model RNA were investigated to ascertain if this requirement is due to the presence of a specific metal ion binding site in the antiterminator. Spectroscopic analysis identified the presence of a hydrated, diffuse Mg(2+) binding site. The results indicate that the mechanistic requirement for divalent metal ions is not due to Mg(2+)-induced pre-formation of a functional antiterminator receptor; rather, Mg(2+) binds in a helical region of high phylogenetic sequence conservation adjacent to the tRNA binding site.

Reliability of finger stick capillary blood for the lymphocyte micronucleus assay.

Compared with peripheral blood sampling, capillary blood collecting by finger stick is less traumatic and more convenient. To assess the sensitivity and reliability of capillary blood for the lymphocyte micronucleus (MN) assay, this study was performed in three sample groups, i.e. healthy donors (n = 3), cancer patients before treatment (n = 7), and cancer patients who were undergoing fractionated partial-body radiotherapy (n = 9). For each group, we measured three intra-individual variables, i.e. micronucleus (MN) frequency, binucleate (BN) index, and micronucleated BN index of lymphocytes obtained from capillary blood and the corresponding peripheral blood. Our results indicated that in all three sample groups, the differences in these variables between capillary blood and peripheral blood either before or after ex vivo 137Cs irradiation (2 Gy) were insignificant. Since capillary blood is more accessible than peripheral blood, we believe that it is a reliable source for the lymphocyte MN assay especially when venipuncture is not convenient.

Assessment of two protocols for the human lymphocyte cytokinesis-blocked micronucleus assay.

This study indicated that by adding cytochalasin B (6 micrograms/ml) at 24 h, the lymphocyte culture time for micronucleus (MN) assay could be shortened to 64 h. In both unirradiated and ex-vivo irradiated (2 Gy) lymphocytes from three populations, we found that the differences in MN yield obtained by our modified cytokinesis-blocked time frame and that recommended by Fenech and Morley (1985) were insignificant (P = 0.66-0.87). We believe that the shorter assay time may enhance the applicability of MN assay for the rapid assessment of ionizing radiation overexposures.

Preservation of cytoplasm in the human lymphocyte micronucleus assay.

A major limitation in the quantitative accuracy of the human lymphocyte micronucleus (MN) assay is preservation of the cytoplasm during the cell harvesting. In this short communication, an improved method for cytoplasm preservation in a cytokinesis-blocked, whole-blood microculture (0.3 ml) technique is described. We believe that the timing of the hypotonic treatment, speed of centrifugation, handling of the cell suspension and proper Giemsa staining are important variables in the human peripheral lymphocyte MN assay.