Q-314. Studying the Association of Malachite Green and Anti-Malachite Green Aptamers

X. Zhang1, A. Potty1, G. Jackson2, V. Stepanov1, Y. Liu1, U. Strych1, G. Fox1, R. Willson1;
1Univ. of Houston, Houston, TX, 2BioTex Inc., Houston, TX.

Malachite green (MG) is used primarily as an antiseptic for the prophylactic treatment of fungal and bacterial infections of fish in aquaculture. Increasing evidence suggests that MG is mutagenic and carcinogenic, and it has thus drawn significant attention as a potential food contaminant. The development of ligands and highly sensitive molecular sensors for MG is thus desirable. In previous work, insertion into Vibrio proteolyticus 5S rRNA was shown to stabilize 13- to 50-nucleotide “guest” RNA sequences expressed in E. coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing MG into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely-related control molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and raises the possibility of direct selection for improved aptamers by their ability to protect E. coli from MG toxicity. In order to test the effect of MG on E. coli cells, we prepared cultures with various concentrations of MG and scored for growth, and determined an MIC of 10 µM for MG in E. coli. Preliminary experiments conducted to test the effect of expressing a plasmid-borne, inducible 5S rRNA-MG-aptamer construct in E. coli growing in medium with MG at concentration above its MIC were promising.