Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green

Abstract

In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13-50 nucleotide "guest" RNA sequences for expression in Escherichia 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 a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF "ribosomal RNA aptamer." Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA 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 may open an approach to screening aptamer libraries in vivo.

In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

“…Two additional scaffolds have recently been described: the V. proteolyticus 5S rRNA scaffold (denoted by us as V5) (Zhang, et al, 2009), and the Φ29 RNA three-way junction motif (denoted by us as F29) (Shu, et al, 2014). V5 is similar to the endogenous bacterial 5S RNA except a specific stem loop structure was removed to provide an insertion point for aptamers (Zhang, et al, 2009). F29 contains a three-way junction, which is an RNA motif seen in naturally occurring riboswitches and viral RNAs (Shu, et al, 2014).…”

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

“…The DNA construct is recognized as essentially a normal RNA coding region and the resulting transcript is processed into a single RNA species of defined sequence and length. The strong expression of chimeric 3× pen aRNA here and in nearly twenty other 5S rRNA/insert chimeras examined in previous studies [36,56] demonstrates that the 5S rRNA scaffold is robust and can accommodate many RNA inserts of diverse sizes.…”

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

“…Once expressed in cells, aptamers are susceptible to RNA degradation and often fail to fold (39). Misfolding can result from competing folding pathways, thermal instability, or dependence on high magnesium ion concentrations that are not normally found in cells (49,67). In addition, adjacent sequences in a target RNA into which an aptamer is inserted can interfere with folding (39, 57).…”