RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides

Abstract

Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio.

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…As an alternative to the eIF4A based BiFC system, a small viral peptide based BiFC system, was developed. Yiu et al fused HTLV-Rex peptide and lN peptide to two halves of EGFP and successfully imaged target RNA sequences with HTLV-Rex aptamer and lN aptamer in bacteria cells 46 .…”

Aptamers in analytics

“…203 Other RNA elements that have been employed for monitoring gene expression in real time include the 25 nt RNA hairpin loop that binds the dimeric PP7 bacteriophage coat protein, 204 the λ phage transcriptional anti-termination signal, 15nt box B RNA, which forms a stem-loop that binds a 22 aa arginine-rich fragment of the λN protein, 205, 206 a 21 nt RNA element that binds a fragment of the splicing protein U1Ap, 207 a 29 nt hairpin E. coli transcriptional anti-terminator that binds the BglG protein, 208, 209 and the viral HTLV-Rex, HIV-Rev RRE, and HIV TAT-TAR protein binding elements. 210, 211 Many copies of the RNA binding elements are encoded in RNAs that are expressed on activation of a chosen promoter in a cell that also constitutively expresses the protein target of the RNA element fused with a fluorescent protein, which is usually GFP. Promoter activation results in RNA transcription and the RNA elements bind the target-GFP fusion proteins and concentrate them over the RNA.…”

“…210-215 Although BiFC systems have lower backgrounds and therefore increased signal/noise, cell lines must be first created that express the fluorescent proteins. This requirement and the additional energy expended by the cells to synthesize the fluorescent fusion proteins and the requirement for oxygen for these proteins to mature also limits their application to certain cell types and cell culture environments.…”

Aptamer-based molecular imaging

“…To achieve good signal/noise ratio, the optimal expression level of the target RNA and the ligand-fused fluorescent protein needs to be empirically determined. To decrease background, a nuclear localization signal is usually attached to the fluorescent (Lim and Peabody, 1994), λN22 aptamer (Austin et al, 2002), HTLV-1 Rex aptamer (Yiu et al, 2011), U1A aptamer (Zeng and Hall, 1997) and eIF4A aptamer (Oguro et al, 2003).…”