About three-fourths of our genome is transcribed into RNA, and these RNAs are involved in essentially every cellular process. Like us, RNA cannot do its job if it is not in the right place at the right time, so the transport of RNA to its proper place is critical for cellular function. Since the 1990s, scientists have figured out how to add fluorescent tags to RNA so that we can watch the RNA move around in the cell in real time under a fluorescence microscope which makes investigating complex research questions easier to pursue. Riboglow, a collaboration between the Palmer and Batey labs in the Department of Biochemistry at CU Boulder, is a new take on these RNA-imaging tools based on naturally occurring RNA sequences, called riboswitches, that fold robustly in the cellular environment and bind their ligands tightly and specifically. Like many other RNA-imaging systems, Riboglow is a two-part system made up of an RNA component and a fluorescent component. The sequence of the RNA component, the aptamer, can be fused to the 3' end of the gene of interest, similar to the addition of a FLAG tag or GFP to a protein of interest. When transcribed, the final RNA includes the RNA of interest and the aptamer. The fluorescent component, or probe, contains the ligand for the riboswitch sequence that the aptamer is based on, a linker, and a fluorophore. Then, this aptamer can tightly and specifically bind the fluorescent probe, completing the fluorescent tag on the RNA of interest.

 

Riboswitch schematicCurrent questions include:

  • Can we engineer orthogonal Riboglow systems for two-color imaging?
  • Can we evolve riboswitch-based RNAs to bind commercially-available fluorescent probes or dyes?
  • Can we alter the fluorescent probe to make it brighter, more photostable, or more membrane permeable?
  • Can we design aptamer and probe pairs to enhance fluorescence upon binding?
  • Can Riboglow be used to see single molecules of RNA inside living cells?
  • Does Riboglow affect the basic biology (transcription, processing, export, trafficking, translation, decay) of the RNA of interest?
  • Can we optimize the aptamer sequence to make it more amenable to cells or to make it easier for us to use Riboglow?