In the 2010s, the Human Genome project revealed that up to 80% of the DNA genome istranscribed into RNA while only 2% is eventually translated into proteins. This discovery painted RNA as a central molecule with crucial functions in the cell. To perform these roles, RNA molecules must be in the proper location at the proper time. Further, the time-dependent localization of RNA molecules is essential to the RNA’s downstream protein function, cellular function, organism development, and organism health. In response, many tools have been created to visualize RNA molecules in live cells over time. One tool, Riboglow — developed as a collaboration between the Palmer and Batey Labs at CU Boulder — is a two-part tool comprised of a cobalamin riboswitch RNA aptamer and a small cobalamin-containing fluorescent probe. The Riboglow aptamer binds the Riboglow probe tightly and specifically, and the first generation of this tool enables the tracking of fluorescent RNA molecules in live cells. Now, colleagues and I seek to improve Riboglow.

In this thesis, I demonstrate the improvement of Riboglow along two axes. One axis improved the interaction between the aptamer and the probe, by installing a functional peptide nucleic acid (PNA) linker designed to hybridize to a single-stranded region of the Riboglow aptamer. The addition of this secondary binding motif increased the brightness of the Riboglow probe, increased its interaction with the Riboglow aptamer, and increased Riboglow’s performance in live cells. The other axis improved the Riboglow array, a section of aptamers linked in series for sufficient signal in live-cell imaging, through synonymization of the aptamer by mining natural cobalamin riboswitch sequences. The synonymization of the Riboglow array increased ease-of-use during molecular cloning and did not negatively impact Riboglow’s performance in cells. These improvements significantly increased the usefulness of Riboglow and its overall function in cells, solidifying Riboglow as a top contender in the RNA-imaging toolbox.