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bioRxiv · 10.64898/2026.09.29.755477

Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions

Coulson, T. L.·Valdez, G. A.·Widom, J. R.
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Abstract

Fluorescent base analogues have proven to be valuable probes of RNA structure. Pyrrolocytosine (pC), an analogue of cytosine, exhibits highly environment-sensitive fluorescence within RNA and is strongly quenched in hydrogen-bonded contexts. In this work, we incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to determine if this quenching mechanism could allow for sensing of RNA-small molecule interactions. We utilized complementary fluorescence spectroscopy methods to under-stand the functional impacts and ligand-sensitivity of pC substitutions distal to and within the binding pocket. We found that pC substitution within a static structural region exhibits the expected ligand-independent fluorescence properties. When placed in the ligand binding pocket, pC exhibits a moderately decreased affinity for the ligands guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. We found that pC behaves as a turn-off sensor, with distinct changes in fluorescence upon G or Hx binding. These differences are dependent on the number of hydrogen bonds formed with pC and are resolvable in both time-dependent and steady-state fluorescence measurements. Our results show that pC can be used to study RNA-small molecule interactions and can discriminate between similar ligands even when they have identical binding modes.

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Coulson, T. L., Valdez, G. A., Widom, J. R.. 2026-09-30. Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions. https://doi.org/10.64898/2026.09.29.755477

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