Pyrrolocytosine as a Fluorescent Sensor of RNA–Small Molecule Interactions
- 1Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 97403, USA
- 2Oregon Center for Optical, Molecular and Quantum Science, University of Oregon, Eugene, OR 97403, USA
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 understand 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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BibTeX
@article{coulson2026pyrrolocytosine,
title = {{Pyrrolocytosine as a Fluorescent Sensor of RNA–Small Molecule Interactions}},
author = {Taylor L. Coulson and Gavin A. Valdez and Julia R. Widom},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27219651},
url = {https://www.mdpi.com/1422-0067/27/21/9651},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Pyrrolocytosine as a Fluorescent Sensor of RNA–Small Molecule Interactions
AU - Taylor L. Coulson
AU - Gavin A. Valdez
AU - Julia R. Widom
PY - 2026
DA - 2026/10/28
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27219651
UR - https://www.mdpi.com/1422-0067/27/21/9651
AB - 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 understand 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.
SN - 1422-0067
ER -