Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions
- 1University of Oregon
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.
Related
- A Fluorescent Aptamer Sensor Based on Cu-TCPP/rGO for Homogeneous Screening of G-Quadruplex Ligands
- A label-free fluorescent sensor for miR-21 based on sequentially coupled CHA-HCR dual-stage amplification and synergistic signal regulation by graphene oxide and SYBR Green I.
- Computational assay of hERG ion permeation and its modulation by small-molecule compounds
- Quantifying structure and performance diversity for sets of small molecules comprising small-molecule screening collections
- A small molecule inhibitor of human ARV1 sterol-binding protein blocks lipid binding
BibTeX
@misc{coulson2026pyrrolocytosine,
title = {{Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions}},
author = {T. L. Coulson and G. A. Valdez and J. R. Widom},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.29.755477},
url = {https://www.biorxiv.org/content/10.64898/2026.09.29.755477v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions
AU - T. L. Coulson
AU - G. A. Valdez
AU - J. R. Widom
PY - 2026
DA - 2026/09/29
PB - bioRxiv
DO - 10.64898/2026.09.29.755477
UR - https://www.biorxiv.org/content/10.64898/2026.09.29.755477v1
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 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.
ER -