Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions
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…
Pyrrolocytosine (pC), an analogue of cytosine, has emerged as a sensitive fluorescent probe for studying RNA structure. In this research, scientists incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to investigate its potential in sensing RNA-small molecule interactions. Utilizing complementary fluorescence spectroscopy methods, the researchers examined the functional impacts and ligand-sensitivity of pC substitutions both distal to and within the binding pocket.
The study revealed that pC substitution within a static structural region exhibited the expected ligand-independent fluorescence properties. However, when pC was placed in the ligand binding pocket, it displayed a moderately decreased affinity for guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. The researchers found that pC functions as a turn-off sensor, displaying distinct changes in fluorescence upon binding with G or Hx.
These differences were dependent on the number of hydrogen bonds formed with pC and were discernible in both time-dependent and steady-state fluorescence measurements.
The findings demonstrate that pC can be effectively employed to study RNA-small molecule interactions and differentiate between similar ligands even when they possess identical binding modes.
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