Strong Binding and Off–On Signaling Functions of Deep‐Red Fluorescent TO‐PRO‐3 for Influenza A Virus RNA Promoter Region

Abstract

The RNA promoter region of the influenza A virus has recently attracted much attention as an RNA target for the development of anti‐influenza drugs. However, there are very few reports on small RNA‐binding ligands targeting this region. In this work, it is reported that TO‐PRO‐3, a thiazole orange analogue with a trimethine bridge, exhibits strong and selective binding to the internal loop structure of the influenza A virus RNA promoter. This binding accompanies the remarkable light‐up response of TO‐PRO‐3 in the deep‐red spectral region. By virtue of these binding and fluorescence signaling functions, TO‐PRO‐3 can act as a useful indicator for the assessment of the binding capabilities of various test compounds for this RNA region, with a view toward the development of anti‐influenza drug candidates.

Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule

“…The obtained titration curve was well fitted by a 1:1 binding model (inset, Figure ), yielding the K d value of 107 ± 9.4 nM ( N = 3). Significantly, tFIT-DPQ showed stronger binding affinity by 2 orders of magnitude compared to DPQ ( K d > 25 μM) . While the binding affinity of tFIT-DPQ is reduced compared to that at acidic pH (Figure S4), this clearly shows the significance of triplex formation by the tFIT unit for the binding to target RNA at neutral pH.…”

“…The binding of tFIT-DPQ was first examined for the model RNA containing the IAV RNA promoter region (Figure A) , in pH 7.0 buffer by fluorescence spectroscopy. As shown in Figure , the TO base surrogate of the conjugate (100 nM) exhibits negligible emission in the absence of target RNA through nonradiative energy loss because of free rotation of the benzothiazole and quinolone rings (fluorescence quantum yield (Φ free < 0.001)).…”

“…Prior to this study, carboxytetramethylrhodamine (TAMRA)-tagged paromomycin (CRP) was developed as the fluorescence indicator to target the IAV RNA promoter region (λ em = 580 nm, K d = 2.0 μM), in which the change in the fluorescence anisotropy was analyzed to monitor the displacement event with test compounds. We have also reported that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, functioned as a deep-red fluorescence indicator for the IAV RNA promoter region (λ em = 663 nm, K d = 2.9 μM) . In addition to the off-on signaling ability (Φ free < 0.0002, Φ bound = 0.18) applicable to high-throughput screening (HTS), the deep-red emission of TO-PRO-3 is of great importance to reduce “compound optical interference” that is frequently found in fluorescence-based screening. , However, the binding affinity of TO-PRO-3 is so moderate that the relatively high concentration of target RNA (1.0 μM) was needed for the FID assay .…”

“…We have also reported that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, functioned as a deep-red fluorescence indicator for the IAV RNA promoter region (λ em = 663 nm, K d = 2.9 μM) . In addition to the off-on signaling ability (Φ free < 0.0002, Φ bound = 0.18) applicable to high-throughput screening (HTS), the deep-red emission of TO-PRO-3 is of great importance to reduce “compound optical interference” that is frequently found in fluorescence-based screening. , However, the binding affinity of TO-PRO-3 is so moderate that the relatively high concentration of target RNA (1.0 μM) was needed for the FID assay . This would fail to achieve the stringent screening condition.…”

“…It was demonstrated that IR-1X was potentially applicable to fluorescence detection of IAV RNAs. These TFPs (IR-1 and IR-1X) developed by the Chen group feature the sequence- and structure-selective recognition of the panhandle structure, which is advantageous for the selective analysis of IAV RNA compared to small molecules capable of binding to the internal loop structure. − Stronger binding of TFPs over small molecules also benefits the analytical applications. However, targeting the overall panhandle structure by simple TFP-mediated recognition seems not to be straightforward given its global structure is highly bent owing to the disruption of the normal A-formed helix by the internal loop. , In addition, it is highly desirable to improve the fluorescence signaling property of the probes in order to achieve the sensitive detection of the IAV RNA promoter.…”