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BBT Nucleotide Fluorescence

Probing viral replication
   



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Diseases evoked by flaviviral infections still impose a major challenge on medical research. In many cases such as FSME, Yellow fever and Dengue fever there are no causative therapeutics available. The propagation of flaviviruses, however, necessarily requires the replication of genomic viral RNA by RNA-dependent RNA polymerases (RdRPs), making these enzymes highly promising drug targets.

To study kinetic[1-3] and pharmaceutical features[4] of the Dengue RdRP in vitro, 2'-(2-benzothiazoyl)-6'-hydroxybenzothiazole-(BBT)-modified nucleotides have been proposed as well-suited fluorescent probes. In the fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA, see Scheme 1), BBT-nucleotides permit the highly sensitive screening for antiviral compounds under high throughput conditions.

Scheme 1: Workflow of the BBT-based FAPA assay. Genomic viral RNA ((+)ssRNA) is subjected to RdRP-dependent replication in the presence of BBT-modified nucleotides, thereby releasing non-fluorescent BBT pyrophosphate (BBTppi). After stopping the replication reaction at a time point of choice, calf intestinal alkaline phosphatase (CIP) is added to convert BBTppi into fluorescent BBT for subsequent readout.

Jena Bioscience now offers the complete set of BBT-modified ribonucleotides:

Compound

Cat. No.

Amount

Price (€)

γ-(BBT)-CTP

NU-933

1 mg

126,08

γ-(BBT)-UTP

NU-934

1 mg

126,08

γ-(BBT)-ATP

NU-1700

1 mg

126,08

γ-(BBT)-GTP

NU-1703

1 mg

126,08


Selected References:
[1] Niyomrattanakit et al. (2011) A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase. J. Biomol. Screen. 16 (2):201.
[2] Niyomrattanakit et al. (2015) Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening. Antiviral Res. 119:36.
[3] Tay et al. (2016) The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production. PLoS Pathog. 12 (9):e1005886.
[4] Yokokawa et al. (2016) Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design. J. Med. Chem. 59 (8):3935.

   
   
   
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