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Design of deep-red emissive forced intercalation-induced light-up peptide as an indicator for the HIV-1 TAR RNA-ligand assay: integration of benzo[c,d]indole-quinoline (BIQ) cyanine dye into Tat peptide.

Akunna Francess UjuaguYusuke SatoEn Ting Tabitha LeeSeiichi Nishizawa
Published in: Analytical sciences : the international journal of the Japan Society for Analytical Chemistry (2024)
We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[c,d]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off-on signaling ability for TAR RNA (λ em  = 660 nm, I/I 0  = 130-fold, Φ free  = 0.0009, Φ bound  = 0.052), and the dissociation constant K d reaches ca. 1 nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λ em  = 541 nm) would cover the examination of a wide range of fluorescent test compounds.
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