Design of forced intercalation-induced light-up Rev peptide probes for HIV RRE RNA

The interaction of the HIV-1 Rev protein with the Rev response element (RRE) RNA is essential for viral replication and represents an attractive target for ligand screening and RNA detection. Here, we developed forced intercalation-induced light-up peptide (FiLuP) probes by incorporating thiazole orange (TO) as an amino acid surrogate at four positions within the RRE-binding Rev_34-50 peptide. Among the resulting probes, E₄₇ exhibited the largest fluorescence enhancement upon RRE binding, with a 58-fold light-up response and a bound-state fluorescence quantum yield (Φ _bound) of 0.31. Fluorescence titration afforded an apparent K _d of 0.84 ± 0.70 nM under the conditions employed. The fluorescence response of E₄₇ to RRE RNA was more than 2-fold compared to those of the tested non-cognate model RNAs. Its selectivity profile was comparable to that of an N-terminal rhodamine B-labelled Rev control probe, indicating that internal TO incorporation largely retained the inherent RRE preference of the Rev-derived sequence. E₄₇ also functioned as an indicator in a fluorescence indicator displacement assay. Furthermore, E₄₇ enabled direct concentration-dependent detection of an RRE RNA model with limits of detection of 190 pM in buffer and 220 pM in 10% serum. These results demonstrate that forced intercalation can convert an RNA-binding peptide into a fluorogenic probe for ligand-displacement analysis and direct RNA detection.