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Forced intercalation-induced light-up peptides as fluorogenic indicators for the HIV-1 TAR RNA-ligand assay.
- Source :
-
The Analyst [Analyst] 2024 Aug 05; Vol. 149 (16), pp. 4179-4186. Date of Electronic Publication: 2024 Aug 05. - Publication Year :
- 2024
-
Abstract
- Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans -activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9-mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto a Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λ <subscript>em</subscript> = 541 nm, Φ <subscript>free</subscript> = 0.0057, and Φ <subscript>bound</subscript> = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP maintains strong binding affinity ( K <subscript>d</subscript> = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between "competitive" and "noncompetitive" inhibitors when used in the fluorescence indicator displacement (FID) assay. The FID assay under stringent screening conditions is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.
- Subjects :
- Ligands
Benzothiazoles chemistry
Quinolines chemistry
Humans
Peptides chemistry
Intercalating Agents chemistry
HIV-1
RNA, Viral
Fluorescent Dyes chemistry
tat Gene Products, Human Immunodeficiency Virus chemistry
tat Gene Products, Human Immunodeficiency Virus metabolism
HIV Long Terminal Repeat
Subjects
Details
- Language :
- English
- ISSN :
- 1364-5528
- Volume :
- 149
- Issue :
- 16
- Database :
- MEDLINE
- Journal :
- The Analyst
- Publication Type :
- Academic Journal
- Accession number :
- 38860915
- Full Text :
- https://doi.org/10.1039/d4an00530a