Screening helix-threading peptides for RNA binding using a thiazole orange displacement assay

Abstract

The fluorescent intercalator displacement assay using thiazole orange has been adapted to the study of RNA-binding helix-threading peptides (HTPs). This assay is highly sensitive with HTP-binding RNAs and provides binding affinity data in good agreement with quantitative ribonuclease footprinting without the need for radiolabeling or gel electrophoresis. The FID assay was used to define structure activity relationships for a small library of helix-threading peptides. Results of these studies indicate their RNA binding is dependent on peptide sequence, alpha-amino acid stereochemistry, and cyclization (vs linear peptides), but independent of macrocyclic ring size for the penta-, tetra- and tri-peptides analyzed.

Figure 1

(A) Structure of thiazole orange; (B) Secondary structures of RNA A, B with putative intercalation site boxed and control RNA C used for the displacement assay, nucleotides highlighted in outlined text are present in the in vitro selected aptamer (RNA A) and human pre-miRNA 23b (RNA B); (C) Fold stimulation of thiazole orange (2.6 µM) fluorescence with RNA A, B, and C at 2 µM concentration each; (D) Titration of RNA A (5 µM) with thiazole orange.

Figure 2

(a) Schematic representation of thiazole orange displacement assay; (b) Binding of HTP 2 to RNA A causes displacement of thiazole orange resulting in decrease in fluorescence; (c) Structure of macrocyclic HTP 2.

Figure 3

Chemical structures of HTPs and aminoglycosides analyzed by thiazole orange displacement assay. The K_D values obtained from the displacement assay are indicated below the structures for each ligand.

Figure 4

Chemical structures of macrocyclic HTPs used in the displacement assay. The K_D values obtained from the displacement assay are indicated below the structures for each HTP.

Scheme 1

Synthesis of 2-phenylquinoline carboxylic acid 12. (a) Methyl anthranilate, Ph₂O, MW, 240 °C, 20 min, 83%.; (b) (i) POCl₃, reflux,1 h, 87%; (ii) NBS, AIBN, cyclohexane, reflux, 50 min, 77%; (iii) Vinyl tributyltin, Pd(PPh₃)₄, DMF, 45 °C, 2 h, 73%; (iv) aniline, SnCl₄, CH₃CN, rt , 1 h and 86%; (c) LiOH.H₂O, THF-H₂O, rt, 24 h, 87%.