A turn-on fluorescent PCNA sensor.

Abstract

The solvatochromic amino-acids 4-DMNA or 4-DAPA, were separately introduced at position 147, 150 or 151 of a short p21 peptide (141-155) known to bind sliding clamp protein PCNA. The ability of these peptides, 1a-3a and 1b-3b, to act as a turn-on fluorescent sensor for PCNA was then investigated. The 4-DMNA-containing peptides (1a-3a) displayed up to a 40-fold difference in fluorescence between a polar (Tris buffer) and a hydrophobic solvent (dioxane with 5 mM 18-crown-6), while the 4-DAPA-containing peptides (1b-3b) displayed a significantly enhanced (300-fold) increase in fluorescence from Tris buffer to dioxane with 18-crown-6. SPR analysis of the peptides against PCNA revealed that the 151-substituted peptides 3a and 3b interacted specifically with PCNA, with K_D values of 921 nM and 1.28 μM, respectively. Analysis of the fluorescence of these peptides in the presence of increasing concentrations of PCNA revealed a 10-fold change in fluorescence for 3a at 2.5 equivalents of PCNA, compared to only a 3.5-fold change in fluorescence for 3b. Peptide 3a is an important lead for development of a PCNA-selective turn-on fluorescent sensor for application as a cell proliferation sensor to investigate diseases such as cancer.