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 KD 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.