Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity

Abstract

The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.

Figure 1

Design of DNA-based light-up fluorescent probes for adenine excision by MUTYH. (a) Neighboring DNA bases quench the emission of a fluorescent adenine analogue, which is excised by MutY/MUTYH, resulting in a turn-on signal. (b) Fluorescent adenine analogues designed and tested in this study.

Figure 2

Kinetics of fluorescent FMART probe responses: (a) probes 1–3 and 5–6 or (b) probes 4 and 7–10 (440 nM) incubated with mMUTYH (220 nM), 20 mM Tris HCl pH 7.5, 10 mM ethylenediaminetetraacetic acid (EDTA), and 30 mM NaCl at 37 °C, measured at their emission maxima listed in Table S1. (c) Fluorescence emission spectra of probe 7 before and after 5 h of incubation with mMUTYH. (d) Kinetics of probes 1–6 (600 nM) incubated with Escherichia coli MutY (300 nM), 20 mM Tris HCl pH 7.5, 10 mM EDTA, and 30 mM NaCl at 37 °C, measured at their emission maxima. (e) Absorption and emission spectra of new nucleosides A1 and A3–5 (10 μM) in PBS (pH 7.4, room temperature). (f) Images of solutions of new nucleosides A1 and A3–5 (10 μM) in PBS (pH 7.4, room temperature) above a transilluminator at two excitation wavelengths.

Figure 3

Application of FMART probes to small-molecule screening. (a) Mammalian repair enzyme activity data from screening a small-molecule library using probe 7 in a 384-well plate (excitation 355 nm, emission monitored at 460 nm). Compounds (20 μM) were incubated with mMUTYH (200 nM) at 37 °C for 10 min before probe 7 (400 nM) was added. See details in Supporting Information. (b) Validation of 13 inhibitor candidates at 5 μM. (c) Structures of representative small-molecule modulator candidates. (d) Dose–response curve of CAY10657 with mMUTYH, measured with probe 7.

Figure 4

(a) Time course of FMART probe 10 response (500 nM) in HeLa cell lysates. (b) Imaging of HeLa cells with FMART probe 10. Note selective fluorescence signals in WT cells as compared with those lacking the MUTYH protein. See Figure S5 for larger images.

Figure 5

(a) Time course of FMART probe 10 response (500 nM) in human blood serum with or without competing substrate 11 (2 μM) or 12 (2 μM). (b) Time course of FMART probe 10 response (500 nM) in human blood leukocyte lysate with or without competing substrate 11 (2 μM) or 12 (2 μM). The fluorescence was monitored at 460 nm (355 nm excitation). Error bars show standard deviation from three replicates.