Fluorescent Probes of DNA Repair

Abstract

DNA repair is now understood to play a key role in a variety of disease states, most notably cancer. Tools for studying DNA have typically relied on traditional biochemical methods which are often laborious and indirect. Efforts to study the biology and therapeutic relevance of DNA repair pathways can be limited by such methods. Recently, specific fluorescent probes have been developed to aid in the study of DNA repair. Fluorescent probes offer the advantage of being able to directly assay for DNA repair activity in a simple, mix-and-measure format. This review will summarize the distinct classes of probe designs and their potential utility in varied research and preclinical settings.

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses

“…We validated the ability of probe 13 to assay ALKBH2 inhibitors by measuring the IC 50 of pyridine 2,4‐dicarboxylic acid (PDCA), a broad‐spectrum inhibitor of 2‐OG‐dependent enzymes (Figure ). The assay was conducted in a 60 μL reaction volume on a 384‐well plate, yielding an IC 50 of 2.43±0.12 μ m , fully consistent with reported values . To our knowledge, probe 13 enables the first continuous real‐time assay of ALKBH2 activity, making it likely the most practical assay available for high‐throughput screens.…”

“…Recently, fluorescent DNA probes have been developed for the direct analysis of DNA repair . Herein, we describe the first fluorogenic probes for ALKBH2 activity, affording rapid and real‐time measurements of repair by ALKBH2 in vitro and in human cell extracts using only a fluorescence microplate reader.…”

An Excimer Clamp for Measuring Damaged‐Base Excision by the DNA Repair Enzyme NTH1

“…Indirect output probes are also likely to have limited utility in biological environments owing to the requirement of multiple steps for signal generation. Recently, fluorescent DNA probes for the direct analysis of other DNA repair activities have been developed based on the fluorescence quenching effect of neighboring bases . Such methods, while effective for certain enzymes, are limited to damaged base/fluorophore combinations that yield effective quenching.…”

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses

“…The assay was conducted in a 60 µL reaction volume on a 384-well plate, yielding an IC 50 of 2.43 ± 0.12 µ m , fully consistent with reported values. [18] To our knowledge, probe 13 enables the first continuous real-time assay of ALKBH2 activity, making it likely the most practical assay available for high-throughput screens.…”

“…Recently,f luorescent DNAp robes have been developed for the direct analysis of DNArepair. [18] Herein, we describe the first fluorogenic probes for ALKBH2 activity,a ffording rapid and real-time measurements of repair by ALKBH2 in vitro and in human cell extracts using only af luorescence microplate reader.T he optimized probe 13 is highly specific for ALKBH2 demethylation and is used to directly quantify elevated ALKBH2 activity in ac ancer cell line treated with the drug TMZ. Probe 13 is also well suited to conducting highthroughput screens of potential ALKBH2 activators/inhibitors.…”