Simple and Convenient G-Quadruplex-Based Turn-On Fluorescence Assay for 3′ → 5′ Exonuclease Activity

Abstract

A selective, oligonucleotide-based, label-free, turn-on fluorescence detection method for 3′ → 5′ exonuclease activity has been developed using crystal violet as a G-quadruplex-binding probe. The assay is highly simple and rapid, does not require the use of gel-based equipment or radioisotopic labeling, and is amenable to high-throughput and real-time detection. A proof-ofconcept of this assay has been demonstrated for prokaryotic ExoIII and human TREX1.Enzymes that contain 3′ → 5′ exonuclease activities play important roles in a variety of key cellular and physiological processes, such as DNA proofreading.1 , 2 3′ → 5′ exonuclease inhibitors have the potential to act synergistically with anticancer drugs, increasing their cytotoxic effects against cancer cells by inhibiting DNA repair.2 , 3 Commonly used 3′ → 5′ exonuclease activity assays are gel-based and/or require the use of radioisotopes such as 3 H or γ-[ 32 P]ATP-labeled DNA.4 However, these protocols tend to be unwieldy and timeconsuming, and necessitate stringent safety measures to control radiographic exposure. Therefore, the development of an efficient detection method for 3′ → 5′ exonucleolytic activity amenable to high-throughput screening would greatly facilitate the identification of exonuclease modulators for potential therapeutic applications. We describe herein the first selective, label-free, high-throughput G-quadruplex-based turn-on fluorescence assay for 3′ → 5′ exonuclease activity.The prokaryotic 3′ → 5′ exonuclease III was chosen to demonstrate the proof-of-concept of our approach. Exonuclease III (ExoIII) catalyzes the stepwise hydrolysis of mononucleotides from the 3′-terminus of double-stranded DNA.5 However, ExoIII is unable to catalyze the removal of bases from a single-stranded substrate. We thus designed an unlabelled oligonucleotide hairpin sequence G 55 = [5′-AG 3 (T 2 AG 3 ) 3 CAGA 2 G 2 AT 2 A(C 3 TA 2 ) 3 C 3 T-3′] consisting of a 22-bp G-quadruplexforming sequence at the 5′-terminus and its complementary cytosine-rich sequence at the 3′-terminus connected by a 11-bp flexible linker ( Figure S1a). The oligonucleotide G 55 was hybridized by annealing at 95 °C for 10 min and slowly cooling to room temperature, forming a stem-loop secondary DNA structure ( Figure S1b) . We used the human telomeric G-quadruplex sequence [5′-AG 3 (T 2 AG 3 ) 3 -3′] due to the strong fluorescent response of crystal violet (CV) to this G-quadruplex.6 CV is an inexpensive, commonly available triphenylmethane dye that has been demonstrated to display significant selectivity for the G-quadruplex secondary structure over single-stranded and double-stranded DNA.6 Celada and coworkers have measured the 3′ → 5′ exonucleolytic activity of TREX1 employing SYBR Green as a probe to monitor the doublestranded to single-stranded DNA transition of a short duplex.4a However, this "turn-off" fluorescence assay for exonuclease activity is readily subject to false positives due to fluorescence quenching by a variety of interfering mechanisms. Secondly, the p...

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress

“…Because ssDNA is more sensitive than G4DNA to degradation by cytoplasmic nucleases such as TREX1, a scrambled sequence with phosphorothioate (PS) at the ends of the oligonucleotide to prevent degradation was also used for comparison. G4DNA is refractory to the nuclease activity of TREX1 (39). Although Cy3-ssDNA was visible in the cytosol, the lack of cytoplasmic TIA1 foci indicated that stress granules were not present (Fig.…”

G-quadruplexes for luminescent sensing and logic gates

“…Our research group has reported a G-quadruplex-based strategy for the detection of 3′ → 5′ exonucleases (29), which are enzymes that play important roles in a variety of key cellular and physiological processes such as DNA proofreading (68). In this study, we designed an unlabelled DNA hairpin sequence [5′-AG 3 (T 2 AG 3 ) 3 CAGA 2 G 2 AT 2 A(C 3 TA 2 ) 3 C 3 T-3′] consisting of a 22-bp G-quadruplex-forming sequence at the 5′-terminus, a 11-bp loop region, and a 22-bp complementary C-rich sequence at the 3′-terminus.…”

Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe

“…In recent years, a lot of effort has been directed toward the development of fluorescence assays for 3 0 ? 5 0 exonuclease activity [10][11][12][13][14][15]. Most of the reported fluorescence methods for exonuclease activity needed fluorescent dyes to label DNA probes.…”