A Quadruplex‐Based, Label‐Free, and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

Hu, Dan; Pu, Fang; Huang, Zhenzhen; Ren, Jinsong; Qu, Xiaogang

doi:10.1002/chem.200902166

Cited by 77 publications

(50 citation statements)

References 32 publications

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“…We envisage that this high-throughput assay may be readily adapted for the rapid screening of exonuclease activity modulators. The emission intensity of CV decreased slowly after reaching a maximum value (Figure S7), a phenomenon that was also observed in the RNase assay by Ren and co-workers 7. The real-time CD analysis suggests that the G-quadruplex is not a substrate for the exonuclease, as evidenced by the CD signal at 295 nm reaching a stable value (Figure S5a).…”

supporting

confidence: 68%

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

et al. 2010

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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...

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confidence: 68%

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

et al. 2010

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“…Further modifications in terms of enhancing the sensitivity and selectivity of the assays, possibly through the use of more specific substrates or the incorporation of some form of amplification mechanism, may be required to realize the in vivo detection of protein or enzyme activities. Furthermore, besides the novel G-quadruplex probes described, other G-quadruplex-based assays utilizing previously reported G-quadruplex probes such as NMM have also been developed for the detection of UDG and reverse transcriptase (Hu et al, 2010(Hu et al, , 2011. G-Quadruplex Probes for the Detection of Nucleic Acids A vast amount of effort has been devoted to the development of probes for nucleic acids.…”

Section: G-quadruplex Probes For Sensing Proteins and Enzyme Activitymentioning

confidence: 99%

Recent Developments in G-Quadruplex Probes

Zhang

Wang

et al. 2015

Chemistry & Biology

164

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Accumulating evidence has linked G-quadruplex structures to a number of biological processes in vivo, including DNA replication, transcription, and genomic maintenance. However, the precise function and mechanism of G-quadruplex formation in mammalian cells remains poorly defined. Therefore, the development of small-molecule G-quadruplex probes has attracted significant attention in recent years. This review highlights examples of G-quadruplex probes that have been reported over the last few years, some of which have been employed for cellular imaging or for use in the construction of G-quadruplex-based analytical sensing platforms.

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“…Ren and co-workers (20) have designed a DNA–RNA hybrid duplex motif for the label-free detection of RNase H activity. RNase H is a ribonuclease that specifically degrades the RNA strand of an RNA–DNA duplex (66).…”

Section: Case Studiesmentioning

confidence: 99%

G-quadruplexes for luminescent sensing and logic gates

Chan

Leung

et al. 2013

Nucleic Acids Research

153

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G-quadruplexes represent a versatile sensing platform for the construction of label-free molecular detection assays owing to their diverse structures that can be selectively recognized by G-quadruplex-specific luminescent probes. In this Survey and Summary, we highlight recent examples of the application of the label-free strategy for the development of G-quadruplex-based luminescent detection platforms with a view towards the potential application of tetraplex structures in the design of DNA logic gates.

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A Quadruplex‐Based, Label‐Free, and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

Cited by 77 publications

References 32 publications

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

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

Recent Developments in G-Quadruplex Probes

G-quadruplexes for luminescent sensing and logic gates

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