Nanoparticles-Free Fluorescence Anisotropy Amplification Assay for Detection of RNA Nucleotide-Cleaving DNAzyme Activity

Abstract

Fluorescence anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large fluorescence anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced fluorescence anisotropy assay for detection of DNAzyme activity. A Pb 2+ -dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5′-end with the DNAzyme strand containing an extended three G bases at the 3′-end. By this design, we demonstrate that both fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb 2+ -induced large fluorescence anisotropy change (|Δr| = 0.168). The limit of detection for Pb 2+ is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we show an extended assay for evaluation of the interactions of Pb 2+ with cysteine and glutathione by the detection of GR5 DNAzyme activity. Collectively, we developed a novel fluorescence anisotropy amplification assay, enabling us to detect DNAzyme activity and associated cofactors and inhibitors and to characterize the Pb 2+ -chelation capability of free thiols.O ne class of functional nucleic acids with catalytic activity is DNAzymes, which have potential applications in diagnosis, therapeutics, and bioanalysis. 1−4 Various DNAzymes have been in vitro selected to catalyze a wide range of reactions, such as RNA cleavage, DNA cleavage, RNA ligation, DNA ligation, DNA phosphorylation, cleavage of the phosphoramidate bond, and porphyrin metalation. 5,6 One type of DNAzymes can cleave RNA. These RNA-cleaving DNAzymes are composed of a DNA enzyme strand and a DNA substrate strand containing a single RNA linkage (rA), which serves as the cleavage site. 7 This class of DNAzymes has been applied in nucleic acid detection, 8 metal ions sensing, 3,4,6,7,9−11 and molecular logic gate constructing. 4,12,13 The enzymatic activities play central roles in any extended analytical applications of DNAzymes. A number of biochemical and biophysical methods have been carried out for measuring DNAzyme activities, especially for RNA-cleaving DNAzymes. These methods include polyacrylamide gel electrophoresis (PAGE), 14 MALDI-TOF mass spectrometry, 15 fluorescence resonance energy transfer (FRET), 16,17 and fluorescence anisotropy (FA). 18−22 Among known techniques for detection of DNAzyme activity, FA has a number of advantages. It is a simple, realtime, and truly homogeneous analysis. Moreover, it does not require the separation of substrates and the cleaved products. This technique also presents several other attractive features, such as the requirement of only one fluorescent label and relative insensitivity to the photobleaching of the fluorophore and to instrument parameters due to its ratiometric nature. In fact, F...

Recent advances in fluorescence anisotropy/polarization signal amplification

“…These FA strategies allow for analysis of proteins, small molecules, metal ion and DNAzyme activity, showing high sensitivity and selectivity. 5,20,36,44,104–106 Peyrin et al designed a FA aptamer sensing platform dedicated to small molecule detection that relied on enhanced fluctuations of segmental motion dynamics of the aptamer tracer mediated by an unlabelled, partially complementary oligonucleotide. 107 Furthermore, by using the accumulation of a lot of DNA in fluorescence indicator-graphene QDs to enhance FA, Hosseini group achieved DNA methyltransferase activity detection.…”

“…First, FA and FP values are intrinsic parameters, and the ratiometric FA and FP techniques are tolerant to photobleaching of the fluorophore and instrumental parameters. 5–8 Second, FA and FP assays are homogeneous; that is, there is no need for separation of substrates. 3,9 Third, because of its simplicity and speed, FA/FP assays are especially suitable for an automated high throughput format assay.…”

Paper‐based point‐of‐care test with xeno nucleic acid probes

“…Due to the excellent fluorescence quenching property and the characteristic of less affinity for double stranded DNA (dsDNA) or secondary single stranded DNA (ssDNA). This paper‐based microfluidic chip integrated GO sensors which associated with fluorescence‐labeled functional aptamer (D. Zhang, Fu, Zhao, Rong, & Wang, ). Another novel design in multiplex paper‐based detection is using target‐responsive DNA hydrogel.…”

DNA-templated Au nanoclusters coupled with proximity-dependent hybridization and guanine-rich DNA induced quenching: a sensitive fluorescent biosensing platform for DNA detection

“…21 The uorescence quenching phenomenon is usually called guanine (G) quenching. Due to its cost-effective and easy design, G quenching of uorescencelabeled DNA probes has been used as a powerful tool for the detection of metal ions, [23][24][25] DNA, 26,27 protein, 28 adenosine triphosphate, 29,30 exonuclease/endonuclease activity, 31,32 and so on. However, most of these methods usually require complicated uorescent dye labelling.…”