Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Selenium-Substituted Monomethine Cyanine Dyes as Selective G-Quadruplex Spectroscopic Probes with Theranostic Potential.

The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect.

A G-quadruplex/hemin structure-undamaged method to inhibit peroxidase-mimic DNAzyme activity for biosensing development.

Damaging the structure of the G-quadruplex (G4) to prevent the formation of the G4/hemin complex is presently the only available method to inhibit the activity of the peroxidase-mimic DNAzyme. In this study, a unique intramolecular inhibitory effect of the adjacent base-pair (InE(N:N)), by installing a rationally adjacent base-pair of the G4 core sequence, is proposed for the inhibition of the DNAzyme activity, which eliminates the need to damage the entire G4 structure. Various base pairs show different abilities to inhibit DNAzyme activity. The adjacent adenine: thymine pair possesses the best inhibitory efficiency (17 times). Through detailed investigations of the InE(N:N), it was revealed that the adjacent adenine: thymine pair downregulated the formation of compound I in the catalytic process, thus inhibiting the G4 DNAzyme activity. The mechanism of inhibition indicated that the carbonyl group on the hexatomic ring of the complementary base played an important role. To further reflect the advantages of the proposed strategy, two InE(N:N)-based biosensors were developed for DNA analysis and Uracil-DNA glycosylase (UDG) detection. Compared with existing DNAzyme-based methods, the application of InE(N: N) facilitates the real-time assay and simplifies the design difficulty. Therefore, InE(N:N) provides new insights into the regulation of the DNAzyme activity and offers an efficient approach for the future application of DNAzyme.

Distribution of Conformational States Adopted by DNA from the Promoter Regions of the VEGF and Bcl-2 Oncogenes.

We employed a previously described procedure, based on circular dichroism (CD) spectroscopy, to quantify the distribution of conformational states adopted by equimolar mixtures of complementary G-rich and C-rich DNA strands from the promoter regions of the VEGF and Bcl-2 oncogenes. Spectra were recorded at different pHs, concentrations of KCl, and temperatures. The temperature dependences of the fractional populations of the duplex, G-quadruplex, i-motif, and coiled conformations of each promoter were then analyzed within the framework of a thermodynamic model to obtain the enthalpy and melting temperature of each folded-to-unfolded transition involved in the equilibrium. A comparison of the conformational data on the VEGF and Bcl-2 DNA with similar results on the c-MYC DNA, which we reported previously, reveals that the distribution of conformational states depends on the specific DNA sequence and is modulated by environmental factors. Under the physiological conditions of room temperature, neutral pH, and elevated concentrations of potassium ions, the duplex conformation coexists with the G-quadruplex conformation in proportions that depend on the sequence. This observed conformational diversity has biological implications, and it further supports our previously proposed thermodynamic hypothesis of gene regulation. In that hypothesis, a specific distribution of duplex and tetraplex conformations in a promoter region is fine-tuned to maintain the healthy level of gene expression. Any deviation from a healthy distribution of conformational states may result in pathology stemming from up- or downregulation of the gene.

Dimethyl sulfoxide (DMSO) is a stabilizing co-solvent for G-quadruplex DNA.

We report the effect of dimethyl sulfoxide (DMSO) on the stability of the four-stranded structures formed by the oligodeoxyribonucleotides d[5'-AGGG(TTAGGG)3-3'] (HTel), d[5'-(GGGT)3GGG-3'] (G3T), d[5'-GGTTGGTGTGGTTGG-3] (TBA), d[5'-GGGGTTTTGGGG-3'] (Oxy-1.5), and d[5'-TGGGGT-3'] (TG4T). In these measurements, influence of the co-solvent was assessed by the change in the mid-point of the heat-induced unfolding, Tm, by monitoring the change in the UV absorption of the sample. Increasing concentrations of DMSO led to an increase in the Tm from the folded to unfolded states. We have also studied the effect of the denaturant urea and mixtures of urea and DMSO on the stability of the intramolecular HTel and the intermolecular TG4T G-quadruplexes. Consistent with earlier data, we found that urea destabilized the folded G-quadruplex structure; the Tm decreases with increasing urea concentration. However, in solutions containing both urea and DMSO, we observed that the two co-solvents off-set the destabilizing and stabilizing effect, respectively, of one another. That is, in solutions containing urea, increasing concentrations of DMSO led to the increase of the Tm of the G-quadruplex structure. This effect is observed in solutions containing sodium, potassium, or ammonium as the ion that stabilizes the folded G-quadruplex structure. The complementary effect of the two co-solvents presumably arises from differential interactions between urea and DMSO and the oligonucleotide or the cations involved in the stabilization of the G-quadruplexes. These results highlight the importance of co-solutes and co-solvents in systems containing guanine-rich DNA, particularly experimental processes that require DMSO.

Ginsenoside Compound K Assisted G-Quadruplex Folding and Regulated G-Quadruplex-Containing Transcription.

Ginsenoside compound K (CK) is one of the major metabolites of the bioactive ingredients in Panax ginseng, which presents excellent bioactivity and regulates the expression of important proteins. In this work, the effects of CK on G-quadruplexes (G4s) were quantitatively analyzed in the presence and absence of their complementary sequences. CK was demonstrated to facilitate the formation of G4s, and increase the quantity of G4s in the competition with duplex. Thermodynamic experiments suggested that the electrostatic interactions were important for G4 stabilization by CK. CK was further found to regulate the transcription of G4-containing templates, reduce full-length transcripts, and decrease the transcription efficiency. Our results provide new evidence for the pharmacological study of ginsenosides at the gene level.

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity.

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor-acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π-π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

The effect of hairpin loop on the structure and gene expression activity of the long-loop G-quadruplex.

G-quadruplex (G4), a four-stranded DNA or RNA structure containing stacks of guanine tetrads, plays regulatory roles in many cellular functions. So far, conventional G4s containing loops of 1-7 nucleotides have been widely studied. Increasing experimental evidence suggests that unconventional G4s, such as G4s containing long loops (long-loop G4s), play a regulatory role in the genome by forming a stable structure. Other secondary structures such as hairpins in the loop might thus contribute to the stability of long-loop G4s. Therefore, investigation of the effect of the hairpin-loops on the structure and function of G4s is required. In this study, we performed a systematic biochemical investigation of model G4s containing long loops with various sizes and structures. We found that the long-loop G4s are less stable than conventional G4s, but their stability increased when the loop forms a hairpin (hairpin-G4). We also verified the biological significance of hairpin-G4s by showing that hairpin-G4s present in the genome also form stable G4s and regulate gene expression as confirmed by in cellulo reporter assays. This study contributes to expanding the scope and diversity of G4s, thus facilitating future studies on the role of G4s in the human genome.

Targeting a Potential G-Quadruplex Forming Sequence Found in the West Nile Virus Genome by Complementary Gamma-Peptide Nucleic Acid Oligomers.

In the United States, West Nile virus (WNV) infects approximately 2500 people per year, of which 100-200 cases are fatal. No antiviral drug or vaccine is currently available for WNV. In this study, we designed gamma-modified peptide nucleic acid (γPNA) oligomers to target a newly identified guanine-rich gene sequence in the WNV genome. The target is found in the NS5 protein-coding region and was previously predicted to fold into a G-quadruplex (GQ) structure. Biophysical techniques such as UV melting analysis, circular dichroism spectroscopy, and fluorescence spectroscopy demonstrated that the target RNA indeed folds into a moderately stable GQ structure at physiological temperature and potassium concentration. Successful invasion of the GQ by three complementary γPNAs was also characterized by the above-mentioned biophysical techniques. The γPNAs showed very strong binding to the target with low femtomolar affinity at physiological temperature. Targeting this potential guanine quadruplex forming sequence (PQS) and other related sequences with γPNA may represent a new approach for inhibiting both WNV replication and transcription, thereby representing a generally useful antiviral strategy.

Temperature-robust and ratiometric G-quadruplex proximate DNAzyme assay for robustly monitoring of uranium pollution and its microbial biosorbents screening.

Uranium pollution in environment and food chain is a serious threat to public security and human health. Herein, we proposed a temperature-robust, ratiometric, and label-free bioassay based on G-quadruplex proximate DNAzyme (G4DNAzyme), accommodating us to precisely monitor uranium pollution and biosorption. The proximity of split G-quadruplex probes was proposed to sense UO22+-activated DNAzyme activity, thus eliminating the use of chemically labeled nucleic acid probes. And the simultaneous monitoring of G-quadruplex and double-stranded structures of DNAzyme probes contributed to a ratiometric and robust detection of UO22+. Particularly, the separation of enzymatic digestion and fluorescence monitoring endued a robust and highly responsive detection of UO22+ upon the temperature of enzymatic digestion process ranged from 18° to 41 °C. Consequently, G4DNAzyme assay allowed a robust, label-free and ratiometric quantification of uranium. We demonstrated the feasibility of G4DNAzyme assay for estimating uranium pollution in water and aquatic product samples. Ultimately, G4DNAzyme assay was adopted to serve as the platform to screen bacterial species and conditions for uranium biosorption, promising its roles in uranium associated biosafety control.

Sensitive and label-free chemiluminescence detection of malathion using exonuclease-assisted dual signal amplification and G-quadruplex/hemin DNAzyme.

Malathion is one of the most commonly used organophosphorus pesticides that can cause serious harm to the ecological environment and human health. Herein, we demonstrated a label-free chemiluminescent aptasensor for the sensitive detection of malathion based on exonuclease-assisted dual signal amplification and G-quadruplex/hemin DNAzyme. Upon the addition of malathion, the aptamer probe specifically bound to the target to form a complex malathion-S3, leaving a duplex S1-S2. The complex malathion-S3 was digested by exonuclease I and the target was released. The released target was recycled to perform exonuclease I-assisted signal amplification. Furthermore, after treatment with exonuclease III, the duplex S1-S2 was converted into the secondary target ST. The secondary target ST interacted with the hairpin H1 to form a complex H1-ST, which was further digested by exonuclease III and released the secondary target. The released secondary target was recycled to perform exonuclease III-assisted signal amplification. After complete amplification, large numbers of G-quadruplex/hemin DNAzymes were generated. Under the optimal experimental conditions, the prepared aptasensor showed an excellent linear response to malathion with a detection limit of 0.47 pM. The relative standard deviations were in the range of 4.2-6.9%. Moreover, the aptasensor was successfully applied to detect malathion in spiked food and traditional Chinese medicine samples.

Targeting RNA G-Quadruplex in SARS-CoV-2: A Promising Therapeutic Target for COVID-19?

The COVID-19 pandemic caused by SARS-CoV-2 has become a global threat. Understanding the underlying mechanisms and developing innovative treatments are extremely urgent. G-quadruplexes (G4s) are important noncanonical nucleic acid structures with distinct biofunctions. Four putative G4-forming sequences (PQSs) in the SARS-CoV-2 genome were studied. One of them (RG-1), which locates in the coding sequence region of SARS-CoV-2 nucleocapsid phosphoprotein (N), has been verified to form a stable RNA G4 structure in live cells. G4-specific compounds, such as PDP (pyridostatin derivative), can stabilize RG-1 G4 and significantly reduce the protein levels of SARS-CoV-2 N by inhibiting its translation both in vitro and in vivo. This result is the first evidence that PQSs in SARS-CoV-2 can form G4 structures in live cells, and that their biofunctions can be regulated by a G4-specific stabilizer. This finding will provide new insights into developing novel antiviral drugs against COVID-19.

A G-quadruplex nanoswitch in the SGK1 promoter regulates isoform expression by K+/Na+ balance and resveratrol binding.

BACKGROUND: High sodium intake can up-regulate the level of renal serum- and glucocorticoid-inducible kinase-1 (SGK1), which plays a pivotal role in controlling blood pressure via activation of the epithelial sodium channel (ENaC), which can lead to salt-sensitive hypertension. Increased potassium intake, or a vegetarian diet, counteracts salt-sensitive hypertension, but the underlying mechanisms are not fully understood. METHODS: Bioinformatics and molecular modeling were used to identify G-quadruplex (G4) and their conformations in the SGK1 promoter. CD spectra and UV melting dynamics were measured to study the stability of G4 as influenced by potassium/sodium balance and resveratrol. RT-PCR and Western blot were employed to study the effects of potassium and resveratrol on the SGK1 isoform expression. RESULTS: The SGK1 gene encodes a G4 structure in the proximal upstream of promoter-2; the G4 structure is stabilized by potassium or resveratrol, but destabilized by sodium. Super-physiological levels of sodium stimulate the transcription of all SGK1 isoforms, whereas resveratrol or potassium supplementation inhibits the transcription of iso-2 and iso-3, but not iso-1. CONCLUSIONS: Stabilizing the G4 by potassium or resveratrol induces alternative promoter usage and/or pre-mRNA splicing in the transcription of SGK1. GENERAL SIGNIFICANCE: Potassium/sodium ion balance or resveratrol binding can act to regulate G4 molecular switches for controlling SGK1 gene expression, thereby presenting a new avenue for drug development.

YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression.

The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.

Metal-induced G-quadruplex polymorphism for ratiometric and label-free detection of lead pollution in tea.

Lead pollution are critical concerns for food safety and human health. Herein, a ratiometric metal-induced G-quadruplex polymorphism was introduced to construct aptamer probes, enabling label-free and ratiometric detection of lead in tea, thus is promising for on-site detection of lead pollution. The key feature of the aptamer probe is the synergistic utilization of the dual-wavelength fluorescent signal outputs from a G-quadruplex specific dye and a DNA intercalation dye under a single-wavelength excitation, leading to a more stable and reliable recognition of Pb2+ than that of analyses based on single fluorescent reporter. The aptamer probe allowed to a mix-and-read, rapid, cost-effective detection of Pb2+ with high specificity and accuracy. Pb2+ analysis in tap water and tea exhibited good performance with recovery rates of 92.3%-109.0%. The adoption of ratiometric metal-induced G-quadruplex polymorphism would be a compelling design strategy for constructing robust aptasensor, facilitating the translation of aptamer for food safety control.

Fishing antitumor ingredients by G-quadruplex affinity from herbal extract on a three-phase-laminar-flow microfluidic chip.

A new method for fishing antitumor ingredients by G-quadruplex recognition from Macleaya cordata seeds extracts was established using a three-phase-laminar-flow-chip (TPL chip). The TPL chip integrated the separation of drugs from the complex ingredients and organic solvent extraction, simplifying pretreatment processes and reducing reagents and time. In addition, the chip method showed a lower false negative result, owing to the gentle membrane-free filtration process based on diffusion separation in microchannel. Four ligands with high content in alkaloids of Macleaya cordata seeds were selected, those are chelerythrine (CHE), sanguinarine (SAN), protopine (PRO), and allocryptopine (ALL), which demonstrated affinity with G-quadruplex and were potential for antitumor.

[Screening of G-quadruplex ligands from Macleaya cordata extract by contrast ultrafiltration with liquid chromatography-mass spectrometry and molecular docking].

G-quadruplex DNA has become an important target for tumor therapy and anti-tumor development. Modern pharmacology has proved that Macleaya cordata has anti-inflammatory, antibacterial, anti-tumor and other pharmacological effects. Affinity ultrafiltration method can screen active ingredients from compounds rapidly, but G-quadruplex DNA ligands are difficult to dissociate, which is a key step in conventional ultrafiltration method. In this paper, the filtrates after ultrafiltration were determined by HPLC-MS in substitution. The peaks with 20% reduction of MS response from the incubation vs control were considered to be ligand components to G-quadruplex. Two of the peaks with the relative abundance above 30% were identified as sanguinarine(SAN) and chelerine(CHE). Their circular dichroism conformations further proved that SAN and CHE are active ligands of HT4. In addition, another two gradients with high relative abundance were identified as protopine(PRO) and allpcryprotopine(ALL). The binding rate of SAN, CHE, PRO and ALL was calculated according to the HPLC-MS results, and the results showed a consistency with that of the molecular docking method. The proposed method can be used to screen active components from mixture.

Multicolor colorimetric detection of ochratoxin A via structure-switching aptamer and enzyme-induced metallization of gold nanorods.

Colorimetric aptasensors have been intensively studied for the ochratoxin A (OTA) detection, but they mostly exhibit just one-color change, resulting in poor visual resolution and limited use for semi-quantitative analysis. Thus, we designed a high-resolution colorimetric assay on the basis of aptamer structural switching and enzyme-induced metallization of gold nanorods (AuNRs). DNA-alkaline phosphatase (ALP)-immobilized magnetic beads were prepared. The aptamer bounded to OTA to form G-quadruplexes, releasing ALP-labelled complementary DNA (cDNA-ALP). After magnetic separation, cDNA-ALP catalyzed the decomposition of ascorbic acid 2-phosphate to ascorbic acid that reduced Ag+, forming an Ag shell on the surface of AuNRs. This caused a blue-shift of the longitudinal local surface plasmon resonance peak of the AuNRs and a naked eye visible multicolor change. Under optimal conditions, the assay exhibited a 9.0 nM detection limit for OTA, with high specificity. This method is promising for the on-site visual semi-quantitative detection of mycotoxins in foods.

Dual-amplified strategy for ultrasensitive electrochemical biosensor based on click chemistry-mediated enzyme-assisted target recycling and functionalized fullerene nanoparticles in the detection of microRNA-141.

Rapid and efficient detection of tumor marker at the early stages is one of the crucial challenges in cancer diagnostics and therapy. In this study, an ultrasensitive electrochemical biosensor was fabricated by dual-amplified strategy for the detection of ultra-trace microRNA-141 (miRNA-141). Firstly, two split sequences contained G-quadruplex were connected by click chemistry-mediated nucleic acid strands self-assembly and the obtained complete G-quadruplex was complementary with miRNA-141 to formed DNA-RNA hybrid duplexes. Subsequently, the formed DNA-RNA hybrid duplexes were specifically recognized by duplex-specific nuclease (DSN), and the DNA part of the duplexes were cleaved and the miRNA-141 were released to trigger next cycle, which acquired a primal signal amplification by enzyme-assisted target recycling (EATR). Moreover, amino and thiol group multi-labeled functionalized fullerene nanoparticles (FC60) with a larger surface active sites and better biocompatibility, were designed rationally to modify the Au electrodes, which produced multiply-enhanced amplified signal. This dual-amplified sensing system exhibited a remarkable analytical performance for the detection of miRNA-141 in concentrations ranging from 0.1 pM to 100 nM and the detection limit of 7.78 fM was obtained. Compared with the biosensor with single amplification strategy such as EATR, this electrochemical biosensor based on dual-amplified strategy exhibited an excellent discrimination capability and higher analytical performance. Therefore, this electrochemical biosensor might hold a great potential for further applications in biomedical research and early clinical diagnosis.