Search Results (6)

MicroRNA-155-3p (miR-155-3p) is an important biomarker in various pathological conditions, including cancer, making the development of sensitive and specific detection methods crucial. Here, we present a molecular beacon (MB-G4) that underwent a conformational switch upon hybridization with miR-155-3p, enabling the formation of a G-quadruplex (G4) structure. This G4 was recognized by the fluorogenic ligand N-methyl mesoporphyrin IX (NMM), producing a fluorescence signal proportional to the target concentration, making it a new detection method. The conformational dynamics of MB-G4 were characterized through circular dichroism (CD) spectroscopy and native polyacrylamide gel electrophoresis (PAGE), confirming the transition from a hairpin structure to an RNA–DNA hybrid duplex that facilitated G4 formation. The optimization of the experimental conditions, including the potassium chloride (KCl) and NMM concentrations, ensured selective detection with minimal background signal. The detection limit (LOD) was determined to be 10.85 nM, using a linear fluorescence response curve, and the specificity studies demonstrated a clear distinction between miR-155-3p and miR-155-5p. Furthermore, MB-G4 was studied with total RNA extracted from the lung cancer cell line A549 to evaluate its detection in a more complex environment and was able to detect its target, validating its potential for biological sample analysis. Full article

Lead (Pb²⁺) pollution is a serious food safety issue, rapid detection of Pb²⁺ residual in food is vital to guarantee food quality and safety. Here we proposed ratiometric aptamer probes, allowing robust Pb²⁺ supervision in food samples. Pb²⁺ specific aptamer can bolster a transition of G-quadruplex structural response to Pb²⁺; this process can be monitored by N-methyl mesoporphyrin IX (NMM), which is highly specific to G-quadruplex. Particularly, the utilization of G-quadruplex specific dye and terminal-labeled fluorophore allowed to endue ratiometric signal outputs towards Pb²⁺, dramatically increase the robustness for lead detection. The ratiometric G-quadruplex assay allowed a facile and one-pot Pb²⁺ detection at room temperature using a single-stranded DNA aptamer. We demonstrated its feasibility for detecting lead pollution in fresh eggs and tap water samples. The ratiometric G-quadruplex design is expected to be used for on-site Pb²⁺ testing associated with food safety. Full article

Guanine quadruplexes (G4s) serve as regulators of replication, recombination and gene expression. G4 motifs have been recently identified in LTR retrotransposons, but their role in the retrotransposon life-cycle is yet to be understood. Therefore, we inserted G4s into the 3′UTR of Ty1his3-AI retrotransposon and measured the frequency of retrotransposition in yeast strains BY4741, Y00509 (without Pif1 helicase) and with G4-stabilization by N-methyl mesoporphyrin IX (NMM) treatment. We evaluated the impact of G4s on mRNA levels by RT-qPCR and products of reverse transcription by Southern blot analysis. We found that the presence of G4 inhibited Ty1his3-AI retrotransposition. The effect was stronger when G4s were on a transcription template strand which leads to reverse transcription interruption. Both NMM and Pif1p deficiency reduced the retrotransposition irrespective of the presence of a G4 motif in the Ty1his3-AI element. Quantity of mRNA and products of reverse transcription did not fully explain the impact of G4s on Ty1his3-AI retrotransposition indicating that G4s probably affect some other steps of the retrotransposon life-cycle (e.g., translation, VLP formation, integration). Our results suggest that G4 DNA conformation can tune the activity of mobile genetic elements that in turn contribute to shaping the eukaryotic genomes. Full article

The monitoring and control of mycotoxins has caused widespread concern due to their adverse effects on human health. In this research, a simple, sensitive and non-label fluorescent aptasensor has been reported for mycotoxin ochratoxin A (OTA) detection based on high selectivity of aptamers and amplification of non-enzyme hybridization chain reaction (HCR). After the introduction of OTA, the aptamer portion of hairpin probe H1 will combine with OTA to form OTA-aptamer complexes. Subsequently, the remainder of the opened H1 will act as an initiator for the HCR between the two hairpin probes, causing H1 and H2 to be sequentially opened and assembled into continuous DNA duplexes embedded with numerous G-quadruplexes, leading to a significant enhancement in fluorescence signal after binding with N-methyl-mesoporphyrin IX (NMM). The proposed sensing strategy can detect OTA with concentration as low as 4.9 pM. Besides, satisfactory results have also been obtained in the tests of actual samples. More importantly, the thermodynamic properties of nucleic acid chains in the monitoring platform were analyzed and the reaction processes and conditions were simulated before carrying out biological experiments, which theoretically proved the feasibility and simplified subsequent experimental operations. Therefore, the proposed method possess a certain application value in terms of monitoring mycotoxins in food samples and improving the quality control of food security. Full article

An enzyme- and label-free aptamer-based assay is described for the determination of thrombin. A DNA strand (S) consisting of two parts was designed, where the first (Sa) is the thrombin-binding aptamer and the second (Se) is a G-quadruplex. In the absence of thrombin, Sa is readily adsorbed by graphene oxide (GO), which has a preference for ss-DNA rather than for ds-DNA. Upon the addition of the N-methyl-mesoporphyrin IX (NMM), its fluorescence (with excitation/emission at 399/610 nm) is quenched by GO. In contrast, in the presence of thrombin, the aptamer will bind thrombin, and thus, be separated from GO. As a result, fluorescence will be enhanced. The increase is linear in the 0.37 nM to 50 µM thrombin concentration range, and the detection limit is 0.37 nM. The method is highly selective over other proteins, cost-effective, and simple. In our perception, it represents a universal detection scheme that may be applied to other targets according to the proper choice of the aptamer sequence and formation of a suitable aptamer-target pair. Full article

Taking advantage of the high selectivity of aptamers and enzyme-free catalyzed hairpin assembly (CHA) amplification strategy, we herein describe a label-free and enzyme-free sensitive fluorescent and colorimetric strategy for thrombin detection in this paper. In the presence of target, the corresponding aptamer of the partial dsDNA probes will bind to the target and liberate the initiation strand, which is artfully designed as the “on” switch for hairpin assembly. Moreover, the displaced initiation strand partakes in a multi-cycle process and produces numerous G-quadruplexes, which have a remarkable enhancement in fluorescent/colorimetric signal from NMM (N-methyl-mesoporphyrin IX) and TMB (3,3′,5,5′-tetramethylbenzidine), respectively. The proposed amplification strategy for thrombin detection is of high sensitivity, down to 2.4 pM, and also achieves colorimetric signals that are able to be distinguished by naked eye. More importantly, the thermodynamics of interacting DNA strands used in our work, and the process of toehold strand displacement-driven assembly are simulated before biological testing, verifying the feasibility theoretically, and simplifying the subsequent actual experiments. Therefore, our approach and simulation have a certain potential application in biomarker detection and quantitatively monitor for disease diagnosis. Full article