Engineering of An Aptamer-Functionalized Fluorescent DNA Sensor for Cu(II) Responding in Living Tumor Cells

Abstract

Copper ions play vital roles in regulating life processes and being closely involved in several diseases such as cancer. Although detection methods based on fluorescent sensors or other strategies have been developed, it still remains a challenge to simultaneously realize the convenience, specificity, and accuracy in intracellular copper ion analysis. Herein, we propose an aptamer-functionalized DNA fluorescent sensor (AFDS) for accurate and specific detection of Cu(II) both in vitro and in cells by engineering the linkage of two DNA aptamers, namely, Lettuce aptamer and AS1411 aptamer, to achieve the manner of recognition response. Taking advantage of the functions of each aptamer, the tumor cell recognition capability and the high-contrast detection performance are simultaneously equipped in the AFDS. Furthermore, the AFDS shows high specificity and selectivity in Cu(II) response to avoid interference from common metal ions, chelators, and reactants by being associated with the irreversible interaction between nucleobases and Cu(II), which can destroy the topological structures and switch off the fluorescence of the AFDS. It also enables a sensitive in vitro detection of Cu(II) with a detection limit as lower as 0.1 μM and a wide detection linear range from 0.1 to 300 μM. The feasibility and superiority of the AFDS provide an opportunity to reveal both concentrationdependent and time-dependent intracellular Cu(II) responses in living cells. Therefore, the AFDS has achieved the novel detection performance of Cu(II) to exhibit great potential in exploring copper-related biological and pathological research.

Binary split fluorescent biosensor based on lettuce DNA aptamer for label-free and enzyme-free analysis of hepatitis B viral DNA

“…Meanwhile, the inconspicuous G4 topology could be detected in HDs, demonstrating G4 topology formation in the triplex DNA structure, further forming the G4 cavity for DFHO insertion. 24,28,35 In addition, a G4 ligand named 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4) for competing with DFHO and urea reagent for disrupting G4 cavity were severally adopted in bsFDA. 36,37 As shown in Fig.…”

“…Currently, optical biosensing assays based on uorescent light-up aptamers (FLAPs) have been regarded as facile, programmable, and label-free detection toolkits for the evaluation of multiple targets and sensing of, for example, metal ions, metabolites, and nucleic acids. [24][25][26][27][28] Among them, the genetically encoded RNA FLAPs such as spinach, broccoli, and corn have been created for specic binding and lighting up the protein chromophore (HBI) derivatives, leading to a robust antiinterference capability and specicity to serve as uorescent protein mimics, which are being applied in cellular imaging. [29][30][31] To further avoid their susceptibility to degradation and reduce the misfolding in RNA FLAPs, the lettuce aptamer, a guanine (G) base-rich DNA sequence, has been selected and optimized.…”

“…32 Besides, compared with the RNA FLAPs, it presents preferable superiority in terms of high structural stability, portability, and low cost in molecule detection. 24,33 Notably, the sequence alignment of lettuce aptamer can be further optimized to explore the target-responsive fluorescence signals after understanding the three-dimensional (3D) architecture of the lettuce aptamer and the principle of its specific molecular binding mode. 34 Thus, it can be assumed that rational sequence transformation in the lettuce aptamer can be designed to realize certain HBV DNA genome segment activation.…”

Recent Advances in Biological Applications of Aptamer-Based Fluorescent Biosensors

“…Mou et al reported an aptasensor that can detect cancer cells as well as analyze Cu 2+ ions, an important trace element in living cells, by combining the AS1411 aptamer, which can specifically recognize cancer cells, and the Lettuce aptamer, an FLAP that induces the fluorescence of DFHBI-1T [48]. The AS1411 aptamer has a G-quadruplex structure with a G-rich sequence.…”

Intracellular Expression of a Fluorogenic DNA Aptamer Using Retron Eco2

“…3,13 For example Lettuce, a DNA FLAP that mimics the fluorescent properties of GFP holds great promise for in pathogen detection or as intracellular sensors for tumor cell recognition. 14–16 However, since most DNA aptamer studies focus on their development and characterization in vitro, their potential for applications inside living cells remains largely unexplored. A particular challenge in this context is the difficulty of biosynthesising DNA aptamers in cells.…”