X. Cui,Y. Zhou,Y. Zheng,L. Cao,L. Gao,J. Duan,H. Yin,S. Ai
N 6 -methyladenosine (m 6 A) is the most abundant and dynamic reversible modification of mRNA in eukaryotes discovered in the mid-1970s, which is widely involved in various physiological processes. Fat mass and obesity-associated protein (FTO protein) is not only a demethylase for m 6 A, but also a carcinogenic factor for many human cancers. Therefore, it is very important to build a simple and sensitive platform for simultaneous detection of m 6 A and FTO protein. To achieve this aim, a novel photoelectrochemical biosensor was constructed with the antibody-free detection strategy of DTT mediated –SH functionalization of m 6 A triggered by FTO protein catalyzed m 6 A oxidation . To achieve high detection sensitivity, CdS/Bi 2 MoO 6 /MoSe 2 system with matched energy gap was employed as photoactive material. To achieve high detection selectivity , the –CH 3 of m 6 A in the captured RNA sequence (m 6 A RNA) on the electrode surface was oxidized by FTO protein to form hydroxymethyl, followed with the covalent reaction of DTT with hydroxymethyl, making the labeling of m 6 A with –SH. Finally, a CdS nanoparticle was modified on the electrode through the specific covalent reaction between –SH and Cd, achieving the improvement of the photocurrent with the matched energy band . Under the optimal experimental conditions, the biosensor showed linear range from 0.001 to 50 nM and 0.0005–500 μg/L with the detection limit of 0.37 pM and 0.034 ng/L (S/N = 3) for m 6 A RNA and FTO protein, respectively. The applicability of the developed method was assessed by investigating the effect of entacapone on FTO protein activity.
Genetic engineering faces persistent challenges in achieving precise Deoxyribonucleic acid (DNA) cleavage, especially with the limitations associated with current enzyme-based methods, exemplified by issues in CRISPR technologies. This study introduces a groundbreaking approach: utilizing reactive oxygen species (ROS) generated by Multiphoton Absorption (MPA)-excited Black Phosphorus Quantum Dots (BPQDs) under femto-second laser irradiation. This innovative method not only allows excitation with lower energy but also enhances overall efficiency. The integration of complementary RNA sequences facilitates high-efficiency, site-selective DNA cleavage in the system, named “TADPOLE” (Targeted DNA Precision Oriented Laser Excision). Beyond its precision in targeting arbitrary DNA sequences using quantum dots, TADPOLE harnesses the unique multiphoton absorption property of BPQDs, enabling excitation with lower-energy light sources suitable for in vivo applications in the future. Moreover, the approach integrates guiding RNA and ultrafast laser technology to provide precise control over local ROS generation and minimal heat production. This guarantees site-specific DNA cleavage while mitigating the risk of damage to non-targeted sequences. In summary, this study catalyzes advancements in enzyme-free DNA cleavage technologies, with transformative implications for genetic engineering, biotechnology, and medicine. The holistic precision, versatility, and endurance presented by TADPOLE open new avenues for targeted gene therapies and transformative applications in related fields.
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