RESUMO
Regulation of gene expression is highly important in the area of cell biology. In this work a novel convenient and versatile strategy is reported which permits both gene regulation and imaging in living cells. An oligonucleotide-based biomimetic probe was designed to target an RNA-induced silencing complex (RISC) and served as an agent for the modulation of c-Myc protein expression in living cells through regulating the RNA interference (RNAi) pathway. In this probe, a DNA strand (Strand1) serving as the frame was immobilized on a AuNP with a thiol group at the 5' end. Strand2, designed to recognize the target RISC with an RNA fragment, was hybridized with the complementary sequence of Strand1. In the original state, the fluorescence of the Cy3 modifier at the 5' end of Strand2 was quenched by both the AuNP and BHQ2, which labelled the 3' end of Strand1. In the presence of RISC, Strand2 was cleaved, resulting in a shorter oligo part with a corresponding lower melting temperature than that of the original full-length Strand2. The shorter oligonucleotide strand containing the Cy3 fluorophore was released, accompanied by a recovered fluorescence signal. Through evaluating the fluorescence intensity, the competition for RISC was dynamically monitored in single cells. Furthermore, capturing RISC by this probe resulted not only in restored fluorescence intensity but also increased c-Myc oncogene expression. Hence, gene expression could be selectively and precisely regulated and imaged via the RISC targeting probe. The synthetic method for the biomimetic probe is universally applicable, and facilitates the fundamental study of RNAi pathways, or development of a gene regulation strategy without cytokine activation. The gene regulation and imaging strategy will accelerate the unveiling of the basic role of the RISC cleavage interaction, the mystery of RNA-silencing and therapeutic monitoring of cancer.
RESUMO
Bimetallic Au@Pt@Au triple-layered core-shell nanoparticles consisting of a Au core, Pt inner shell, and an outer shell composed of Au protuberances on graphene oxide (GO) nanosheets were successfully prepared by a galvanic replacement and reagent reduction reaction. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and cyclic voltammetry (CV) were employed to characterize the GO-supported Au@Pt@Au (GO/Au@Pt@Au) nanocomposites. The as-prepared catalyst has peroxidase-like activity, allowing it to express high electrocatalytic ability in hydrogen peroxide (H2O2) oxidation and reduction, thus leading to a highly sensitive H2O2 bi-directional amperometric sensing. The bi-directional sensor showed a linear range from 0.05 µM to 17.5 mM with a detection limit of 0.02 µM (S/N = 3) at an applied potential of +0.5 V and a linear range from 0.5 µM to 110 mM with a detection limit of 0.25 µM (S/N = 3) at an applied potential of -0.3 V. The proposed sensor was tested to determine H2O2 released from living cells and shows good application potential in biological electrochemistry.