Sensitive Interrogation of Enhancer Activity in Living Cells on a Nanoelectroporation-Probing Platform

Abstract

Enhancers involved in the upregulation of multiple oncogenes play a fundamental role in tumorigenesis and immortalization. Exploring the activity of enhancers in living cells has emerged as a critical path to a deep understanding of cancer properties, further providing important clues to targeted therapy. However, identifying enhancer activity in living cells is challenging due to the double biological barriers of a cell cytoplasmic membrane and a nuclear membrane, limiting the sensitivity and responsiveness of conventional probing methods. In this work, we developed a nanoelectroporation-probing (NP) platform, which enables intranuclear probe delivery for sensitive interrogation of enhancer activity in living cells. The nanoelectroporation biochip achieved highly focused perforation of the cell cytoplasmic membrane and brought about additional driving force to expedite the delivery of probes into the nucleus. The probes targeting enhancer activity (named "PH probe") are programmed with a cyclic amplification strategy and enable an increase in the fluorescence signals over 100-fold within 1 h. The platform was leveraged to detect the activity of CCAT1 enhancers (CCAT1, colon cancer-associated transcript-1, a long noncoding RNA that functions in tumor invasion and metastasis) in cell samples from clinical lung cancer patients, as well as reveal the heterogeneity of enhancers among different patients. The observations may extend the linkages between enhancers and cancer cells while validating the robustness and reliability of the platform for the assay of enhancer activity. This platform will be a promising toolbox with wide applicable potential for the intranuclear study of living cells.

A Nano‐Electroporation‐DNA Tensioner Platform Enhances Intracellular Delivery and Mechanical Analysis Toward Rapid Drug Assessment

“…When performing nano-electroporation, the top electrode (a silver sheet) was assembled with the rest of the structure by the fixation of a support. [35] For assembly of the DNA tensioner in the mechanic's detection chamber, the DNA tensioner (Sequence: 5′-cholesterol&FAM-AAAAAGGGTTAACATCTAGATTCTATTTTTAGAATCTAGATGTTAACCCAA-AAA-BHQ-1-3′) was annealed in a water bath at 95 °C for 10 min, which allows the sequence to fully open its secondary or tertiary structures. Then, the sequence was slowly cooled to room temperature to fold in the least energetic way.…”

“…When performing nano‐electroporation, the top electrode (a silver sheet) was assembled with the rest of the structure by the fixation of a support. [ 35 ]…”

Cell Membrane Perforation: Patterns, Mechanisms and Functions

“…Their work amplified the signal 100 times within 1 h while leaving the cells alive, which the traditional method of cell lysis cannot reach, guiding the decipherer of genetic disorders in cancer. [173] Chen et al constructed carbon nanocoil-based magnetic nanorobots, which targeted and perforated the cancer cell membrane mechanically and then detected the intracellular Raman signals using surface-enhanced Raman spectroscopy (SERS) technology. The intensity of obtained spectra was much higher than without nanorobots, providing a feasible method for bio-sensing and cellular biology measurement [174] (Figure 18).…”