Endogenous Adenosine Triphosphate-Assisted Three-Dimensional DNA Walker Assembled on Soft Nanoparticles for Intracellular MicroRNA Imaging

Abstract

DNA walkers, a sophisticated type of nanomachines, exhibit intelligent application in biosensing with high programmability and flexibility but usually need additional auxiliary driving force, particularly when walking on hard surfaces. Herein, we construct a three-dimensional (3D) DNA walker on the soft surface of DNA nanospheres (DSs) by using a single-stranded DNA (ssDNA), which is powered by endogenous adenosine triphosphate (ATP) of live cells, so as to sensitively image microRNA (miRNA) in the tumor microenvironment. When the DS walker enters into live cells, miR-21, a general overexpressed biomarker in cancer cells, binds with the blocking strand (B), releasing the walking strand (W) and triggering an ATPpropelled walking reaction. The walking of the DS walker then generates an increasing Cy3 fluorescence signal that indicates the content of miR-21 with about 2.73-fold increase in sensitivity and about 157-fold decrease in the detection limit. Notably, the assembly of the DS walker on soft nanoparticles needs just an easy hybridization process, which facilitates the operation. Meanwhile, this endogenous ATP-powered 3D DNA walker walking on the soft surface performs real-time in situ imaging of miR-21 in live cells, which not only avoids the complex cell treatment and signal error induced by additional auxiliary factors, but also shows high promise of designing programmable DNA nanomachines.

Advances in DNA walking nanomachine‐based biosensors

“…Liang and co-workers developed a 3D DNA walker by employing singlestranded DNA on the surface of DNA nanospheres (DS). 97 This unique system, powered by endogenous ATP obtained from living cells, enabled sensitive imaging of miR-21 in the tumor microenvironment (Figure 6A). Upon entering a living cell, the DS walker bound to the intended target, released the walking strand, and initiated an ATP-driven walking response.…”

“…101 This investigation achieved ultrasensitive detection of HBV DNA within a linear range of 100 aM to 10 pM, with a LOD as low as 62.1 aM. Impressively, this innovative approach involved a novel aluminium metal-organic backbone that exhibited excellent ECL properties, characterized by high intensity and exceptional stability even 97 Copyright 2023, American Chemical Society. (B) DNA walking nanomachine-based fluorescent biosensors for tumor exosome detection.…”

Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells

“…37−42 Among them, the DNA walker is more suitable for intracellular imaging, and it can perform autonomous movement along the trajectory to achieve cascade signal amplification. 43,44 Three-dimensional (3D) DNA walker assembled from a combination of nanomaterials and functional nucleic acids displays a large surface-to-volume ratio and high DNA loading capacity, endowing them with high amplification efficiency and high signal output. 45 An ideal 3D DNA walker requires appropriate driving forces to achieve the efficient sensing of target analytes within living cells.…”

“…In order to overcome these issues, endogenous substance (e.g., ATP)driven DNA nanomachine has been engineered. 43 The utilization of cellular ATP as a driving force simplifies the operation of DNA nanomachines, but the weak interaction of ATP with its aptamer may impair the operational efficiency of DNA nanomachines inside living cells, restricting their applications in rapid intracellular imaging. As an alternative, endogenous apurinic/apyrimidinic endonuclease 1 (APE1) can hydrolyze apurinic/apyrimidinic (AP) sites in the base excision repair pathway.…”

“…Although DNAzymes and nucleases , with rapid cleavage capability can significantly increase the walking speed of DNA walker, enzyme-powered DNA walkers generally involve the transfection of auxiliary metal ions and exogenous enzymes, complicating the cell treatment and making it difficult to manipulate the spatiotemporal distribution of core components of DNA walker in live cells, and consequently increasing the risk of signal bias. In order to overcome these issues, endogenous substance (e.g., ATP)-driven DNA nanomachine has been engineered . The utilization of cellular ATP as a driving force simplifies the operation of DNA nanomachines, but the weak interaction of ATP with its aptamer may impair the operational efficiency of DNA nanomachines inside living cells, restricting their applications in rapid intracellular imaging.…”