Direct visualization of the reaction transformation and signal amplification in a DNA molecular machine with total internal reflection fluorescence microscopy.

Rui Ren,Rui Liu,Shusheng Zhang,Haiyan Wang

doi:10.3389/fchem.2013.00023

Abstract

In this study, as a proof of concept, the signal amplification in an artificial DNA molecular machine was directly visualized via total internal reflection fluorescence microscopy (TIRFM). The molecular machine brought about obvious morphology change in DNA nanostructures as well as signal amplifications. On one hand, through a triggered and autonomically repeated RCA, a DNA nano-complex featuring a “locked” circular DNA template (serving as raw feed) was converted into a long periodically repeated strand, i.e., the RCA products. On the other hand, this RCA was repeated in three controllable reaction phases, bring about progressive signal amplification. It was testified that the RCA products (presented as long thread-like fluorescent objects) can be easily distinguished from the inputted DNA probes (presented as fluorescent dots), thus the transformation in reaction can be visualized. Also, by quantitive counting of the aforementioned fluorescence objects, the progress of the reaction through the phases, along with time, and over the lysozyme concentration can be demonstrated through TIRFM visualization. Overall, it was demonstrated that TIRFM is an efficient approach to quantitatively visualize the biochemical processes at single-molecule level.

Highlights

In recent years, artificial DNA molecular machines have attracted extensive attention for they provided effective signal amplification, which is always highly desirable in bioanalysis
As for nicking-polymerization cycle (NPC) (Weizmann et al, 2006; Beissenhirtz et al, 2007; Shlyahovsky et al, 2007) developed by Itamar Willner’s group, a double-stranded DNA with a nicking site served as the starting structure; in the presence of polymerase and nicking endonuclease that is specific to the nicking site, ssDNA strands can be repetitively produced and released
There was target-displacement polymerization (TDP), in which the starting structures included complex of a non-nucleic acid target and its aptamer strand and a primer that binds to the former; and in the polymerization that use the aptamer strand as the template, the target was replaced and released, so that it could be recycled (Ren et al, 2012); two or more DNA molecular machines can be interconnected: the product or the intermediate of one machine can act as the “trigger” of another, resulting in dual signal amplification: the downstream cycle poses a further amplification upon the amplification in the upstream cycle (Connolly and Trau, 2010; Ren et al, 2012; Zhao et al, 2013)

Summary

Introduction

Artificial DNA molecular machines have attracted extensive attention for they provided effective signal amplification, which is always highly desirable in bioanalysis. When the stimuli are inputted nucleic acid strands or other biological molecules (to be detected), and when the output was multiplied DNA segments that served as signal, the molecular machine fulfilled the signal amplification function. There was target-displacement polymerization (TDP), in which the starting structures included complex of a non-nucleic acid target and its aptamer strand and a primer that binds to the former; and in the polymerization that use the aptamer strand as the template, the target was replaced and released, so that it could be recycled (Ren et al, 2012); two or more DNA molecular machines can be interconnected: the product or the intermediate of one machine can act as the “trigger” of another, resulting in dual signal amplification: the downstream cycle poses a further amplification upon the amplification in the upstream cycle (Connolly and Trau, 2010; Ren et al, 2012; Zhao et al, 2013)

Methods

Results

Conclusion