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

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.

Keywords: DNA molecular machine; cycling amplification reaction; direct visualization; rolling circle amplification; total internal reflection fluorescence microscopy.

Scheme 1

The scheme of the DNA molecule machine with three phases. Half-arrows on the strands indicate the extending direction of the polymization. Small arrows in the structure of the species indicate the potential nicking site.

Figure 1

Products of the molecular machine at three phases. (A) TIRFM visualization: (a) Phase A, simple RCA (rolling-circle amplification); (b) Phase B, RCA cycled and amplified by TDP (target-displacement polymerization); (c) Phase C, the full molecular machine, RCA cycled by TDP which was further cycled and amplified by NPC (nicking-polymerization cycle). (B) Counting results of the dots and thread-like objects.

Figure 2

Products of the molecular machine when different lysozyme concentrations were applied. (A) TIRFM visualization: (a) blank, (b) 10⁻¹³ M (c) 10⁻¹² M (d) 10⁻¹¹ M (e) 10⁻¹⁰ M (f) 10⁻⁹ M; (B) Counting results of the dots and thread-like objects.

Figure 3A

Figure 3A. Products of the molecular machine when different reaction time. (A) 0.5h, (B) 1.0h, (C) 2.0h, (D) 3.0h.

Figure 3B

Figure 3B. Products of the molecular machine when different reaction time. (A) TIRFM visualization. (a) 0.5h, (b) 1.0h (c) 2.0h (d) 3.0h; (B) Counting results of the dots and thread-like objects.