Detection of Specific Nucleic Acids Utilizing DNA Nano-Tweezers Structures
Authors: Funabashi Hisakage , Yoshinaga Keisuke , Shigeto Hajime , Nakatsuka Keisuke , Kuroda Akio 
Published: February 2020

We have developed signal-transducing molecules for detecting specific nucleic acids in a homogeneous assay format. A DNA nano-tweezers (DNA-NT) structure, self-assembled from three single-stranded DNAs, was employed as a main component that alters its structure from an open state to a closed state in recognition of target nucleic acids. This dynamic structural change was utilized for generating a target-specific sensing signal.

 A fluorescence resonance energy transfer (FRET)-paired fluorescent dyes were modified onto a DNA-NT to generate a target-specific sensing signal. This FRET-based DNA-NT alters its structure in response to target nucleic acids, resulting in the closing of the distal relation of FRET-paired fluorescent dyes. The FRET-based DNA-NT generates the FRET signal only when it recognizes target nucleic acids, and thus the target detection can be performed in a homogeneous assay format, which does not require washing procedure. Therefore, when the FRET-based DNA-NT is introduced into a cell, the continuous detection of specific target mRNA inside living cells can be realized in a non-destructive manner. Live-cell imaging analysis of drug stimulated cells with the FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those of target mRNA measured with the conventional mass analysis technique of semi-quantitative real-time polymerase chain reaction. Thus, it was concluded that continuous monitoring of specific genes is feasible with a FRET-based DNA-NT, even in a single cell manner.

 As another example of a component for signal generation, a G-quadruplex (Gq) composed a guanine-rich DNA sequence was employed. A Gq is one of the well-characterized DNazymes that exhibits a peroxidase activity with the help of hemin as a co-factor. In our design, the split Gq sequences were added to both ends of a DNA-NT instead of the FRET-paired fluorescent dyes. This split-Gq-based DNA-NT alters its structure from an open state to a closed state, inducing the complementation of the split Gq that exhibits restored peroxidase activity. A model target RNA was specifically recognized by the split Gq-based DNA-NT. The peroxidase activity was measured by following the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the concentration of model target RNA. The split-Gq-based DNA-NT generates a peroxidase activity only when it recognizes target nucleic acids, and thus the target detection can be performed in a homogeneous assay format that is suitable for a portable format. Therefore, the detection of specific nucleic acids with the split Gq-based DNA-NT has great potential for use in a wide variety of applications, such as point-of-care testing and point-of-need testing.

 The DNA-NT-based signal-transducing molecules, enabling the detection of target nucleic acids in a homogeneous assay format, recognize the target without competitive reactions in a bimolecular reaction and directly produce a readily detectable signal. Notably, the target recognition sites of DNA-NT-based signal-transducing molecules can easily be designed without delicate optimization of tweezers structure. Therefore, the DNA-NT-based signal-transducing molecules are expected to become a versatile tool for detecting specific nucleic acids.