Design of Split G-quadruplex-based DNA–Bridged Nucleic Acid Chimera Nanotweezers That Recognize Short Nucleic Acids with a Single-base Mismatch

The design and feasibility of split G-quadruplex-based DNA nanotweezers (split Gq-based DNA-NTs) that can recognize short nucleic acids with a single-base mismatch are discussed. The split Gq-based DNA-NTs consist of three single-stranded DNA sequences forming a tweezers shape with split Gq sequences at the edge of each arm.


Introduction
The point-of-care testing (POCT) of nucleic acids has wide applications in fields such as diagnostics and food safety. (1)(2)(3) Nucleic acids consist of simple combinations of four bases, and thus, a difference of even one base can result in a huge difference in function. Therefore, the discrimination of nucleic acids with a single-base mismatch is an important topic. MicroRNAs (miRNAs), one of the representative nucleic acids that exhibit a significance difference in function based on single-base differences, are noncoding small RNA molecules composed of approximately 20-30 nucleotides. Correlations have been found between many diseases such as cancer (4)(5)(6) and miRNAs in bodily fluids including blood, urine, tears, and saliva. (7,8) Accordingly, miRNAs have attracted attention as promising biomarkers for POCT in many diseases because they can be collected with no or low invasiveness. However, conventional methods for detecting nucleic acids such as reverse transcription polymerase chain reaction (RT-PCR)-based methods are not ideal for POCT because they often require complex procedures and expensive instruments. Thus, it is necessary to develop a simple method for detecting miRNAs, even those with a single-base mismatch in order to use miRNAs as biomarkers in a POCT format.
We have been developing a DNA nanotweezer technique for detecting nucleic acids. (9)(10)(11) A tweezer shape made via the self-assembly of three single-stranded DNA oligonucleotides alters its structure from an open state to a closed state after recognizing a target nucleic acid with two arms, inducing the edges of each arm in a proximity relationship. The split G-quadruplex-based DNA nanotweezers (split Gq-based DNA-NTs) possess split Gq sequences (12)(13)(14)(15) at the edges of each arm. In response to target recognition, the split Gq-based DNA-NTs drag the split Gq sequences in a proximity relationship, and then the split Gq sequences recover their ability to form a Gq/hemin complex, which exhibits peroxidase activity. (16,17) Thus, a simple homogeneous assay with a signal based on the peroxidase activity for the detection of nucleic acids can be realized by utilizing split Gq-based DNA-NTs. (10) Because of a simple structure and principle, the split Gq-based DNA-NTs and their derivatives have also been utilized by other research groups for the detection of a protein (18) and applied to the electrochemical detection of target nucleic acids. (19)(20)(21) However, the target recognition ability of split Gq-based DNA-NTs has not been precisely investigated yet. Specifically, it is unclear whether they can recognize short nucleic acids with a single-base mismatch. Therefore, to apply this DNA-NT technique to the detection of short miRNAs with a single-base mismatch, it is necessary to discuss the design of target recognition sites in its structure.
In this study, we demonstrated the feasibility of DNA-NTs to distinguish miRNAs with a single-base mismatch by controlling the melting temperature (Tm) at the target recognition sites of DNA-NTs utilizing a bridged nucleic acid (BNA). (22) A BNA is an artificial nucleic acid possessing a rigid structure attributed to a bridge between the 2′-and 4′-positions of the ribose, and it exhibits a higher affinity for its complementary base than native nucleic acids such as DNA and RNA. Therefore, by inserting a BNA at the target recognition site of mismatched sequences, it is expected that the affinity of split Gq-based DNA-NTs for a specific miRNA will increase, permitting the discrimination of single-base mismatched miRNAs (Fig. 1). In this study, we designed a strategy to distinguish let-7a and let-7c (23,24) as a single-base mismatched model to conceptually prove the feasibility of the split Gq-based DNA-NT technique for miRNA detection.

Materials
All nucleotide sequences used in this study are listed in Table 1. Synthetic DNA and RNA were purchased from Integrated DNA Technologies (MBL, Nagoya, Japan). DNA-BNA (22) chimera oligonucleotides were synthesized by GeneDesign, Inc. (Osaka, Japan). Among several different types of BNAs, we selected BNA NC because it was expected to exhibit the highest Tm according to the manufacturer. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hemin were purchased from Wako (Osaka, Japan).

Evaluation of effect of Tm at target recognition site on target recognition ability of split Gq-based DNA-NTs
The effect of Tm at the target recognition site on the target recognition ability of split Gqbased DNA-NTs was evaluated by measuring its peroxidase activity. The length of the target recognition site in one arm of the FRET-based DNA-NTs for Hes-1, (9) which was previously identified as valid DNA-NTs, was gradually reduced, after which its peroxidase activity was measured as described elsewhere. The Tm for each target recognition site was estimated using the following equation: Tm = 4 °C × (number of G + C in target recognition site) + 2 °C × (number of A + T in target recognition site).  (9) CTCAACTTTTATAATACAAATACATTTTACGCCTGGTGCC O2 (Hes-1) (9) CCGACCGCAGGATCCTATAAGGCGCAATCCAATAT O2-14 base CCGACCGCAGGATCCTATAAGGCGCAATCCAATA O2-13 base CCGACCGCAGGATCCTATAAGGCGCAATCCAAT O2-12 base CCGACCGCAGGATCCTATAAGGCGCAATCCAA O2-11 base CCGACCGCAGGATCCTATAAGGCGCAATCCA O2-10 base CCGACCGCAGGATCCTATAAGGCGCAATCC O2-9 base CCGACCGCAGGATCCTATAAGGCGCAATC CCGACCGCAGGATCCTATAATaCTACCTCA Target (Hes-1) (9) ATATTGGATTGCGCCTTTGTATTATAAAAGTTGAG Target (let-7a) (23,24) UGAGGUAGUAGGUUGUAU A GUU Target (let-7c) (23,24) UGAGGUAGUAGGUUGUAU G GUU Nucleic acids colored in red in O1 sequences are the target recognition sites hybridized to the underlined red parts in the corresponding targets. Bold lowercase letters in O1 (let-7a) and O1 (let-7c) denote BNAs. The bold characters in Target (let-7a) and Target (let-7c) are mismatched points hybridized to BNAs. Nucleic acids colored in blue in O2 sequences are the target recognition sites hybridized to the underlined blue parts in the corresponding targets. Bold lowercase letters in O2 (let-7a, let-7c) denote BNAs. The green color in O3 indicates the split sequences of the G-quadruplex. The black parts in O1, O2, and O3 were hybridized to form the DNA nanotweezer shape.

Measurement of peroxidase activity (10)
The peroxidase activity of each DNA-NT with target recognition sites of various lengths was measured via a typical colorimetric method utilizing 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS). The DNA-NTs (final, 50 nM), target (final, 50 nM), and hemin (final, 1 μM) were mixed in a working buffer (50 mM Tris-HCl, 150 mM NH 4 Cl, 20 mM KCl, and 0.03% Triton X-100, pH 7.5) to a total volume of 89 μL and incubated for 1 h at 25 °C. Then, 1 μL of ABTS (final, 2 mM) was added to the reaction mixture before transferring the reaction mixture to a 96-well clear-bottomed plate. A colorimetric reaction was started by the addition of 10 μL of H 2 O 2 (final, 2 mM) to a final volume of 100 μL. The absorbance at 420 nm was measured immediately at 10 s intervals using a Spectra Max M5 (Molecular Devices Japan, Tokyo, Japan).

Results and Discussion
Split Gq-based DNA-NTs recognize target nucleic acids with target recognition sites encoded in both arms and exhibit peroxidase activity. To discriminate miRNAs with a single-base mismatch, it is necessary to control the peroxidase activity between the "on" and "off" states according to the single-base difference. Therefore, we first determined the Tm condition at target recognition sites to eliminate their peroxidase activities by shortening the length of one arm in the split Gq-based DNA-NTs for Hes-1 as a model. As shown in Fig. 2, the split Gq-based DNA-NTs did not exhibit peroxidase activity when the Tm of the target recognition site in one arm was lower than 30 °C. This temperature was slightly higher than room temperature. It was suggested that split Gq-based DNA-NTs tend to maintain an open status due to the steric obstacle of the hinge and/or the repulsion of their arms because of the negative charge of the DNA backbone. (10) Thus, a higher Tm, implying a higher affinity, may be needed to bind to a target to maintain the closed status at temperatures exceeding room temperature.  Typically, miRNAs are approximately 20-30 nucleotides in length. Therefore, it is difficult to guarantee that the Tm at the target recognition sites of DNA-NTs will exceed 30 °C because the target should be divided into two sequences that are approximately 10-15 nucleotides in length. In fact, the Tm of the front halves of let-7a and let 7c, which have identical 10-nucleotide sequences, was estimated to be 28 °C. Therefore, we added one BNA to increase the Tm of the target recognition sites of both chimeric DNA-NTs (Fig. 3). Then, we determined whether the DNA-BNA chimera NTs with a BNA at the mismatched point respond to the corresponding target but not to a target with a single-base mismatch. Namely, the DNA-BNA chimera NTs would alter their structure through the hybridization of specific miRNAs with the target recognition site, leading to a closed status that exhibits peroxidase activity ("on"). Moreover, an open status lacking peroxidase activity ("off") would be maintained against other miRNAs with a single-base mismatch because of the lower affinity. In fact, as shown in Figs  The two DNA-BNA chimera NTs exhibited a difference in peroxidase activity. This might be caused by the difference in the type of inserted BNA base that recognizes each mismatched point in the targets. The 19th base of let-7a is adenine (A), whereas that of let-7c is guanine (G). Accordingly, the BNAs introduced into the target recognition sites were thymine (T) and cytosine (C), respectively. Generally, the affinity between G and C is higher than that between A and T in the case of Watson-Crick base pairing. Thus, the Tm against let-7c is higher than that against let-7a in the case of the DNA-BNA chimera NTs. This difference in Tm was considered to explain the difference in peroxidase activity against the corresponding targets. Nevertheless, it has been successfully demonstrated that split Gq-based DNA-BNA chimera NTs have a potential to discriminate miRNAs with a single-base mismatch.

Conclusions
In this study, we have conceptually proved the feasibility of detecting miRNAs with a singlebase mismatch using split Gq-based DNA-BNA chimera NTs that offer a convenient method for detecting nucleic acids using peroxidase activity in a homogeneous assay format. Although the detection sensitivity should be further considered, many methods that are capable of amplifying the number of specific nucleic acids under isothermal conditions are now available. (25)(26)(27)(28) Also, in addition to a colorimetric method, other methods such as electrochemical detection with split Gq-based DNA-NTs were reported. (19)(20)(21) Therefore, by the combination of these methods, it is expected that split Gq-based DNA-BNA chimera NTs will be applicable to the development of POCT devices for the detection of a wide variety of nucleic acids as target recognition and signal generation elements.