2021 年 3 巻 3 号 p. 102-105
Simple, rapid, and accurate diagnosis is indispensable for broadercontrol measures, search for routes of infection, and understanding treatment status of emerging infectious diseases. Although PCR and LAMP are commonly used, in cases where the pathogen is an RNA virus, there are issues in terms of simplicity and speed, as multi-step operations including reverse transcription (RT) and many reagents, as well as time and expertise in optimizing primers and probes, are required. To solve these issues, we propose the artificial chaperone-enhanced MNAzyme (ACEzyme), which combines the functions of the multicomponent nucleic acid enzyme (MNAzyme) and cationic copolymers. PLL-g-Dex has artificial nucleic acid chaperone activity and can act as a molecular sensing system. ACEzyme resulted in an approximately 2,700-fold increase in MNAzyme activity. In this study, we showed that ACEzyme was also useful when working with RNA targets with robust high-order structures, and had high selectivity for single nucleotide mutation detection.
• MNAzyme has attracted attention as a new nucleic acid-based RNA-detection method, and PLL-g-Dex, a cationic copolymer, improved the activity of MNAzyme by approximately 1,000-fold.
• PLL-g-Dex maintained MNAzyme activity against RNA targets with robust higher order structures.
• Functionally enhanced MNAzyme by PLL-g-Dex showed high sequence selectivity toward single-nucleotide mutations.
The spread of emerging infectious diseases, such as coronavirus infection (COVID-19), has caused enormous damage to people’s health and livelihoods. A simple, rapid, and accurate diagnosis is indispensable for broader control measures, search for routes of infection, and understanding the treatment status of emerging infectious diseases [1, 2]. Nucleic acid-based detection methods have been developed as methods that can respond more quickly than immunoassay-based methods requiring the establishment of antibodies; PCR- and LAMP-based methods have also been utilized [3, 4]. However, in cases where the pathogen is an RNA virus, multi-step operations including RNA isolation and reverse transcription (RT) as well as many reagents are required, which complicates the test protocol and leads to contamination [5, 6]. In addition, low stability of protein reagents, such as reverse transcriptase and DNA polymerase, during the storage is also an issue. Since the design of primer nucleic acids and probe nucleic acids requires time and a high degree of expertise, further progress is required for enhanced rapidity. Therefore, establishment of a method based on new principles is urgently needed.
MNAzyme (Fig. 1A) [7] is an allosteric nucleic acid enzyme, which is activated by binding to a target nucleic acid, and has been studied as a new nucleic acid-based detection method. It consists of two partzymes, each containing a substrate-binding arm (S-arm), a partial catalytic core, and a target-binding arm (T-arm). When a couple of the partzymes binds to the target and substrate at the T-arm and S-arm, respectively, an active MNAzyme capable of catalytically cleaving the substrates is assembled (Fig. 1A). The cleavage of substrates can be detected isothermally using fluorophotometry. MNAzyme can amplify the RNA signal without using the reverse transcription reaction, and its storage stability is high as labile reagents, such as protein enzymes and unstable chemicals, are not required. In addition, designing of an MNAzyme is simple, owing to which an MNAzyme against an emergent pathogen can be readily prepared, making it a promising tool for RNA detection.
MNAzyme activity toward RNA targets with robust intramolecular structures. (A) Sequence and location of fluorophore and quencher on 10–23 MNAzyme used in the experiment. Target intramolecular structure and Tm were calculated using DINAmelt; calculation conditions: RNA, 1 M Na+, 0 M Mg2+, and 37°C. (B) Chemical structure of PLL-g-Dex. (C) Percent substrate cleavage to E6-33 (33 nt, green line) or E6-82 (82 nt, orange line) with time in the absence (N/P=0, dotted line) and presence (N/P=2, solid line) of PLL-g-Dex. Experimental conditions: 20 nM MNAzymes, 100 nM substrate, 1 nM E6-33 or E6-82, PLL-g-Dex N/P=0 or 2, 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, and 37°C.
Further improvement in the reactivity and thereby the sensitivity of MNAzymes is desired. We have previously developed a cationic copolymer, PLL-g-Dex (Fig. 1B), with nucleic acid chaperone activity. The copolymer improved the activity of MNAzyme by nearly 1,000-fold with only trace amount added to the reaction solution [8]. In addition, we further optimized the MNAzyme structure and observed that the detection sensitivity and selectivity can be improved in the presence of PLL-g-Dex [9, 10]. Incorporation of LNA, an artificial nucleic acid, to the T-arm, and shortening the S-arm of the MNAzymes, cooperatively improved its activity with the aid of the copolymer. In addition, the shortening of the substrate-binding arm led to lowering of the optimal temperature for MNAzyme to close to the ambient temperature, indicating the possibility of an on-site detection deployment. Such functionally enhanced MNAzyme (Artificial Chaperone-Enhanced MNAzyme (ACEzyme)) is promising as a sensing molecular system for simple, convenient, and sensitive detection platforms that can also respond immediately to RNA viruses, such as SARS-CoV-2.
In this study, the possibility of using ACEzyme for RNA detection was investigated. ACEzyme has already been shown to be able to detect short RNAs such as microRNAs (miRNAs) [9, 10]. Here, we examined whether it can also detect longer RNA targets with a more robust higher order structure than miRNAs. Sequence variation in pathogenic RNAs also leads to changes in symptoms and infectivity, as well as response to therapeutic agents and vaccine resistance [11, 12]. Therefore, identification of mutant pathogens is also important for an accurate diagnosis of infectious diseases. Hence, we also evaluated the ability of ACEzyme to discriminate between single nucleotide mutations.
Poly (L-lysine hydrobromide) (PLL-HBr, Mw=7.5 × 103) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextran (Dex, Mw=8.0 × 103–1.2 × 104) was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Sodium hydroxide, sodium chloride, and manganese (II) chloride tetrahydrate were purchased from Wako Pure Chemical Industries (Osaka, Japan). HEPES (2-[4-(2-Hydroxyethyl) piperazin-1-yl]ethanesulfonic acid) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). LNA-modified DNA was purchased from GeneDesign, Inc. (Osaka, Japan), and other unmodified DNAs and RNAs were purchased from Fasmac Co., Ltd. (Kanagawa, Japan). All oligonucleotides were purified using HPLC.
Synthesis of poly (L-lysine)-graft-dextranPoly (L-lysine)-graft-dextran (PLL-g-Dex) was synthesized by reductive amination of PLL and dextran according to a previously reported protocol [13]. The obtained copolymers were purified by ion exchange, dialyzed, and freeze-dried. Subsequently, the product characteristics were confirmed by 1H NMR (Avance 400, Bruker, Billerica, MA, USA) at 60°C as well as by GPC (Gel Permeation Chromatography) (865-CO RI-4030 PU-2080, Jasco, Tokyo, Japan). In this study, PLL-g-Dex composed of 10 wt% PLL and 90 wt% dextran (11.5 mol% of the lysine units of PLL were modified with dextran) was used.
MethodsBased on previous reports, the catalytic core structure of 10–23 MNAzyme was used in this study [6,7,8]. Substrate cleavage by MNAzyme was analyzed using the Förster resonance energy transfer (FRET) method. First, the substrate (100 nM), MNAzyme (20 or 2 nM), and target RNA (1 or 20 nM) were lysed in the reaction buffer (50 mM HEPES, 150 mM NaCl, pH 7.3) and incubated at 37°C for 5 min (final concentration is in parentheses). After 5 min of incubation, PLL-g-Dex (N/P=2) was added so that the molar ratio (N/P) between the amino group of the copolymer and the phosphate group of the nucleic acid was 2. In the absence of PLL-g-Dex, MilliQ was added instead of PLL-g-Dex (N/P=0). MnCl2 (5 mM) was added after incubation for an additional minute. Substrates were modified with fluorescent molecules, fluorescein, and the quenching molecule, BHQ-1; the increase in the fluorescence intensity upon cleavage of the substrates by MNAzyme was measured (λex: 494 nm; λem: 520 nm). Time-course changes in fluorescence intensity were measured using an FP-6500 spectrofluorometer (Jasco), and the cleavage rate of the substrate was calculated using the following equation.
Substrate cleavage (%) =[(It−I0 )/(I∞−I0)]×100
wherein, It is the fluorescence intensity at an arbitrary reaction time t, I∞ is the fluorescence intensity when the reaction is saturated, and I0 is the initial fluorescence intensity before the addition of MnCl2. The reaction rate constant kobs was calculated from the reaction curves obtained by monitoring the cleavage of the first 20% of the substrate, using the following formula:
The RNA targets (E6-33 and E6-82) used in this experiment were derived from human papillomavirus type 18 E6; E6-33 and E6-82 were 33 and 82 nucleotides long, respectively (Fig. 1A). The MNAzyme with 33 nt T-arms complementary to the RNA targets was designed (Fig. 1A). The intramolecular structures of RNA were estimated using DINAmelt. It was found that E6-82 (Tm: 70.0°C) has a stronger intramolecular structure than E6-33 (Tm: 58.4°C) (Fig. 1A) (calculation conditions: RNA, 1 M Na+, 0 M Mg2+, and 37°C). The robust higher order structure of RNA targets could inhibit binding of the targets to MNAzyme and cause a reduction in MNAzyme activity. However, the reactivities of the MNAzymes targeting E6-33 and E-82 showed more than a 300-fold increase after the addition of PLL-g-Dex (Fig. 1C). Although the reaction rate for E6-82, a long target, was slightly lower than that for E6-33, a short target, 1 nM E6-82 could be detected within 1 min. Note that, without the annealing treatment, which facilitated binding of the target RNAs to MNAzymes by heating and cooling, ACEzyme activity remained high even for the RNA target with stable intramolecular structures.
Selectivity of ACEzyme toward single-base mismatched targetsBased on the sequence of miR-21, a microRNA associated with cardiovascular disease, eight types of target RNAs in which a single nucleotide mutation was introduced, were prepared (Fig. 2A). The T-arm of MNAzyme used was complementary to miR-21, and LNA was introduced (Fig. 2A). Compared with the full-match miR-21, the reactivity of MNAzyme toward mis (1) with a mutation at the 5′-end was slightly reduced. In contrast, the reactivities of mis (8) and mis (11), whose mutations were located inside the target sequences, decreased to 1/10 and 1/40, respectively. The closer the mutated site of the target RNAs was to the middle of the sequence, the lesser was the activity (Fig. 2B). The mismatched base pair located closer to the catalytic core deformed the catalytic core structure of MNAzyme, leading to a larger loss in the activity. In contrast, mis (9) showed mutant site-independent activity. This is probably due to the formation of a G-U pair, which is a wobble base pair with a relatively stable bond. Mis (6) showed higher activity than mis (5) because the binding stability was improved by the LNA-modification of the T-arm. It was shown that a single-base mismatch could be detected by designing the T-arm such that the mutant base site was located near the catalytic core. These results indicate that an ACEzyme with high discrimination ability against single nucleotide mutations can be readily prepared.
MNAzyme activity against mismatched targets. (A) Sequence and location of fluorophore and quencher on 10–23 MNAzyme used in this experiment (LNA modification is underlined). Sequences of miR-21 and its mutant targets (Blue letter: single mutation). (B) Reaction rates for single-base mismatched targets. Experimental conditions: 2 nM MNAzyme, 100 nM substrate, 20 nM target, PLL-g-Dex N/P=2, 50 mM HEPES, 150 mM NaCl, pH 7.3, 5 mM MnCl2, and 37°C.
The ACEzyme method was able to accommodate RNA targets with robust intramolecular structures without any requirement for annealing. We also found that the ACEzyme method showed high selectivity toward single-nucleotide mismatched targets. These findings indicate the possibility of using ACEzyme as a sensing molecule for simple, rapid, and accurate detection of RNAs.
The authors have nothing to disclose.
This work was financially supported by JSPS KAKENHI (21H03816 to A. M.), the Center of Innovation Program (JPMJCE1305 to A. M.), Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP, JPMJTR21U2 to A. M.) from the Japan Science and Technology Agency (JST), the cooperative research program of the “Network Joint Research Center for Materials and Devices” (to A. M.), a Grant-in-Aid for Transformative Research Areas “Molecular Cybernetics” (20A40, to N. S.) from MEXT, and the Research Program on Emerging and Re-emerging Infectious Diseases (JP 19fk0108152h0001) of AMED (to A. M.).
