XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification
Graphical abstract
XNA probe mediated methylation-specific polymerase chain reaction and CRISPR/Cas12a-powered flexible electrochemical and fluorescent dual-mode assay for sensitive detection of N6-methyladenosine in RNA.
Introduction
Eukaryotic RNAs contain a variety of epigenetic modifications. Among them, N6-methyladenosine (m6A) is the most common and abundant methylation modification, especially in messenger RNA (mRNA) and long-stranded noncoding RNA (lncRNA) [1]. Recent studies have shown that m6A plays an important role in regulating DNA replication [2,3], gene expression [[4], [5], [6]], protein translation [7,8],and other life processes [9]. However, the specific regulatory mechanisms of methylation in various diseases (such as tumorigenesis [2,10,11], myeloid differentiation [12,13], pathogenic infections [[14], [15], [16]] and drug resistance regulation [[17], [18], [19]]) are still unclear. Therefore, the construction of effective detection methods for site-specific m6A modifications is important to promote further research on RNA epigenetic modifications in precision medicine.
Partial methylation modifications of RNA (e.g., m1A, m3U, m1G, etc.) are located at the Watson-Crick edge and have a greater impact on the base structure, and such modified RNAs are blocked or base mismatches occur to some extent during the reverse transcription reaction phase, which makes it relatively easy to achieve specific detection [[20], [21], [22]]. In contrast, the m6A-modified methyl group is located at the N6 position of adenine, which has less influence on the base structure and hardly interferes with Watson-Crick base pairing [23]. Moreover, the methyl information would be lost by the use of conventional amplification methods (e.g., polymerase chain reaction or nucleic acid isothermal amplification strategies) because of the irreproducibility of m6A. Therefore, it cannot be directly and effectively detected by sequencing and nucleic acid hybridization alone. In addition, the methylation rate of m6A modification sites often ranges from 1% to 80% and is mostly in a hypomethylation state (less than 20%) in biological lncRNA and mRNA samples [24]. Previously, we constructed a xeno nucleic acid (XNA) probe mediated methylation-specific reverse transcription polymerase chain reaction (MsRT-PCR) for the single-base-resolved detection of site-specific m6A modifications. In this study, we found that the constructed method had good detection linearity and analytical performance in the range of the 10%–100% methylation fraction [25]. However, the difference in the number of PCR amplification cycles between hypomethylation fraction samples and unmethylated samples was not significant, and it was difficult to effectively detect the differences in these hypomethylation sites simply with PCR amplification curves. Therefore, a strategy combining signal amplification is needed to improve the sensitivity and accuracy of hypomethylated RNA site detection.
Recently, the rapid development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) was selected as a series of programmable genetic engineering biology tools that provide new ideas for biosensing constructs [26,27]. The cleavage effect was triggered by nonspecific single-stranded DNA/RNA or double-stranded DNA (dsDNA) complementary to CRISPR-derived RNA (crRNA). Among them, CRISPR/Cas12a was favored for its relatively low cost, excellent nucleic acid targeting specificity and high reaction efficiency compared with other CRISPR/Cas-based enzymatic cleavage systems [28,29]. Importantly, the Cas12a/crRNA complex specifically recognizes target dsDNA and then activates cis-cleavage (to target DNA) and trans-cleavage (to ssDNA) activities. In this study, we intend to integrate CRISPR/Cas12a technology and design complementary crRNA targeted m6A site-specific amplification fragments of PCR reaction products. Exploring the reaction regularity of PCR combined with CRISPR/Cas12a and optimizing the reaction kinetic procedure can suppress background interference caused by inefficient PCR amplification. The specificity and sensitivity of the method can be greatly improved by dual specific recognition of the XNA probe and crRNA, and combined signal amplification of MsRT-PCR and CRISPR/Cas12a.
Electrochemical and fluorescent signal outputs are the two most commonly used modalities for the detection of analytes relevant to clinical diagnosis and monitoring treatment of disease, based on their unique detection sensitivity, stability, portability [[30], [31], [32], [33]]. Due to the difference in detection sensitivity and stable linear range between electrochemistry and fluorescence, we extend the application of CRISPR-Cas12a to the development of a dual-mode biosensing system, and validate the detection performance of m6A sites with different methylation fraction ranges by the output of electrochemistry and fluorescence signals. The combination of dual-mode signal output methods enables accurate and sensitive detection of samples with different methylation fraction ranges.
In this study, we integrated the MsRT-PCR reaction with the CRISPR/Cas12a system to amplify the detection signal between different methylation fractions and proposed an ultrasensitive m6A modification detection method. The crRNA of CRISPR/Cas12a was designed to complement one strand of the double stranded PCR products. In this way, the CRISPR/Cas12a cleavage system was activated by m6A-specific PCR products and realized signal amplification output. The output of the fluorescence and electrochemical signal allows the quantification of the double-stranded products of PCR and thus the indirect quantification of the abundance of m6A at specific RNA sites. Subsequently, 1% of the m6A sites were sensitively detected by integrating the MsRT-PCR and CRISPR/Cas12a signal amplification systems. Furthermore, multiple hypomethylated sites in mRNA and lncRNA extracted from HEK293T cells were also successfully quantified.
Section snippets
Materials and reagents
All primers, probes and modified nucleic acids used in these experiments were synthesized and HPLC purified by TaKaRa Biotechnology (Dalian, China) (see Table S1 for sequences). RT-qPCR one-step kits, EnGen® Lba Cas12a (Cpf1), and NEB buffer (2.1) were purchased from New England Biolabs (New England, China). TranscriptAidTM T7 High Yield Transcription Kit, RNeasy MinElute Cleanup Kit, DNase I, DMEM medium, fetal bovine serum, penicillin, streptomycin, and ribonuclease inhibitors were purchased
Principle of detection based on MsRT-PCR and CRISPR/Cas12a signal amplification
The methylation rates of m6A-modified sites in actual samples of lncRNAs and mRNAs mostly ranged from 1% to 80%, and the vast majority of sites were below 20% the majority. In our previous study, we proposed a simple xeno nucleic acid (XNA) as a block probe mediated methylation specific reverse transcription quantitative polymerase chain reaction assay single-base resolution analysis of m6A-RNA. Strand displacement reactions (SDR) selectively initiated between the reverse transcription primer
Conclusions
In this study, the preconstructed MsRT-PCR and CRISPR/Cas12a systems were integrated skillfully to analyze site-specific RNA modifications. The XNA probes were used to selectively amplify the site specificity of m6A-RNA and quantify the difficult m6A modification in DNA fragments. The crRNA was cleverly designed to target the m6A site-specific amplification fragment, and the CRISPR/Cas12a system was activated by double-stranded products of MsRT-PCR for cleavage of reporter probes. Therefore,
Credit author statement
The contribution of each author to this work as following. Qinli Pu: Design of this work, performing the experiments and writing. Yuanyuan Ye: Performing the experiments and writing. Juan Hu: Performing the experiments and data collection. Cong Xie: Design of this work and reviewing. Xi Zhou: Design of this work and reviewing. Hongyan Yu: Reviewing and editing. Fangli Liao: Reviewing and editing. Song Jiang: Data collection and processing. Linshan Jiang: Data collection. Guoming Xie: Reviewing,
Declaration of competing interest
We have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was financially supported by grants from the National Natural Science Foundation of China, China (No. 81873971), the China Postdoctoral Science Foundation, China (No. 2022M710561), the Graduate Scientific Research and Innovation Project of Chongqing, China (No. CYB19162) and the Science and Technology Commission Foundation of Chongqing, China (No. Cstc2016jcyA0264).
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