Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
G-wire-based self-quenched fluorescence probe combining with target-activated isothermal cascade amplification for ultrasensitive microRNA detection
Graphical abstract
We reported a novel G-wire-based self-quenched fluorescence probe and its application in ultrasensitive microRNA detection by combining with target-activated isothermal cascade amplification. The as-proposed platform is the strong candidate for homogeneous quantification and early clinical diagnosis.
Introduction
DNA G-quadruplexe structures are utilized to be the universal building biomaterials, which greatly benefit fluorescence sensing probe [1] as well as nanomachine design [2]. Diverse G-quadruplexes are generated using various G-rich sequences, such as antiparallel-, parallel-, or hybrid-stranded types [3], [4]. Interestingly, parallel G-quadruplexes allow for the spontaneous assembling to the nanostructures based on long, continuous quadruplex generally termed guanine nanowires (G-wires) [5], [6], [7]. For example, the telomeric oligonucleotide d(AGGGTGGGG), also known as cellular myelocytomatosis oncogene (c-myc), is able to form parallel-stranded G-quadruplex in the presence of K+. Besides, when there is Mg2+, the G-wire nanostructure that contains a large number of G-quadruplex repeat units is prepared [8], [9]. In the G-wire, many DNA strands and G-tetrads are under well control and aligned regulation, therefore, it shows a potential application in molecular electronics and optical devices [10], [11]. Herein, this work first synthesized a terminal single fluorescein (FAM)-labeled self-quenched signal probe based on the formation of G-wire that brought proximity to FAM.
MicroRNA (miRNA) is the small, non-coding RNA (ncRNA) molecule with around 18–25 nucleotides (nt) [12]. MiRNAs play regulating roles in many crucial cell events, such as cell proliferation, apoptosis, differentiation, and hematopoiesis [13], [14], [15], [16]. Accumulating evidence has indicated that aberrant miRNA expression predicts human tumor genesis [17], [18]. To take an example, over expression of miRNA-21 can be detected among 80% of cancer cases [19], whereas the down-regulation of miRNA-143 and miRNA-145 can be detected in colorectal cancer (CRC) cases [20], [21]. Therefore, miRNAs are identified to be the next-generation biomarkers used to classify, diagnose and predict the prognosis of cancer [22], [23], [24], [25]. As miRNAs of the same family are similar in their sequences and have low levels, it is still challenging to construct a detection system with ultrahigh sensitivity for miRNA [26].
Up to now, many isothermal DNA amplification methods have attracted wide attention in the detection of miRNA, such as ligase chain reaction (LCR) [27], [28], rolling circle amplification (RCA) [29], [30], strand displacement amplification (SDA) [31], [32], [33] and exponential amplification reaction (EXPAR) [34], [35]. Among these methods, target-activated isothermal cascade amplification, which exhibits high sensitivity and specificity, has been widely used to rapidly detect miRNAs. For example, Xia et al. reported ultrasensitive detection of specific miRNAs through quadratic isothermal amplification of the target oligonucleotide [36]. Zou et al. developed a single-tube strategy with ultrahigh sensitivity and specificity for miRNA assay at sub-attomole level by triple isothermal cascade amplification reaction [37].
Based on this background, the present work introduced the G-Wire-based self-quenched fluorescence probe in the cascade amplification reaction, so as to establish an “on” mechanism for miRNA detection by constructing cycles of target recycling, digestion-replication reaction, and formation-unravelling of G-wire nanostructures. To the best of our knowledge, the G-Wire-based self-quenched fluorescence probe was integrated into the cascade amplification reaction for the first time, which thus combined target recognition-amplification and the fluorescent “on” mechanism in a single process. Meanwhile, the effect of different random tails on the fluorescence self-quenching was studied, and the single-mismatched miRNA detection was also conducted.
Section snippets
Materials
We purchased Nt.BbvCI NEase and Klenow Fragment (KF, 3′ → 5′ exo-) from New England Biolabs (USA) Ltd. In addition, we obtained deoxynucleotide solution mixture (dNTPs), miRNAs, diethylpyrocarbonate (DEPC)-exposed deionized (DI) water, RNase inhitor, and DNA oligonucleotides after HPLC purification in Sangon Inc. (Shanghai, China). The remaining chemicals were of analytical grade, provided by Aladdin (Shanghai, China) and utilized as received. Human serum samples were kindly provided by Huzhou
Characterization of G-wire-based self-quenched fluorescence probe
The proposed signal probe contained a terminal single-fluorescein (FAM)-labeled G-rich oligonucleotide (c-myc), which was able to form the G-wire nanostructures (G-wires) based on head-to-tail parallel G-quadruplex. In the presence of K+, unimolecular parallel G-quadruplex structure of c-myc formed through one strand containing three double-chain-reversal loops around the G-tetrads core. And then in the presence of Mg2+, higher-order G-wires were developed with parallel G-quadruplex repeat
Conclusions
To sum up, this work prepared the terminal-single-FAM-labeled G-wire-based fluorescence signal probes, Moreover, this work demonstrated the universal miRNA sensing platform based on the proximate FAMs self-quenching effect and the target-activated isothermal cascade amplification. The model target miRNA-21 initiated the cascade recycling processes to release massive triggers, which later hybridized with the self-quenched probe to achieve signal-on amplification. Due to the KF polymerase
CRediT authorship contribution statement
Qingyou Cai: Conceptualization, Methodology, Resources, Writing – original draft, Visualization. Fanfan Wang: Validation, Formal analysis, Investigation, Data curation. Jingying Ge: Validation, Data curation. Zhiguo Xu: Data curation, Visualization. Mei Li: Conceptualization, Methodology, Resources, Writing – review & editing. Hui Xu: Supervision. Hua Wang: Project administration.
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 supported by the National Natural Science Foundation of China (No. 22074079), the Natural Science Foundation of Shandong Province (ZR2020MB075), the China Postdoctoral Science Foundation (2018M633301), Department of Education of Zhejiang Province (GH2021425), Huzhou University (JG202111), and Huzhou College (2022HXKM06, 2022CXCY27 and 2022CXCY37).
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