Herein, DNA nanostructures were prepared via a palindromic padlock probe-based rolling circle amplification (called P-RCA) and then employed to implement the sensitive and specific detection of let-7a miRNA extracted from cancer cells without chemical modification. The presence of target let-7a miRNA as a polymerization primer can trigger the P-RCA process, generating a long tandemly repetitive DNA strand. The resulting products can fold into nanostructures via self-hybridization of palindromic regions and possess numerous double-stranded fragments. In this case, the strong fluorescent signal is detected upon exposure to SYBR Green I. As a result, in homogeneous solution, target miRNA can be detected down to 6.4 pM with a wide dynamic range. A high specificity was demonstrated by the excellent discrimination between let-7 miRNA family members, while the applicability of this sensing system in complex biological environments was confirmed by the analysis of target miRNAs extracted from HeLa cells. It should be noted that increasing numbers of palindromic fragments in padlock probe further increases signal amplification efficiency. The experimental results indicate that the newly proposed P-RCA DNA nanostructures have potential to become a promising analytical platform in biomedical research and clinical diagnosis for the miRNA detection with high sensitivity and good specificity.

MicroRNAs (miRNAs) are regarded as promising cancer-related biomarkers. Here, we developed a simple, sensitive and specific fluorescence method based on the combination of rolling circle amplification (RCA) and multifunctional molecular beacon-based strand-displacement amplification (MMB-SDA) for the amplification detection of let-7a miRNA. Specifically, a multifunctional molecular beacon (MMB) was proposed to execute several functions, including the signal reporter, primer and polymerization template. To achieve RCA reaction, 3' and 5' terminal bases of padlock probe were designed as the recognition regions capable of hybridizing with let-7a miRNA. Moreover, this miRNA could serve as the primer to trigger RCA reaction after ligation, producing tens and hundreds of tandemly repeated copies of cycle. Subsequently, the RCA product hybridized with MMB and initiated the repetitive SDA reaction, opening a significant amount of MMBs and causing an amplified signal. As a result, let-7a miRNA can be specifically detected down to 51 pM, and the single-base difference between let-7a miRNAs was easily detected. The potential application was demonstrated via evaluating the let-7a miRNA level in real samples (total RNA extracted from HeLa cells). Therefore, the developed sensing strategy would provide a powerful platform for early clinical diagnostics. (c) 2017 Elsevier B.V. All rights reserved.

Highly sensitive detection of K-ras gene is of great significance in biomedical research and clinical diagnosis. Here, we developed a colorimetric biosensing system for the detection of proto-oncogene K-ras based on enhanced amplification effect of DNA molecular machine, where dual isothermal circular strand-displacement amplification (D-SDA) occurs on two arms in one-to-one correspondence. Specifically, we designed a primer-locked hairpin probe (HP) and a primer-contained linear polymerization template (PPT). In the presence of target gene, HP can hybridize with PPT, forming a DNA molecular machine with dual functional arms (called DFA-machine). Each of the two probes in this machine is able to be extended by polymerase on its counterpart species. Moreover, with the help of nicking endonuclease, the dual isothermal polymerization is converted into dual circular strand-displacement amplification, generating a large amount of anti-hemin aptamer-contained products. After binding to hemins, the aptamer/hemin duplex, horseradish peroxidase (HRP)-mimicking DNAzyme, was formed and catalyzed the oxidation of colorless ABTS by H2O2, producing a visible green color. The proposed colorimetric assay exhibits a wide linear range from 0.01 to 150nM with a low detection limit of 10pM. More interestingly, the mutations existing in target gene are easily observed by the naked eye. It should be noted that this colorimetric system was proved by the analysis of K-ras gene of SW620 cell lines. The simple and powerful DFA-machine is expected to provide promising potential in the sensitive detection of biomarkers for cancer diagnosis, prognosis and therapy.

K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. (C) 2016 Elsevier B.V. All rights reserved.

Mutations in Kras gene may be used as a diagnostic marker and a target for treatment of the broad spectrum of human cancers. In this study, we developed a new class of amplification assay, double-hairpin molecular beacon (DHMB)-based cascade rolling circle amplification (RCA), for ultrasensitive and selective detection of Kras gene in a homogenous solution. Specifically, target DNA can hybridize with DHMB and activate cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NMDP). The resulting nicked/displaced fragments substantially outnumber target DNA and cause the cascade rolling circle amplification (C-RCA) and nicked fragment-induced strand-displacement polymerization (NFDP). Even if four amplification processes are designed, only DHMB, padlock probe and polymerization primer are involved. Under optimized conditions, this screening system exhibits a linear range of 5 orders of magnitude (from 100fM to 20nM), and the detection limit is down to 16fM. Moreover, the developed biosensing system offers a high assay specificity for perfectly matched target DNA, and the measured data from practical samples demonstrated the potential application in the cancer diagnoses. As a proof-of-concept genetic assay, the novel signaling strategy, as well as desirable analytical capability, would significantly benefit the development of versatile amplification gene profiling platforms, revealing great promise in biological studies and medical diagnostics.