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Abstract MicroRNAs (miRNAs) play an important role in the regulation of various biological processes and have been used as potential biomarkers for biomedical research and clinical diagnosis. Here, nicking-mediated rolling circle amplification (N-RCA)/symmetric isothermal circular strand-displacement amplification (S-SDA)-integrated combined cascade amplification (rs-CCA) was proposed for let-7a miRNA detection. Via introducing a palindromic fragment-integrated recognition sites for nicking endonuclease Nt.AlWI into the padlock probe, the hybridization of target miRNA can induce N-RCA and continuously generate the nicked fragments (NFs). Because the released NFs each have a palindromic sequence and daughter nicking site at the 3' end, they hybridize with each other, followed by alternately extension by polymerase and nicking by endonuclease. This leads to S-SDA effect, and the released single-stranded products in turn hybridize with NFs, accomplishing the rs-CCA process. When the Sybr Green I dyes intercalate into double-stranded DNA products, the amplified fluorescence signal is achieved. Thus, the target miRNA can be detected down to 5 pM. Importantly, the rs-CCA system is capable of distinguishing the single base difference between target miRNAs, indicating the high sequence specificity. Moreover, its potential application in disease diagnosis was demonstrated via detecting target miRNA in complex biological matrix and analyzing the total RNAs extracted from HeLa cells. As a proof-of-concept building, the impressive rs-CCA scheme is expected to provide a valuable insight into constructing powerful signal amplification strategies via sophisticated combination of biotechnologies available for nucleic acid manipulation and significantly benefit biomedical research and disease diagnostics. Graphical abstract Image 1 Highlights • Nt.AlWI binding site/palindromic sequence-integrated combined cascade amplification (rs-CCA) strategy was proposed. • Along this line, a sensitive and specific fluorescent assay was developed for miRNA detection based on N-RCA and S-SDA. • The let-7a miRNA can be detected down to 5 pM with the linear response range over three orders of magnitude. • The single base difference between target miRNAs is easily discriminated. • The let-7a miRNA can be detected down to 5 pM with the linear response range over three orders of magnitude. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Palindromic DNA sequences and RCA were used to create new DNA nanostructures that can execute the amplification detection of target miRNA (let-7a miRNA) in vitro. fx1 Highlights • Palindromic DNA sequences and RCA were used to create new DNA nanostructures for target miRNA detection. • This as-prepared P-RCA nanostructures achieved a high sensitivity for let-7a miRNA detection. • The difference between let-7 miRNA family members was able to be readily distinguished. • The P-RCA DNA nanostructures could be applied for the analysis of let-7a miRNA extracted from HeLa cells. [ABSTRACT FROM AUTHOR]

In this study, a biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) was proposed for the highly sensitive detection of cancer-related let-7a microRNA (miRNA). The sensing system consists of a padlock probe (PP), which contains a target recognition sequence and two binding sites for nicking endonuclease (NEase), and molecular beacon (MB) as reporting molecule. Upon hybridization with let-7a, the PP can be circularized by ligase. Then, the miRNA acted as polymerization primer to initiate rolling circle amplification (RCA). With the assistance of NEase, RCA products can be nicked on the cyclized PP and are displaced during the subsequent duplication process, generating numerous nicked fragments (NFs). These NFs not only induce another RCA reaction but also open the molecular beacons (MBs) via hybridization, leading to significantly amplified fluorescence signal. Under the optimized conditions, this method exhibits high sensitivity toward target miRNA let-7a with a detection limit of as low as 10 pM, a dynamic range of three orders of magnitude is achieved, and its family member is easily distinguished even with only one mismatched base. Meanwhile, it displays good recovery and satisfactory reproducibility in fetal bovine serum (FBS). Therefore, these merits endow the newly proposed N-RCA strategy with powerful implications for miRNA detection.A biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) for the highly sensitive detection of cancer-related let-7a microRNA [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Developing highly efficient and reliable methods for simultaneous imaging of microRNAs in living cells is often appealed to understanding their synergistic functions and guiding the diagnosis and treatment of human diseases, such as cancers. In this work, we rationally engineered a four-arm shaped nanoprobe that can be stimuli-responsively tied into a Figure-of-Eight nanoknot via spa tial c onf i nement-b a sed dua l - c atalytic h airpin a ssembly (SPACIAL-CHA) reaction and applied for accelerated simultaneous detection and imaging of different miRNAs in living cells. The four-arm nanoprobe was facilely assembled from a cross-shaped DNA scaffold and two pairs of CHA hairpin probes (21HP-a and 21HP-b for miR-21, while 155HP-a and 155HP-b for miR-155) via the "one-pot" annealing method. The DNA scaffold structurally provided a well-known spatial-confinement effect to improve the localized concentration of CHA probes and shorten their physical distance, resulting in an enhanced intramolecular collision probability and accelerating the enzyme-free reaction. The miRNA-mediated strand displacement reactions can rapidly tie numerous four-arm nanoprobes into Figure-of-Eight nanoknots, yielding remarkably dual-channel fluorescence proportional to the different miRNA expression levels. Moreover, benefiting from the nuclease-resistant DNA structure based on the unique arched DNA protrusions makes the system ideal for operating in complicated intracellular environments. We have demonstrated that the four-arm-shaped nanoprobe is superior to the common catalytic hairpin assembly (COM-CHA) in stability, reaction speed, and amplification sensitivity in vitro and living cells. Final applications in cell imaging have also revealed the capacity of the proposed system for reliable identification of cancer cells (e.g., HeLa and MCF-7) from normal cells. The four-arm nanoprobe shows great potential in molecular biology and biomedical imaging with the above advantages. [Display omitted] • Four-arm-shaped nanoprobe can be used for simultaneous imaging of different miRNAs. • The SPACIAL-CHA-based system exhibits a high reaction speed and sensitivity. • Arched DNA protrusions have provided the probe an enhanced nuclease resistance. • The technique has applications in molecular diagnosis and biomedical studies. [ABSTRACT FROM AUTHOR]

The microRNAs (miRNAs) play a critical role in many biological processes and are essential biomarkers for diagnosing disease. However, the sensitive and specific quantification of microRNAs (miRNAs) expression in living cells still faces a huge challenge. Our study designed a multifunctional linear DNA nanostructure (MLN) as a carrier of molecular beacons (MB-21) for detecting and intracellular imaging miRNA-21. The MLN-MB consists of three parts: aptamer, MLN, and MB-21. The aptamer (AS1411) could media MLN-MB enter live cells without additional transfection reagents. Once inside the cells, the intracellular miRNA-21 could hybridize the MB-21s, resulting in significantly enhanced fluorescence signals. The whole process was enzyme-free, autonomous, and continuous, which avoided the necessity of adding external fuel strands or enzymes. We demonstrated that the MLN-MB could be used to screen the miRNA-21 with a detection limit of 320 pM in a short time (10 min) and show high specificity toward miRNA-21 against other miRNAs. Moreover, the proposed MLN-MB could detect the miRNA-21 in complex matrixes stably. With its outstanding stability, dual recognition, and biocompatibility, MLN-MB is capable of delivering into living cells to identify specific cancer cells. Therefore, our sensing approach, with high sensitivity, specificity, and simplicity advantages, holds great potential for early cancer diagnosis. [Display omitted] • The well-design MLN-MB can enter live cells without additional transfection reagents. • The MLN-MB system exhibits high sensitivity, excellent specificity, and a wide linear detection range in vitro. • It can improve cancer detection accuracy by offering a double-check platform to identify specific cancer cells. • The MLN-MB system can be used to image different levels of intracellular miRNA in cancerous cells and normal cells. • The unique method holds potential for biomedical research and molecular diagnostic applications. [ABSTRACT FROM AUTHOR]

Highly sensitive and specific imaging of low-level microRNAs (miRNAs) in cytoplasm is vital for early diagnosis of cancers. In this work, we have developed the amplification strategies for miRNA-155 detection based on the combination the nicked rolling circle amplification (N-RCA) and catalyzed hairpin assembly (CHA). In this system, the target miRNA-155 acts as a polymerase primer to activate N-RCA to produce nicked fragment1 (NF1) and NF2. NF1 acted as new primer could further initiate a new N-RCA reaction over and over. Then, the NF2s could serve as triggers to induce the CHA reaction, and the Y-shaped DNA nanostructure (Y-SDN) was formed. Thus, an amplified fluorescence signal was obtained based on the multiple amplification. Under the optimized experimental conditions, a high sensitivity with a detection limit as low as 1.8 pM at 3σ miRNA-155 and excellent specificity in buffer condition have been achieved by applying this method. Meanwhile, the proposed method enables the application in miRNA-155 detection in human serum. Moreover, we have shown that the method performs well for the intracellular miRNA-155 imaging in cellular environments. Therefore, the present strategy was expected to apply into the clinical disease diagnosis effectively. [Display omitted] • A rapid method for micro-RNA in vitro detection with a limit of detection of 5 pM. • A new strategy can that distinguish the control miRNA from the target miRNA. • Due to its superior sensitivity and selectivity, our method can be further used to detect miRNA-155 in a complex matrix. • The intelligent design was also used for visualization of miRNA-155 in different cell lines.. [ABSTRACT FROM AUTHOR]

Extremely sensitive and visual measurements of microRNA (miRNA) in situ for early detection and monitoring of diseases remains a major challenge. To address this issue, this work reports a rapid, highly sensitive and selective microRNA (miRNA) biosensing strategy based on isothermal circular strand-displacement polymerization (ICSDP), and miRNA imaging was performed inside cells. In this work, a double hairpin DNA probe (HP1/HP2 complex) embedded with a sensing region and polymerase primer region was designed. Briefly, after the specific binding of target miRNA with the HP1/HP2 probe, HP1/HP2 itself can function as a primer to initiate the ICSDP with the help of Klenow Fragment (KF), yielding target miRNA for new rounds of ICSDP. In this process, one target can produce multiple signal outputs (1: n), achieving low abundance of miRNA detection. Under optimized conditions, the proposed strategy showed high sensitivity with a detection limit of 5 pM within 15 min and can also easily distinguish the control miRNA from the target miRNA. This method can be further applied to image the intracellular miRNA of interest in situ inside the cancer cells. Image 1 • A micro-RNA probe based only one enzyme and one DNA segment. • A rapid method for micro-RNA detection. • A new strategy can that distinguish the control miRNA from the target miRNA. • A promise method to detect in situ the intracellular miRNA for diagnosis of cancer cells. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are involved in a variety of biological processes, and the accurate detection of miRNAs is of great importance for early diagnosis of various cancers. Herein, we have developed a highly sensitive method for the intracellular imaging of miRNAs based on a palindromic probe-induced strand displacement amplification (pSDA). The sensing element is a partly complementary hybrid consisting of two DNA components: one fluorescent dye-labeled signaling probe containing a palindromic sequence and loop-based target recognition site and one quencher moiety-attached locking probe. In the presence of target miRNA, the target species can hybridize with the loop site and release the terminal palindromic fragment, initiating the pSDA reaction. Thus, a considerable amount of fluorescent moieties are spatially separated from the quenchers, generating a dramatically enhanced fluorescence signal. As a result, the target miRNAs can be quantified down to 25 pM with the linear response range over four orders of magnitude. The detection specificity is high enough to eliminate the interference from nontarget miRNAs and other biospecies co-existing in samples, and thus the diseased cells are easily distinguished from healthy cells. Strikingly, the pSDA-based system possesses the desirable capability to discriminate tumor cells from healthy cells, indicating a promising diagnostic tool for the detection of cancers and other diseases in early stage. Image 1 • A palindromic probe-induced strand displacement amplification (pSDA) was proposed for the intracellular imaging miRNAs. • The target miRNAs can be detected down to 25 pM with a linear response range over four orders of magnitude. • The pSDA system is suitable for the detection of target miRNAs in a complex biological matrix. • The pSDA strategy can distinguish diseased cells from healthy cells, indicating a tool for the diagnosis of cancers. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are crucial regulators in various pathological and physiological processes, and their misregulation is a hallmark of many diseases. In this study, we introduce an advanced DNA nanomachine using split-type molecular beacons (STMBs) for sensitive detection of miR-21, a key biomarker in cancer diagnostics. Utilizing an innovative STMB-mediated cascade strand displacement amplification (STMB-CSDA) technique, our approach offers a powerful means for the precise quantification of miRNAs, using miR-21 as a primary example. The system operates through target-induced linkage of STMBs, initiating a series of strand displacement amplifications resulting in exponential signal amplification. Coupled with the precision of T4 DNA ligase, this mechanism translates minimal miRNA presence into significant fluorescence signals, offering detection sensitivity as low as 5.96 pM and a dynamic range spanning five orders of magnitude. Characterized by its high specificity, which includes the ability to identify single-base mismatches, along with its user-friendly design, our method represents a significant leap forward in miRNA analysis and molecular diagnostics. Its successful application in examining total RNA from cancer cells and clinical serum samples demonstrates its immense potential as a groundbreaking tool for early cancer detection and gene expression studies, paving the way for the next generation of non-invasive diagnostics in personalized healthcare. [Display omitted] • It enhances detection by combining key reactions, achieving picomolar sensitivity. • Selectively identifies target miRNA, distinguishing single nucleotide variations. • Operates under simple conditions at body temperature, no complex setups needed. • Detects miR-21, differentiates cancerous from healthy cells, used in clinical samples. • Adaptable for various miRNAs, improves diagnostic precision, promising for clinical use. [ABSTRACT FROM AUTHOR]

• Innovates miRNA detection with cholesterol-enhanced 3D DNA Walker, enhancing precision. • Uses micelle structure for nuclease protection, cutting false positives in samples. • Allows cancer cell autonomous miRNA imaging, no need for external cofactors. • Enables precise cancer subtype identification via multiple miRNAs, boosting accuracy. • Advances molecular diagnostics, key for early detection and personalized medicine. Achieving reliable miRNA detection and imaging remains a critical and formidable challenge. To address this challenge, we developed a three-dimensional (3D) DNA walker nanoprobe (DWN), derived from a multifunctional molecular beacon with a lipidic moiety. This innovative structure employs a cholesterol-conjugated multifunctional molecular beacon (Chol-MMB) assembled through hydrophobic interactions, thereby bypassing the cumbersome and inefficient process of immobilizing DNA strands onto rigid surfaces. Once the walker nanoprobe enters cells, the binding between the target miRNA and the nanoprobe triggers an intermolecular polymerization process, leading to a conformational change in DWN. This change liberates a walking strand, initiating the walking reaction. As a result, the 3D DNA walker nanoprobe illuminates the fluorophore, enabling precise quantitative analysis. The efficacy of the DWN was demonstrated by the limit of detection of 0.326 fmol for miR-21 with a reaction duration of 1 h. In addition, this strategy effectively differentiates various cells based on the specific miRNA. Further refinement of this method will lead to an efficacious miRNA-based platform for cell typing and liquid biopsy. [ABSTRACT FROM AUTHOR]

Highlights • A sensitive self-enhanced rolling circle amplification strategy was proposed even if only two probes were involved. • A sensing platform is developed for intracellular miRNA imaging, displaying high sensitivity and desirable universality. • The measured data demonstrates its potential application in clinical diagnosis and other areas relying on miRNA analysis. Abstract MicroRNAs (miRs) are a class of non-coding, small RNA molecules that play important roles in gene expression. The aberrant expression of miRs is associated with various human diseases, and miRs have been regarded as biomarkers and therapeutic targets in cancer treatment. In this work, using miR-21 as model target species, an efficiently self-enhanced rolling circle amplification (seRCA)-based biosensing platform was developed for the ultrasensitive detection of cancer-related miRs. In the presence of target, a linear DNA template can be cyclized by ligase and initiate the first RCA (fRCA) reaction. Without any separation step and chemical modification, the fRCA products (fRCAp) are capable of activating the second exponential RCA and produce a much higher fluorescence signal, causing the self-enhancement effect of RCA reaction. Because two different signal amplification processes are introduced, there are two linear dynamic ranges together over 7 orders of magnitude. Target miR can be detected down to 1.0 fM with very high specificity, eliminating the interference from homologous miRs. If a fluorescently modified oligonucleotide probe is involved, the seRCA-based biosensing strategy is able to be employed for the intracellular imaging of cancer-related miR. Significantly, the comparative evaluation demonstrates that the proposed strategy exhibits the impressive capability to execute the miR imaging within cells higher than classical fluorescence in situ hybridization (FISH) method. The newly-developed powerful sensing strategy for the specific miR imaging would offer a unique opportunity to promote molecular biological research, early diagnosis and treatment of human cancers. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have been identified as important biomarkers with great significance for diagnosis and treatment of various diseases. However, their unique properties, such as small size, high sequence homology, and low abundance, make quantitative analysis of miRNAs extremely challenging. Herein, we reported a cascade catalytic hairpin assembly (CCHA) for sensitive and selective detection of miRNA with three kinds of hairpin probes (HP1, HP2, and HP3). In the presence of target miRNA, a series of toehold-mediated intermolecular DNA strand displacement and hybridization was activated among HP1, HP2, and HP3 to assembly numbers of DNA nanoobjects. During this period, the fluorescence response was greatly intensified to indicate the presence and expression level of interested target miRNA. We have demonstrated that the proposed method exhibits a high assay sensitivity to detect low concentration target and an excellent sequence specificity to distinguish even a single-nucleotide difference in vitro. Moreover, we also demonstrated that our design enables the intracellular imaging of miRNA in live cancer and normal cells. These results showing the promising potential of our CCHA for powerful biosensing, clinic diagnosis, or prognosis. [Display omitted] • We have developed a target-triggered cascade catalytic hairpin assembly (CCHA) using three hairpin probes. • The CCHA system can be used for robustly detection of miRNA-21. • The CCHA system can be applied to image intracellular miRNA in various types of cell lines. • This unique method holds promising potential for biomedical study and clinical molecular diagnostics. [ABSTRACT FROM AUTHOR]