3WJ Structure Research Articles

A single CAA interrupt in a DNA three-way junction containing a CAG repeat hairpin results in parity-dependent trapping.

An increasing number of human disorders are attributed to genomic expansions of short tandem repeats (STRs). Secondary DNA structures formed by STRs are believed to play an important role in expansion, while the presence of nucleotide interruptions within the pure repeat sequence isknown to delay the onset and progression of disease. We have used two single-molecule fluorescence techniques to analyse the structure and dynamics of DNA three-way junctions (3WJs) containing CAG repeat hairpin slipouts, with and without a single CAA interrupt. For a 3WJ with a (CAG)10 slipout, the CAA interrupt is preferentially located in the hairpin loop, and the branch migration dynamics are 4-fold slower than for the 3WJ with a pure (CAG)10, and 3-fold slower than a 3WJ with a pure (CAG)40 repeat. The (CAG)11 3WJ with CAA interrupt adopts a conformation that places the interrupt in or near the hairpin loop, with similar dynamics to the pure (CAG)10 and (CAG)11 3WJs. We have shown that changing a single nucleotide (G to A)in a pure repeat can have a large impact on 3WJ structure and dynamics, which may be important for the protective role of interrupts in repeat expansion diseases.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues.

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures.

We report on a single-tube biosensor for real-time detection of bacterial pathogens with multiplex capabilities. The biosensor consists of two DNA probes, which bind to the complementary fragment of a bacterial RNA to form a three-way junction (3WJ) nucleic acid structure. One of the probes encodes a fluorescent light-up RNA aptamer under T7 promoter. It allows for generation of multiple aptamer copies due to elongation and transcription of the 3WJ structure in the presence of the complementary target. The aptamer coordinates and thereby enhances fluorescence of a cognate fluorogenic dye, allowing for fluorescent detection of the RNA target. Multiple aptamer copies can be produced from a single target-dependent 3WJ structure allowing for amplification and visual observation of the signal. The limit of detection depended on the assay time and was found to be 1.7 nM or 0.6 nM for 30-min or 60-min assay, respectively, when N-methylmesoporphyrin IX (NMM) was used as a fluorescent indicator. The sensor is excellent in analyzing folded RNA targets and differentiating between closely related sequences due to the multicomponent character of the target-interrogating probe. Response to unamplified samples of total bacterial RNA from Mycobacterium tuberculosis complex or Escherichia coli was observed with excellent selectivity within 30 min under isothermal conditions at 50°C in a one-tube one-step assay. Several bacterial species can be detected in multiplex by utilizing biosensors with the template strands encoding different light-up aptamers. The isothermal one-tube-one-step format of the assay and the possibility to monitor the signal visually makes it amenable to use in a point-of-care scenario.

Target-mediated assembly formation of multi-arm DNAzyme nanostructures for sensitive and accurate discrimination of single-nucleotide polymorphism in K-ras gene

Single-nucleotide polymorphism (SNP) is a typical way of genetic variation and commonly used as genetic marker to indicate various types of human diseases. We herein report a non-enzymatic and label-free sensing method for accurate and ultrasensitive differentiation of SNP in K-ras gene. The presence of target mutant K-ras gene (MtDNA) leads to the formation of “three-way junctions” (3WJ) structure with an exposure toehold site, which initiates the toehold strand displacement reaction (TSDR) to form multi-arm DNAzyme nanostructures. These DNAzymes further cyclically cleave the hairpin DNA substrates and release many G-rich fragments to interact with organic dye thioflavin T (ThT) and yield significantly enhanced fluorescence for sensitive discrimination of MtDNA. However, the presence of wild K-ras gene (WtDNA) trigger a base pairing frame shift on the 3WJ structure and result in the closure of the toehold site, thus neither TSDR nor DNAzyme-assistant cleavage reaction is initiated, and no obvious fluorescent signal is observed. Importantly, this strategy shows high sensitivity with a detection limit of 3.63 fM and exhibits high selectivity to discriminate MtDNA from a mixture with a concentration ratio of WtDNA to MtDNA as high as 5000:1. Moreover, this approach can be used to analyze target MtDNA in diluted serum samples, which demonstrates its potential application for future clinical diagnostics.

Development of targeted therapy therapeutics to sensitize triple-negative breast cancer chemosensitivity utilizing bacteriophage phi29 derived packaging RNA

BackgroundTo date, triple-negative breast cancer (TNBC) treatment options are limited because of the loss of target receptors and, as a result, are only managed with chemotherapy. What is worse is that TNBC is frequently developing resistance to chemotherapy. By using small interfering RNA (siRNA)-based therapeutics, our recent work demonstrated X-box-binding protein 1 (XBP1) was linked to human epidermal growth factor receptor 2 positive (HER2+) breast cancer development and chemoresistance. Given the instability, off-target effects, net negative charge, and hydrophobicity of siRNA in vivo utilization and clinical transformation, its use in treatment is hampered. Thus, the development of a siRNA-based drug delivery system (DDS) with ultra-stability and specificity is necessary to address the predicament of siRNA delivery.ResultsHere, we assembled RNase resistant RNA nanoparticles (NPs) based on the 3WJ structure from Phi29 DNA packaging motor. To improved targeted therapy and sensitize TNBC to chemotherapy, the RNA NPs were equipped with an epidermal growth factor receptor (EGFR) targeting aptamer and XBP1 siRNA. We found our RNA NPs could deplete XBP1 expression and suppress tumor growth after intravenous administration. Meanwhile, RNA NPs treatment could promote sensitization to chemotherapy and impede angiogenesis in vivo.ConclusionsThe results further demonstrate that our RNA NPs could serve as an effective and promising platform not only for siRNA delivery but also for chemotherapy-resistant TNBC therapy.

Urinary exosomal mRNA detection using novel isothermal gene amplification method based on three-way junction

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

Three-way junction-induced isothermal amplification for nucleic acid detection

We, herein, describe a three-way junction (3WJ)-induced isothermal amplification (ThIsAmp) reaction for target nucleic acid detection. In this strategy, target nucleic acid induces the formation of 3WJ structure by associating a specially designed ThIsAmp template and ThIsAmp primer. Upon the formation of 3WJ structure, ThIsAmp primer is subjected to continuously repeated extension and nicking reaction by the combined activities of DNA polymerase and nicking endonuclease, consequently producing a large number of trigger strands. The trigger strands then initiate two separate but interconnected pathways by binding to either 3’ overhang of ThIsAmp template within the 3WJ structure or free ThIsAmp template. As a consequence, a large number of final double-stranded DNA products are produced under an isothermal condition, which can be monitored in real-time by detecting the fluorescence intensity resulting from SYBR Green I staining. Based on this principle, we successfully detected target DNA down to 78.1 aM with excellent specificity. The sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling target nucleic acid detection.

Fabrication of electrochemical biosensor composed of multi-functional DNA structure/Au nanospike on micro-gap/PCB system for detecting troponin I in human serum.

Acute myocardial infarction (AMI) is one of the most serious diseases affecting human beings. In this study, in order to rapidly detect AMI disease, the authors fabricated a label-free electrochemical biosensor composed of a multi-functional DNA structure on Au nanospike (AuNS) with a fabricated Au micro-gap electrode which was incorporated with a PCB chip in order to detect cardiac troponin I (cTnI). As a bioprobe, the DNA 3 way-junction (3WJ) was introduced, because the DNA 3WJ has three arms for embodying the multi-functionality. Each piece of DNA was assembled to simultaneously form the DNA 3WJ for cTnI detection, signal transduction, and immobilization, respectively. The assembled DNA 3WJ structure was confirmed by Native-TBM PAGE. Moreover, in order to increase the electrochemical signal sensitivity, AuNS was prepared. The Au micro-gap array is fabricated with a printed circuit board (PCB) chip in order to control each micro-gap electrode panel selectively so as to detect low volumes of cTnI. Then, the DNA strucuture on pAuNS-modified electrode was prepared using the layer-by-layer (LbL) assembly method. FE-SEM and AFM were used to investigate the modified-surface morphology. The cyclic voltammetry (CV) was measured to confirm the cTnI binding to DNA 3WJ-modified electrode. cTnI was detected in the HEPES solution and human serum, respectively. The LOD result exhibited 1.0 pM in HEPES solution and 1.0 pM in 20% diluted human serum, respectively. In addition, the selectivity test was carried out with various proteins as the control experiment. The present study showed label-free, simple fabrication, and easy-to-tailor detection elements for cTnI.

DNA Three-Way Junction for Differentiation of Single-Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles.

A label- and enzyme-free fluorescent sensor for the detection of single-nucleotide polymorphisms (SNPs) at room temperature is proposed, using new copper nanoparticles (CuNPs) as fluorescent reporters. The CuNPs were constructed by using a DNA three-way junction (3WJ) template. In this assay, two complementary adenine/thymine-rich probes can hybridize with the wild-type target simultaneously to construct a 3WJ structure, serving as an efficient scaffold for the generation of CuNPs. However, the CuNPs produce weak fluorescence when the probes bind with a mutant-type target. SNPs can be identified by the difference in fluorescence intensity of the CuNPs. This SNPs detection strategy is straightforward, cost-effective, and avoids the complicated procedures of labeling or enzymatic reactions. The fluorescent sensor is versatile and can be applied to all types of mutation because the probes are programmable. Moreover, the sensor exhibits good detection performance in biological samples.

Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal reaction format. In the presence of target RNA, 3WJ probes (primer and template) are designed to form a 3WJ structure, from which multiple signal primers for the following PG-RCA can be generated by repeating primer extension, nicking and signal primer dissociation. Although this signal primer generation is a linear amplification process, the PG-RCA exponentially can amplify these signal primers and thus even a very small amount of RNA specimen can be detected. After optimizing the structures of 3WJ probes, the detection limit of this assay was 15.9 zmol (9.55 × 103 molecules) of synthetic RNA or 143 zmol (8.6 × 104 molecules) of in vitro transcribed human CD4 mRNA. Further, the applicability of this assay to detect CD4 mRNA in a human mRNA sample was demonstrated.