Chain Reaction-ligation Research Articles

Assembly of ligation chain reaction and DNA triangular prism for miRNA diagnostics

Controllable assembly of DNA nanostructure provides a powerful way for quantitative analysis of various targets including nucleic acid molecules. In this study, we have designed detachable DNA nanostructures at electrochemical sensing interface and constructed a ligation chain reaction (LCR) strategy for amplified detection of miRNA. A three-dimensional DNA triangular prism nanostructure is fabricated to provide suitable molecule recognition environment, which can be further regenerated for additional tests via convenient pH adjustment. Target triggered LCR is highly efficient and specific towards target miRNA. Under optimal experimental conditions, this approach enables ultrasensitive exploration in a wide linear range with a single-base resolution. Moreover, it shows excellent performances for the analysis of cell samples and clinical serum samples.

DNA-Templated Click Ligation Chain Reaction Catalyzed by Heterogeneous Cu2O for Enzyme-Free Amplification and Ultrasensitive Detection of Nucleic Acids.

Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via DNA-templated click ligation chain reaction (DT-CLCR) catalyzed by a heterogeneous nanocatalyst made of Cu2O (hnCu2O). This hnCu2O-DT-CLCR method is built on two cross-amplifying hnCu2O-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCu2O-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCu2O-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCu2O-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCu2O-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.

A split-type electrochemical biosensor using enzyme-linked DNA magnetic beads realizes the detection of BCR/ABLp210 fusion gene in clinical samples: Duplex ligation chain reaction coupled with OR logic gate design

Detection of BCR/ABLp210 transcript levels, allowing for the early diagnosis of chronic myelocytic leukemia (CML) and its minimal residual disease (MRD) monitoring, plays a decisive role in the cure of CML. Herein, to realize the sensitive detection of BCR/ABLp210 transcript by electrchemical DNA (E-DNA) sensors in clinical samples, a split-type strategy using enzyme-linked DNA magnetic beads (MBs) was intelligently provided. Specifically, the utilization of magnetic separation makes the target recognition event separate from electrochemical measurements, avoiding the interference of complex matrices on the sensing interface. Besides, the MBs adopted as dispersible electrodes could improve mass transfer of target compared to that of a planarelectrode. Consequently, the proposed E-DNA sensor combined with the ligation chain reaction (LCR) could achieve an exceptional performance with a low detection limit of 1 aM, a broad linear concentration range, and a remarkable specificity with a single-base resolution. Also, K562 cells in up to a 1000-fold excess of NB4 cells could be successfully detected by this E-DNA sensor. Finally, the integration of duplex LCR and OR logic gate into the E-DNA sensor has enabled the detection of e13a2/e14a2/both isoforms of BCR/ABLp210 transcript in a single assay in both newly diagnosed CML patients and those undergoing or having undergone treatment by tyrosine kinase inhibitors. Therefore, this method offers an alternative for the early diagnosis of CML and its MRD monitoring, and holds a huge potential of clinical translation due to its advantage of low cost, ease of miniaturization and generalizability.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Minimizing Product Inhibition in DNA Self-Replication: Insights for Prebiotic Replication from the Role of the Enzyme in Lesion-Induced DNA Amplification.

Self-replication of nucleic acids in the absence of enzymes represents an important and poorly understood step in the origin of life as such reportedsystems are strongly hindered by product inhibition. Studying one of the few successful examples of enzymatic DNA self-replication based on a simple ligation chain reaction, lesion-induced DNA amplification (LIDA), can shed light on how this fundamental process may have originally evolved. To identify the unknown factors that lead LIDA to overcome product inhibitionwe have employed isothermal titration calorimetryand global fitting of time-dependent ligation data to characterize the individual steps of the amplification process. We find that incorporating the abasic lesion into one of the four primers substantially decreases the stability difference between the product and intermediate complexes compared with complexes without the abasic group. In the presence of T4 DNA ligase this stability gap is further reduced by two orders of magnitude revealing thatthe ligase also helps overcome product inhibition. Kinetic simulations revealthat the intermediate complex stability and the magnitude of the ligation rate constant significantly impact the rate of self-replication, suggesting that catalysts that both facilitate ligation and stabilize the intermediate complex might be a route to efficient nonenzymatic replication.

Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform.

As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (μM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.

Nucleic acid amplification-integrated single-molecule fluorescence imaging for in vitro and in vivo biosensing.

Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption, and visualization capability. Recently, a variety of nucleic acid amplification approaches have been developed to provide a straightforward and highly efficient way for amplifying low abundance target signals. The integration of single-molecule fluorescence imaging with nucleic acid amplification has greatly facilitated the construction of various fluorescent biosensors for in vitro and in vivo detection of DNAs, RNAs, enzymes, and live cells with high sensitivity and good selectivity. Herein, we review the advances in the development of fluorescent biosensors by integrating single-molecule fluorescence imaging with nucleic acid amplification based on enzyme (e.g., DNA polymerase, RNA polymerase, exonuclease, and endonuclease)-assisted and enzyme-free (e.g., catalytic hairpin assembly, entropy-driven DNA amplification, ligation chain reaction, and hybridization chain reaction) strategies, and summarize the principles, features, and in vitro and in vivo applications of the emerging biosensors. Moreover, we discuss the remaining challenges and future directions in this area. This review may inspire the development of new signal-amplified single-molecule biosensors and promote their practical applications in fundamental and clinical research.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

Biocomputing label-free security system based on homogenous ligation chain reaction-induced dramatic change in melting temperature for screening single nucleotide polymorphisms

The development of smart platform with accurate, inexpensive and reliable detection of single-nucleotide polymorphisms (SNPs) has long been concerned in the fields of medical diagnosis and basic research. Here, we present a ligation chain reaction (LCR)-based sensing system for the cost-effective screening of SNPs by simply conducting DNA melting analysis. No chemical modification is required and the signaling operation is accomplished in homogeneous solution, circumventing the complex modification process and possibly compromised enzymatic activity associated with heterogeneous materials, such as quantum dot (QD) and gold nanoparticle (GNP). Due to the enzymatic catalysis and high fidelity of ligase, the system is capable of executing signal amplification, providing a high sensitivity and selectivity. KRAS gene is easily recognized and the site-specific mutation of guanine (G) to adenine (A), thymine (T) or cytosine (C) is accurately screened. Moreover, the excellent reliability was demonstrated by blind test and recovery test. LCR-based signaling mechanism was further used to develop the biocomputing security system, and two logic gates consisting of four single-stranded DNAs (ssDNAs) offer a double insurance to protect the information against illegal invasion, guaranteeing the reliability of output information. Once in the absence of one essential factor, the security system was always locked regardless of target key, serving as a novel strategy to ensure the safety of output information at molecular level. As a proof-of-concept scheme, this contribution introduces new insight into the development of DNA security systems and the exploitation of powerful signal transduction strategy suitable for rapid and convenient disease diagnosis.

Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction

A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol–H2O2–horseradish peroxidase (HRP)-mimicking DNAzyme–fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X⁎, YG and Y⁎) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3′-end of YG and fluorescein at 5′-end of Y⁎. After the LCR, the resulting products of XYG/X⁎Y⁎ with biotin-labeled X⁎ were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol–H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis.

Ultrasensitive detection of DNA and RNA based on enzyme-free click chemical ligation chain reaction on dispersed gold nanoparticles.

An ultrasensitive colorimetric DNA and RNA assay using a combination of enzyme-free click chemical ligation chain reaction (CCLCR) on dispersed gold nanoparticles (GNPs) and a magnetic separation process has been developed. The click chemical ligation between an azide-containing probe DNA-modified GNP and a dibenzocyclooctyne-containing probe biotinyl DNA occurred through hybridization with target DNA (RNA) to form the biotinyl-ligated GNPs (ligated products). Eventually, both the biotinyl-ligated GNPs and target DNA (RNA) were amplified exponentially using thermal cycling. After separation of the biotinyl-ligated GNPs using streptavidin-modified magnetic beads, the change in intensity of the surface plasmon band at 525 nm in the supernatants was observed by UV/vis measurement and was also evident visually. The CCLCR assay provides ultrasensitive detection (50 zM: several copies) of target DNA that is comparable to PCR-based approaches. Note that target RNA could also be detected with similar sensitivity without the need for reverse transcription to the corresponding cDNA. The amplification efficiency of the CCLCR assay was as high as 82% due to the pseudohomogeneous reaction behavior of CCLCR on dispersed GNPs. In addition, the CCLCR assay was able to discriminate differences in single-base mismatches and to specifically detect target DNA and target RNA from the cell lysate.

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR.

Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests.

Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample.

Association of pregnancy-associated plasma protein-A gene polymorphism with ischemic stroke in northern Chinese Han population

Objectives: The pregnancy-associated plasma protein-A (PAPP-A), belonging to the metalloproteinase superfamily, plays an important role in destabilization of atherosclerotic plaques. The aim of this study is to evaluate whether PAPP-A gene polymorphism is associated with the risk of ischemic stroke (IS) in northern Chinese Han populations.Methods: A total of 368 patients with IS and 374 aged-matched healthy controls in a Chinese Han population were included in the case–control study. The single-nucleotide polymorphism (SNP) IVS6+95 (rs13290387) in the PAPP-A gene was analyzed by the polymerase chain reaction–ligation detection reaction (PCR–LDR) method.Results: Compared with the GG+CG genotype, the frequencies of the CC genotype of IVS6+95 (rs13290387) in the patients with IS were significantly higher than those in the controls (P = 0·026). After adjusting the confounding risk factors of IS (age, gender, smoking, alcohol drinking, hypertension, diabetes mellitus, and hyperlipidemia) by the multivariate logistic analysis, this significant correlation still remained (P = 0·010).Discussion: The CC genotype of IVS6+95 (rs13290387) was associated with increased incidence of IS in northern Chinese Han populations. This is an initial study to indicate that PAPP-A (rs13290387) might contribute to IS susceptibility in northern Chinese Han populations.

Ligation Chain Reaction based gold nanoparticle assembly for ultrasensitive DNA detection

A simple and ultrasensitive DNA biosensor was developed utilizing the color and size changes originating from Ligation Chain Reaction (LCR)-based gold nanoparticle assembly as an indicator. Using synthetic DNA of 10-fold serial dilutions as a target, the limit of detection (LOD) was 1.5aM with a range of DNA concentrations from 0.01 to 1000fM by UV–vis absorption and 0.1aM in the range of 0.01–10,000fM by dynamic light scattering (DLS). This developed method is low-cost, rapid, and much lower LOD for the detection of specific short DNA sequences, and the results can be directly determined either by the color or by the DLS. The capability of high-throughput detection with the aid of PCR amplification apparatus can also be realized.

Gold Nanoparticle-Enabled Real-Time Ligation Chain Reaction for Ultrasensitive Detection of DNA

A simple and ultrasensitive colorimetric DNA assay based on the detection of the product of a ligation chain reaction (LCR) and the use of gold nanoparticles (AuNPs) as signal generators has been developed. During LCR, the AuNPs were ligated together, resulting in a distinct color change in real time after a sufficient number of thermal cycles. The cumulative nature of the protocol produced a detection limit of 20 aM with a selectivity factor of 10(3).

Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads

Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.

Colorimetric detection of single-nucleotide polymorphisms with a real-time PCR-like sensitivity

Coupling gold nanoparticle-based ligation to a ligation chain reaction enables colorimetric detection of single-nucleotide polymorphisms with a real-time PCR-like sensitivity. One mutant in 2000 copies of a wild-type gene can be detected in 20-100 pg of PCR-amplified genomic DNA samples.

Gold Nanoparticle-Based Label-Free Detection of BRCA1 Mutations Utilizing DNA Ligation on DNA Microarray

Here we describe a label-free detection strategy for point mutation in breast cancer susceptibility gene BRCA1 utilizing ligation chain reaction (LCR) and zip-code array. We amplified the genomic regions containing mutation sites by polymerase chain reaction (PCR) to prepare the template for LCR. Then we ligated a primary probe extended by zip-code complementary at the 5' end with a 3'-thiol modified secondary probe. The resulting ligated product was labeled with gold nanoparticles and allowed to hybridize with corresponding zip-code sequences on the microarray. Finally, we applied silver enhancement to amplify the signals. Using this strategy, we successfully genotyped BRCA1 mutation, which was detected by naked eye.