Circular Strand-displacement Polymerization Research Articles

One-step self-assembly of quantum dot-based spherical nucleic acid nanostructure for accurate monitoring of long noncoding RNA MALAT1 in living cells and tissues

Simple and sensitive measurement of long non-coding RNA (lncRNA) is critical for early detection of malignancies. Herein, we demonstrate one-step self-assembly of quantum dot (QD)-based spherical nucleic acid (SNA) nanostructure for accurate monitoring of lncRNAs in living cells and tissues. When target lncRNA is present, it binds with a dumbbell probe to expose the complementary domain of Cy5-labeled primer, which subsequently induces cascade primer exchange reaction to produce abundant Cy5-labeled initiators. The Cy5-labeled initiators subsequently hybridize with hairpin probes on the QD surface to activate isothermal circular strand-displacement polymerization reaction, generating the QD-DNA-Cy5 nanostructures and inducing efficient Förster resonance energy transfer (FRET) between donor QD and acceptor Cy5. The obtained FRET signals are accurately quantified by single-molecule imaging. Notably, the single QD-based SNA nanostructure functions not only as a signal transmitter but also as a protector against non-specific amplification. Moreover, this assay utilizes only one DNA polymerase to achieve two-stage amplification, avoiding careful modulation of multiple enzymes. The self-assembly of QD nanosensor can be accomplished in single-step and single-tube manners at room temperature, eliminating precise temperature control and labor-intensive reaction protocols. This QD nanosensor achieves high sensitivity with a limit of detection (LOD) of 65.25 aM, and it is capable of quantifying lncRNA expression at single-cell level, differentiate tumor cells from normal cells, and distinguish breast cancer patients from healthy individuals, providing a versatile paradigm for biomedical research and early clinic diagnostics.

Isothermal circular strand displacement–based assay for microRNA detection in liquid biopsy

Extracellular miRNAs are promising targets for developing new assays for the early diagnosis and prognosis of diseases based on liquid biopsy. The detection of miRNAs in liquid biopsies is challenged by their short sequence length, low concentration, and interferences with bodily fluid components. Isothermal circular strand displacement polymerization has emerged as a convenient method for nucleic acid amplification and detection. Herein, we describe an innovative strategy for microRNA detection directly from biological fluids based on hairpin probe–assisted isothermal amplification reaction. We designed and optimized the assay to detect target analytes in 1 µL of the complex media’s biological matrix using a microfluidic device for the straightforward analysis of multiple samples. We validated the assay to detect circulating miR-127-5p in synovial fluid, recently indicated as a predictive biomarker for osteoarthritis disease. The combined use of a mutant polymerase operating with high yield and a primer incorporating locked nucleic acid nucleosides allowed detection of miR-127-5p with 34 fmol L−1 LOD. We quantified circulating miR-127-5p directly in synovial fluid, thus demonstrating that the assay may be employed for the convenient detection of 4.3 ± 0.5 pmol L−1 concentrated miRNAs in liquid biopsy samples.Graphical abstract

Isothermal chemiluminescent assay based on circular stand-displacement polymerization reaction amplification for cel-miRNA-39-3p determination in cell extracts

A sensitive and specific heterogeneous assay for quantitation of cel-miRNA-39-3p (miRNA-39) was constructed. To improve the assay sensitivity an amplification strategy based on the use of isothermal circular strand-displacement polymerization reaction (ICSDPR), polyperoxidase conjugated with streptavidin and enhanced chemiluminescence was used. The detection limit of the proposed assay was 4 × 10−13 M. The coefficient of variation (CV) for quantitation of miRNA-39 within the working range was below 8%. The study of cross-reactivity of different miRNAs including miRNA-39 demonstrated high specificity of the proposed assay. Comparison of the calibration curves of miRNA-39 dissolved in the buffer and the lysate of MCF-7 cells (prepared by lysis of the cells with phenol/guanidine thiocyanate mixture and purified using silica membrane spin column) has demonstrated a negligible matrix effect. The proposed assay makes it possible to estimate the yield of purification of miRNAs from cells, which is necessary for the quantitative calculation of the intracellular content of miRNAs measured with the isothermal assay coupled with ICSDPR.

Ultrasensitive electrochemical biosensor for attomolar level detection of let 7a based on toehold mediated strand displacement reaction circuits and molecular beacon mediated circular strand displacement polymerization

In this study, an ultrasensitive electrochemical miRNA biosensor based on toehold mediated strand displacement reaction circuits (SDRCs) and molecular beacon mediated isothermal circular strand displacement polymerization reaction (ICSDPR) has been proposed. During the SDRCs module, the cascade strand displacement reaction induces the recycling of the target let 7a and generation of a large amount of strand A (SA). The SA recognition opens the hairpin capture probe immobilized on the gold electrode, thus, varying the distance between the redox molecules and electrode surface. The primer mediated ICSDPR is observed to further generate a large amount of SA, thus, leading to a reduction in the signal. Considering these merits, the proposed method is observed to exhibit a log-linear linearity from 10 aM to 100 pM and ultrahigh sensitivity towards let 7a down to 6.2 aM, with a capability of distinguishing the let 7a family members, thereby, providing a new electrochemical route for early cancer screening.

An integrated target recognition and polymerase primer probe for microRNA detection

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.

Chemiluminescent microplate-based assay of DNA based on isothermal circular strand-displacement polymerization reaction (ICSDPR)

Sensitive microplate-based chemiluminescent assay coupled with isothermal circular strand-displacement polymerization reaction (ICSDPR) for DNA detection was developed. The assay sensitivity was improved using a triple amplification strategy based on employment of ICSDPR, streptavidin-polyperoxidase conjugate and an enhanced chemiluminescent reaction. To increase the accuracy all stages of the assay (one-pot format) were carried out in the same microplate well. The proposed assay detected the target DNA in fM-pM ranges and distinguish target DNA from related mismatched DNAs, demonstrating high sensitivity and high selectivity. This format is highly suitable for the development of sensitive and high-throughput kits of nucleic acids which can be easily automated.

A highly sensitive assay of DNA based on inductively coupled plasma mass spectrometry detection with gold nanoparticle amplification and isothermal circular strand-displacement polymerization reaction

In this work, a simple, sensitive and specific assay for DNA was proposed by combining inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticle (AuNPs) amplification and isothermal circular strand-displacement polymerization reaction (ICSDPR). First, AuNPs were decorated with hairpin-structured DNA (HP-DNA) through Au–S bond to form the AuNPs probe. The ICSDPR was conducted on AuNPs probe in a homogeneous phase to realize the dual amplification and simplify the analytical process at the same time. By using a biotin modified primer, AuNPs were connected with biotins after the ICSDPR, then captured by the streptavidin modified magnetic beads (SA-MBs), and finally detected by ICP-MS. Many key factors including probe volume, hybridization time, polymerase amount, primer concentration, enzyme reaction time, SA-MBs capture time and desorption time were optimized. Under the optimized condition, the proposed method could detect target DNA as low as 45 zmol (8.9 fM in 5 μL) in a relative short time (about 4.5 h) with good specificity, and the linear range of this method is 0.1–10000 pM, the relative standard deviations are in the range of 3.6–6.4%. The proposed method was applied for determination of target DNA in human serum samples, the recovery for the spiked human serum samples is in the range of 84–120%. It demonstrates a good application potential of the developed method for biological studies and clinical diagnosis of human pathogenic diseases.

Robust and highly specific fluorescence sensing of Salmonella typhimurium based on dual-functional phi29 DNA polymerase-mediated isothermal circular strand displacement polymerization.

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP). The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29; thus, amplified detection of the target can be achieved. To our knowledge, this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria. It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3' → 5' proofreading exonuclease activity of phi29, which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension. On the basis of the present amplification strategy, our biosensor exhibits excellent specificity and sensitivity toward S. typhimurium with an excellent detection limit as low as 1.5 cfu mL-1. In addition, the strategy offers the advantages of a simplified operation, shortened analysis time, and highly sensitive detection of pathogens with only a one-step reaction. Furthermore, by redesigning the corresponding binding molecules, the proposed strategy can be easily extended for the detection of a wide spectrum of analytes. Hence, the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.

Branched DNA Junction-Enhanced Isothermal Circular Strand Displacement Polymerization for Intracellular Imaging of MicroRNAs.

The microRNA profiles within living cells are informative for diagnosis and prognosis of human cancers. In the present work, we developed a new sensing strategy based on branched DNA junction-enhanced isothermal circular strand displacement polymerization (B-ICSDP) for the detection and intracellular imaging of microRNAs in living cells of interest. A circular DNA template consisting of three repetitive fragments serves as the scaffold for the self-assembly of sophisticated signaling probes, resulting a shrunk branched DNA junction. Target microRNA triggers the opening of molecular beacon, not only restoring the quenched fluorescence but also activating a circular polymerization-based strand displacement reaction. Thus, patulous branched DNA junction is abundantly formed, generating the amplified signal. It is noteworthy that great heaps of branched product assemblies can be also achieved in living cells, and the intracellular enzymatic assembly based strategy is able to be used to recognize specific microRNA-expressed cancer cells. Moreover, different microRNAs coexisting in the same living cells can be simultaneously screened without any interference from each other by confocal laser scanning microscopy. The measured data from confocal fluorescence imaging of different cancer cells demonstrates that the B-ICSDP-based system is a promising alternative for in vivo analysis of microRNAs in complicated biological samples.

A multiple signal amplified colorimetric aptasensor for antibiotics measurement using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic tags and CSDP target-triggered cycles

An ultrasensitive colorimetric aptasensor was developed for antibiotics detection, with chloramphenicol (CAP) as model target, using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic signal tags and target-triggered circular strand-displacement polymerization (CSDP) for signal amplification. The system consists of two components which can partially hybridize with each other: one is capture probe which was formed through immobilizing hairpin DNA containing aptamer sequence on magnetic beads (MB-cDNA), another is signal tag which was constructed through labeling single strand DNAzyme (G-quadruplex/Hemin) which can partially hybrid with cDNA on platinum nanoparticles functionalized Fe-MIL-88 (MIL-88-Pt-DNAzyme). All components of MIL-88, Pt and DNAzyme in the tag can act as peroxidase mimic to triply catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 into a blue-colored oxidized TMB (oxTMB) for the colorimetric readout. Thus distinctive signal can be observed by naked eye even in presence of 0.02 nM tags. In the presence of target and primer, cDNA loop can open to form cDNA/CAP intermediates, enabling primer to hybridize with the exposed sequences of the cDNA, which initiated target assisted CSDP recycles. Then numerous signal tags were released into supernatant to catalyze TMB for color development. There was a liner relationship between the absorbance and the concentration of CAP in the range of 0.1 pM (0.0323 pg/mL) to 1000 pM (323 pg/mL) with the detection limit of 0.03 pM (0.0097 pg/mL). The ultra-high sensitivity was ascribed to the multiplex catalytic activities from the tags and CSDP based signal amplification. Furthermore, this method can produce signals being observed by naked eye to facilitate in-situ detection and be further extended to detect other antibiotics in food just by simply replacing cDNA on the sensing system.

Droplet Microfluidic Device Fabrication and Use for Isothermal Amplification and Detection of MicroRNA.

Droplet microfluidics combined with the isothermal circular strand displacement polymerization (ICSDP) represents a powerful new technique to detect both single-stranded DNA and microRNA sequences. The method here described helps in overcoming some drawbacks of the lately introduced droplet polymerase chain reaction (PCR) amplification when implemented in microfluidic devices. The method also allows the detection of nanoliter droplets of nucleic acids sequences solutions, with a particular attention to microRNA sequences that are detected at the picomolar level. The integration of the ICSDP amplification protocol in droplet microfluidic devices reduces the time of analysis and the amount of sample required. In addition, there is also the possibility to design parallel analyses to be integrated in portable devices.

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo−), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.

A Biofunctional Molecular Beacon for Detecting Single Base Mutations in Cancer Cells.

The development of a convenient and sensitive biosensing system to detect specific DNA sequences is an important issue in the field of genetic disease therapy. As a classic DNA detection technique, molecular beacon (MB) is often used in the biosensing system. However, it has intrinsic drawbacks, including high assay cost, complicated chemical modification, and operational complexity. In this study, we developed a simple and cost-effective label-free multifunctional MB (LMMB) by integrating elements of polymerization primer, template, target recognition, and G-quadruplex into one entity to detect target DNA. The core technique was accomplished by introducing a G-hairpin that features fragments of both G-quadruplex and target DNA recognition in the G-hairpin stem. Hybridization between LMMB and target DNA triggered conformational change between the G-hairpin and the common C-hairpin, resulting in significant SYBR-green signal amplification. The hybridization continues to the isothermal circular strand-displacement polymerization and accumulation of the double-stranded fragments, causing the uninterrupted extension of the LMMB without a need of chemical modification and other assistant DNA sequences. The novel and programmable LMMB could detect target DNA with sensitivity at 250 pmol/l with a linear range from 2 to 100 nmol/l and the relative standard deviation of 7.98%. The LMMB could sense a single base mutation from the normal DNA, and polymerase chain reaction (PCR) amplicons of the mutant-type cell line from the wild-type one. The total time required for preparation and assaying was only 25 minutes. Apparently, the LMMB shows great potential for detecting DNA and its mutations in biosamples, and therefore it opens up a new prospect for genetic disease therapy.

Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction

A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA–invertase conjugation on the surface of Streptavidin-MNBs. The DNA–invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples.

Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate

In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6×10−10M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.

Ultrasensitive label-free amplified colorimetric detection of p53 based on G-quadruplex MBzymes

A novel label-free DNAzyme molecular beacon (MBzyme) strategy was for the first time developed for colorimetric amplification detection of target nucleic acids. The MBzyme, which is designed to contain peroxidase-mimicking DNAzyme that is locked by a common hairpin, was engineered to form a catalytically active MBzyme through hybridizing with the target p53 DNA. The MBzyme is a multifunctional label-free probe that can act as the target recognition element, catalytic DNAzyme and the primer of polymerization. The target p53 DNA hybridization can induce the isothermal circular strand-displacement polymerization even without any chemical modification and other DNA sequences. This unique amplifying strategy leads to the generation of multiple numbers of active MBzyme molecules even if one hybridization event occurs, achieving a dynamic range of seven orders of magnitude and giving a detection limit down to 25fM which is 3–5 orders of magnitude lower than those of related literature reports. These achievements might be helpful in the design of highly efficient enhancers for G-quadruplex–hemin DNAzymes to be applied on the fundamental research, biotechnology, and biomedical diagnosis.

Hybridization-induced isothermal cycling signal amplification for sensitive electronic detection of nucleic acid

This works reports a new signal-on amplification strategy for sensitive electronic detection of nucleic acid based on the isothermal circular strand-displacement polymerization (ICSDP) reaction. The assay mainly involves a hybridization of ferrocene-labeled hairpin DNA with blocker DNA, a strand-displacement process with target DNA, and an ICSDP-based polymerization reaction. The signal is amplified by the labeled ferrocene on the hairpin probe with target recycling. Upon addition of target analyte, the blocker DNA is initially displaced by target DNA from the hairpin/blocker DNA duplex owing to the difference of the folding free energy, then the newly formed target/blocker DNA duplex causes the ICSDP reaction with the aid of the primer and polymerase, and then the released target DNA retriggers the strand-displacement for target recycling. Numerous ferrocene molecules are close to the electrode surface due to the reformation of hairpin DNA, each of which produces an electronic signal within the applied potentials, thereby resulting in the amplification of electrochemical signal. Under the optimal conditions, the ICSDP-based amplification method displays good electrochemical responses for detection of target DNA at a concentration as low as 0.03pM.

A label-free amplified fluorescence DNA detection based on isothermal circular strand-displacement polymerization reaction and graphene oxide

A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction

We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.