Nucleic Acid Strand Displacement Research Articles

Availability: A Metric for Nucleic Acid Strand Displacement Systems.

DNA strand displacement systems have transformative potential in synthetic biology. While powerful examples have been reported in DNA nanotechnology, such systems are plagued by leakage, which limits network stability, sensitivity, and scalability. An approach to mitigate leakage in DNA nanotechnology, which is applicable to synthetic biology, is to introduce mismatches to complementary fuel sequences at key locations. However, this method overlooks nuances in the secondary structure of the fuel and substrate that impact the leakage reaction kinetics in strand displacement systems. In an effort to quantify the impact of secondary structure on leakage, we introduce the concepts of availability and mutual availability and demonstrate their utility for network analysis. Our approach exposes vulnerable locations on the substrate and quantifies the secondary structure of fuel strands. Using these concepts, a 4-fold reduction in leakage has been achieved. The result is a rational design process that efficiently suppresses leakage and provides new insight into dynamic nucleic acid networks.

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots.

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.

Highly sensitive detection of cancer-related genes based on complete fluorescence restoration of a molecular beacon with a functional overhang

The accurate detection of cancer-related genes is of great significance for early diagnosis and targeted therapy of cancer. In this contribution, an automatically cycling operation of a functional overhang-containing molecular beacon (OMB)-based sensing system was proposed to perform amplification detection of the p53 gene. Contrary to the common molecular beacon (MB), a target DNA is designated to hybridize with a label-free recognition probe (RP) with a hairpin structure rather than OMB. In the presence of a target DNA of interest, the locked primer in RP opens and triggers the subsequent amplification procedures. The newly-developed OMB is not only capable of accomplishing cyclical nucleic acid strand-displacement polymerization (CNDP) with the help of polymerase and nicking endonuclease, but is also cleaved by restriction endonucleases, removing the quencher away from the fluorophore. Thus, the target DNA at an extremely low concentration is expected to generate a considerable amount of double-stranded and cleaved OMBs, and the quenched fluorescence is completely restored, leading to a dramatic increase in fluorescence intensity. Utilizing this sensing platform, the target gene can be detected down to 8.2 pM in a homogeneous way, and a linear response range of 0.01 to 150 nM could be obtained. More strikingly, the mutant genes can be easily distinguished from the wild-type ones. The proof-of-concept demonstrations reported herein are expected to promote the development of DNA biosensing systems, showing great potential in basic research and clinical diagnosis.

Intelligent DNA machine for the ultrasensitive colorimetric detection of nucleic acids.

As DNA is employed to serve as a smart building block, an increasing interest has been devoted to the development of different DNA-based machines for the specific purpose, for example, the exploration of inter- or intramolecular interaction. In the current contribution, we developed an intelligent DNA machine and its operation can be designed to execute the ultrasensitive colorimetric detection of target nucleic acids. The DNA machine consists of a hairpin probe (HP) and an assistant template (AT). Using p53 gene as the target model to trigger the molecular machine operation, cyclic nucleic acid strand displacement polymerization (CNDP) was specifically induced, leading to the DNAzyme mediated catalytic reaction for signal readout. Specifically, with the help of polymerase and nickase, one target molecule was able to drive DNA nano-mechanical devices one-by-one through the hybridization/polymerization displacement cycles, and every initiated machine continued to operate, causing the dramatic accumulation of G-quadruplex-contained products. The G-quadruplex structure after binding to hemin could act as a horseradish peroxidase (HRP)-mimicking DNAzyme and catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by H2O2. As a result, an enhanced color change could be detected because of the generation of oxidation product ABTS•(+). In this way, the DNA machine has no any signal loss and enables the quantitative measurement of p53 DNA with a detection limit of 10fM, indicating great promise for unique application in biomedical research and early clinical diagnosis.

Energy Transfer Assays Using Quantum Dot-Gold Nanoparticle Complexes: Optimizing Oligonucleotide Assay Configuration Using Monovalently Conjugated Quantum Dots.

The energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) represents a popular transduction scheme in analytical assays that use nanomaterials. The impact of the spatial arrangement of the two types of nanoparticles on analytical performance has now been evaluated using a nucleic acid strand displacement assay. The first spatial arrangement (configuration 1) involved the assembly of a number of monovalently functionalized QD-oligonucleotide conjugates around a single central AuNP that was functionalized with complementary oligonucleotide sequences. The assembly of these complexes, and subsequent disassembly via target oligonucleotide-mediated displacement, were used to evaluate energy transfer efficiencies. Furthermore, the inner filter effect of AuNPs on the fluorescence intensity of the QD was studied. AuNPs of three different diameters (6, 13, and 30 nm) were used in these studies. Configuration 2 was based on the placement of monovalently functionalized AuNP-oligonucleotide conjugates around a single QD that was functionalized with a complementary oligonucleotide. The optimal assay configuration, established by evaluating energy transfer efficiencies and inner filter effects, was obtained by arranging at most 15 QDs around the 13 nm AuNP (configuration 1). These assays provided a 2.5-fold change in fluorescence intensity in the presence of target oligonucleotides. To obtain the same response with configuration 2 required the placement of three 6 nm AuNPs around the QD. This resulted in configuration 2 having a 5-fold lower fluorescence intensity when compared to configuration 1. The use of low-cost detection systems (digital camera) further emphasized the higher analytical performance of configuration 1. Response curves obtained using these detection systems demonstrated that configuration 1 had a 10-fold higher sensitivity when compared to configuration 2. This study provides an important framework for the development of sensitive assays using gold nanoparticles and quantum dots.

Cascade DNA nanomachine and exponential amplification biosensing.

DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis.

A sensitive SERS detection of miRNA using a label-free multifunctional probe.

A novel surface enhanced Raman scattering (SERS) detection method is fabricated for miRNA based on a smart multifunctional probe for dual cyclical nucleic acid strand-displacement polymerization (CNDP), achieving high sensitivity, universality, rapid analysis, and good performance in real cell samples.

Discovery of the unique self-assembly behavior of terminal suckers-contained dsDNA onto GNP and novel “light-up” colorimetric assay of nucleic acids

Noble metal nanoparticles are currently of great interest because of their unique optical properties and potential applications in disease diagnostics and cancer treatment. In the present work, a discovery was reported that dsDNA with terminal thiols at its two ends could lie easily flat onto the gold nanoparticle (GNP) surface rather than cross linked different GNPs, indicating an unique self-assembly behavior of newly-designed molecules on GNPs. This could intensively stabilize gold nanoparticles against aggregation even at a high salt concentration. On the basis of this discovery, a novel light-up colorimetric sensing strategy was developed for the detection of p53 gene by combining with the cyclical nucleic acid strand-displacement polymerization (CNDP). For the described colorimetric system, GNPs require no any surface functionalization, and target recognition reaction and CNDP amplification could be conducted under the optimized conditions to achieve a high efficiency. The high detection sensitivity and desirable selectivity are achieved, and the potential practical application was demonstrated. Besides, this sensing system can function in a wide range of salts, making it a suitable platform to cooperate with many biological processes.

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.

Nucleic Acid Chaperone Activity Associated with the Arginine-Rich Domain of Human Hepatitis B Virus Core Protein

Hepatitis B virus (HBV) DNA replication occurs within the HBV icosahedral core particles. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its carboxyl terminus. This ARD domain of HBc 149-183 is known to be important for viral replication but not known to have a structure. Recently, nucleocapsid proteins of several viruses have been shown to contain nucleic acid chaperone activity, which can facilitate structural rearrangement of viral genome. Major features of nucleic acid chaperones include highly basic amino acid residues and flexible protein structure. To test the nucleic acid chaperone hypothesis for HBc ARD, we first used the disassembled full-length HBc from Escherichia coli to analyze the nucleic acid annealing and strand displacement activities. To exclude the potential contamination of chaperones from E. coli, we designed synthetic HBc ARD peptides with different lengths and serine phosphorylations. We demonstrated that HBc ARD peptide can behave like a bona fide nucleic acid chaperone and that the chaperone activity depends on basic residues of the ARD domain. The loss of chaperone activity by arginine-to-alanine substitutions in the ARD can be rescued by restoring basic residues in the ARD. Furthermore, the chaperone activity is subject to regulation by phosphorylation and dephosphorylation at the HBc ARD. Interestingly, the HBc ARD can enhance in vitro cleavage activity of RNA substrate by a hammerhead ribozyme. We discuss here the potential significance of the HBc ARD chaperone activity in the context of viral DNA replication, in particular, at the steps of primer translocations and circularization of linear replicative intermediates. Hepatitis B virus is a major human pathogen. At present, no effective treatment can completely eradicate the virus from patients with chronic hepatitis B. We report here a novel chaperone activity associated with the viral core protein. Our discovery could lead to a new drug design for more effective treatment against hepatitis B virus in the future.

A sensitive quartz crystal microbalance assay of adenosine triphosphate via DNAzyme-activated and aptamer-based target-triggering circular amplification

In this work, a simple and novel quartz crystal microbalance (QCM) assay is demonstrated to selectively and sensitively detect the adenosine triphosphate (ATP). The amplification process consists of circular nucleic acid strand-displacement polymerization, aptamer recognition strategy and nanoparticle signal amplification. With the involvement of an aptamer-based complex, two amplification reaction templates and AuNP-functionalized probes, the whole circle amplification process is triggered by the target recognition of ATP. As an efficient mass amplifier, AuNP-functionalized probes are introduced to enhance the QCM signals. As a result of DNA multiple amplification, a large number of AuNP-functionalized probes are released and hybridized with the capture probes on the gold electrode. Therefore the QCM signals are significantly enhanced, reaching a detection limit of ATP as low as 1.3nM. This strategy can be conveniently used for any aptamer-target binding events with other biological detection such as protein and small molecules. Moreover, the practical determination of ATP in cancer cells demonstrates the feasibility of this QCM approach and potential application in clinical diagnostics.

Label-free optical bifunctional oligonucleotide probe for homogeneous amplification detection of disease markers

Oligonucleotide-based detection schemes that avoid chemical modification possess significant advantages, including simplified design, intrinsic affinity for targets, low cost and ease to extend applications. In this contribution, we developed a label-free self-locked bifunctional oligonucleotide probe (signaling probe) for the detection of different disease markers in parallel. Two signal enhancement techniques based on isothermal circular strand-displacement polymerization reaction, cyclical nucleic acid strand-displacement polymerization (CNDP) and cyclical common (nonnucleic acid) target-displacement polymerization (CCDP), were employed to implement the amplification assay for p53 gene and PDGF-BB, respectively. The attractive assay properties confirmed the effectiveness of isothermal polymerization in common biosensing systems without evolving any chemical modification: PDGF could be detected down to 0.87ng/mL, and a dynamic response range of 8–5000ng/mL was achieved; The capability to screen the p53 gene was also considerably improved, including the detection limit, sensitivity, dynamic range and so on. Moreover, because no any chemical modification of the signaling probe was acquired and different targets were separately detected in homogeneous solution. This interrogating platform exhibits the design flexibility, convenience, simplicity and cost-effectiveness. The success achieved here is expected to serve as a significant step toward the development of robust label-free oligonucleotide probes in biomarker profiling and disease diagnostics.

Highly Sensitive and Selective Bifunctional Oligonucleotide Probe for Homogeneous Parallel Fluorescence Detection of Protein and Nucleotide Sequence

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.

Robustness and modularity properties of a non-covalent DNA catalytic reaction

The biophysics of nucleic acid hybridization and strand displacement have been used for the rational design of a number of nanoscale structures and functions. Recently, molecular amplification methods have been developed in the form of non-covalent DNA catalytic reactions, in which single-stranded DNA (ssDNA) molecules catalyze the release of ssDNA product molecules from multi-stranded complexes. Here, we characterize the robustness and specificity of one such strand displacement-based catalytic reaction. We show that the designed reaction is simultaneously sensitive to sequence mutations in the catalyst and robust to a variety of impurities and molecular noise. These properties facilitate the incorporation of strand displacement-based DNA components in synthetic chemical and biological reaction networks.

RNA Structural Rearrangement via Unwinding and Annealing by the Cyanobacterial RNA Helicase, CrhR

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.

HIV-1 Nucleocapsid Protein as a Nucleic Acid Chaperone: Spectroscopic Study of its Helix-destabilizing Properties, Structural Binding Specificity, and Annealing Activity

Assembly of infectious retroviral particles involves recognition of specific sequences on the viral RNA by the nucleocapsid (NC) domain of the Gag polyprotein, and subsequent stoichiometric binding of the processed NC protein along the entire length of the RNA. NC proteins also act as nucleic acid chaperones. They accelerate nucleic acid hybridization and strand exchange, which may be critical during the initial stages of reverse transcription. In order to better understand these properties, we have studied the nucleic acid helix-destabilizing t m-depressing) and binding activities of HIV-1 NCp7 protein with a variety of substrates, and the real-time kinetics of NC-induced strand exchange. At low ionic strength (0.01 M Na phosphate, pH 7.0) and saturating levels of protein, NCp7 displays moderate helix-destabilizing activity on double-stranded DNA. Saturating levels of NCp7 lowered the t m of a synthetic 28 base-pair 28(+)/28(−) oligonucleotide duplex by about 10 deg. C (51 to 41 °C). The presence of single-stranded calf thymus DNA (equimolar with duplex) eliminated the t m depression, whereas double-stranded calf thymus DNA only altered the t m of the 28-mer duplex by about 2 deg. C. Similar effects were seen with duplexes with single-stranded overhangs or internal single-stranded gaps. Binding experiments utilizing intrinsic tryptophan quenching indicated significant affinity ( K d about 0.1 μM) for both single-stranded and double-stranded forms of the 28-mer in 0.01 M sodium phosphate at 25 °C, although long-chain (calf thymus double-stranded) DNA displayed a much lower affinity. The effects of NCp7 on the kinetics of nucleic acid annealing, strand exchange, and strand displacement were determined by use of oligonucleotides with end-labeled fluorophores serving as donor–acceptor pairs. NCp7 accelerated all these reactions. In the strand exchange reaction, an imperfect duplex, 28(+)/21(−), was reacted with a perfect complement, 28(−). The kinetics of 28(+)/28(−) annealing in this reaction did not conform to a simple bimolecular model, but could be well fit to the sum of two exponential decays. Addition of stoichiometric levels of NCp7 increased the rate constants of both components, and significantly increased the fraction of exchange associated with the rapid process. Increasing levels of 28(−) also increased the rapid fraction, as well as the rapid rate constant. This concentration dependence indicates that, although the kinetic decays appear biexponential, at least one of the steps is bimolecular. Simple annealing reactions, 28(+) with 28(−), could be fit to single-exponential decays, and their magnitudes in the presence of NCp7 were comparable to the rapid step of annealing observed for exchange reactions, suggesting that this step is connected with annealing. Strand dissociation during exchange was monitored by placing the fluorescent acceptor on the 21(−) strand. The results, though complex, suggest that the slow step of exchange is largely associated with the dissociation of the shorter oligonucleotide. Analogous experiments were performed with variants of these oligonucleotides, and the results are in line with the 28(+)/21(−)/28(−) experiments. On the basis of an analysis of the effect of increasing levels of 28(−) on the formation of the perfect 28 bp duplex from the imperfect duplex, we propose that NCp7 forms a ternary complex intermediate with imperfect duplex and 28(−), and suggest several ways by which such an intermediate would facilitate strand exchange.