Nucleic Acid Strand Displacement Research Articles

Genetically Expressed RNA Strand Displacement for Cellular Manipulation.

Nucleic acid strand displacement is a pivotal concept in dynamic nucleic acid nanotechnologies, which has been extensively investigated and applied across various fields. Compared with DNA systems, the genetically expressed RNA strand displacement technology offers unique advantages for construction of genetic circuits in living cells, where RNA expression and modulation may be seamlessly integrated into the genomic network for long-term and stable regulations of diversified biological functionalities. This Concept paper provides an overview of previous efforts on developments of synthetic gene circuits through utilization of RNA strand displacement, including our endeavors in this field. Moreover, future prospects, potential applications and challenges of the genetically expressed RNA strand displacement technology are also discussed.

Preamplification-free viral RNA diagnostics with single-nucleotide resolution using MARVE, an origami paper-based colorimetric nucleic acid test.

The evolution and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgent concerns as they pose the risk of vaccine failure and increased viral transmission. However, affordable and scalable tools allowing rapid identification of SARS-CoV-2 variants are not readily available, which impedes diagnosis and epidemiological surveillance. Here we present a colorimetric nucleic acid assay named MARVE (multiplexed, preamplification-free, single-nucleotide-resolved viral evolution) that is convenient to perform and yields single-nucleotide resolution. The assay integrates nucleic acid strand displacement reactions with enzymatic amplification to colorimetrically sense viral RNA using a metal ion-incorporated DNA probe (TEprobe). We provide detailed guidelines to design TEprobes for discriminating single-nucleotide variations in viral RNAs, and to fabricate a test paper for the detection of SARS-CoV-2 variants of concern. Compared with other nucleic acid assays, our assay is preamplification-free, single-nucleotide-resolvable and results are visible via a color change. Besides, it is smartphone readable, multiplexed, quick and cheap ($0.30 per test). The protocol takes ~2 h to complete, from the design and preparation of the DNA probes and test papers (~1 h) to the detection of SARS-CoV-2 or its variants (30-45 min). The design of the TEprobes requires basic knowledge of molecular biology and familiarity with NUPACK or the Python programming language. The fabrication of the origami papers requires access to a wax printer using the CAD and PDF files provided or requires users to be familiar with AutoCAD to design new origami papers. The protocol is also applicable for designing assays to detect other pathogens and their variants.

Detection of Single Nucleotide Polymorphisms of Circulating Tumor DNA by Strand Displacement Amplification Coupled with Liquid Chromatography.

The detection of multiple single nucleotide polymorphisms (SNPs) of circulating tumor DNA (ctDNA) is still a great challenge. In this study, we designed enzyme-assisted nucleic acid strand displacement amplification combined with high-performance liquid chromatography (HPLC) for the simultaneous detection of three ctDNA SNPs. First, the trace ctDNA could be hybridized to the specially designed template strand, which initiated the strand displacement nucleic acid amplification process under the synergistic action of DNA polymerase and restriction endonuclease. Then, the targets would be replaced with G-quadruplex fluorescent probes with different tail lengths. Finally, the HPLC-fluorescence assay enabled the separation and quantification of multiple signals. Notably, this method can simultaneously detect both the wild type (WT) and mutant type (MT) of multiple ctDNA SNPs. Within a linear range of 0.1 fM-0.1 nM, the detection limits of BRAF V600E-WT, EGFR T790M-WT, and KRAS 134A-WT and BRAF V600E-MT, EGFR T790M-MT, and KRAS 134A-MT were 29, 31, and 11 aM and 22, 29, and 33 aM, respectively. By using this method, the mutation rates of multiple ctDNA SNPs in blood samples from patients with lung or breast cancer can be obtained in a simple way, providing a convenient and highly sensitive analytical assay for the early screening and monitoring of lung cancer.

Nucleic acid strand displacement for indirect determination of foodborne bacteria by capillary electrophoresis and its application in antagonism and bacteriostasis studies.

Foodborne bacteria threaten human's health. Capillary electrophoresis (CE) is a powerful separation means for the determination of bacteria. Direct separation of bacteria suffers from the shortages of low resolution, channel adsorption, and bacterial aggregation. In this work, a method of nucleic acid strand displacement was developed to indirect separate the bacteria by CE. DNA complexes, consisting of probes and aptamers, were mixed with the three bacteria Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The aptamers could specifically bond with bacteria and release the probes. Through the separation of the probes, the bacteria could be indirectly determined by CE. This method avoided the shortages of direct separation of bacteria. Under the optimized conditions, the three probes for the bacteria could be separated and detected within 2.5min by high-speed CE with laser-induced fluorescence detection. The limits of detection for the bacteria were in the range 4.20×106 to 1.75×107 CFU/mL. Finally, the developed method was applied on the study of antagonism of the coexistent bacteria to reveal the relationship between them. Furthermore, the efficiency of bacteriostasis of three traditional Chinese medicines, Coptis chinensis, Schisandra chinensis, and honeysuckle, was also studied by this method.

Enabling programmable dynamic DNA chemistry using small-molecule DNA binders

The binding of small molecules to the double helical structure of DNA, through either intercalation or minor groove binding, may significantly alter the stability and functionality of DNA, which is a fundamental basis for many therapeutic and sensing applications. Here, we report that small-molecule DNA binders can also be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder. The binder-induced nucleic acid strand displacement (BIND) thus enables innovative technologies to accelerate the discovery and characterization of bioactive small molecules. Specifically, we demonstrate the comprehensive characterization of existing and newly discovered DNA binders, where critical parameters for binding affinity and sequence selectivity can be obtained in a single, unbiased molecular platform without the need for any specialized equipment. We also engineer a tandem BIND system as a high-throughput screening assay for discovering DNA binders, through which 8 DNA binders were successfully discovered from a library of 700 compounds.

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

A paper-based assay for the colorimetric detection of SARS-CoV-2 variants at single-nucleotide resolution.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for versatile diagnostic assays that can discriminate among emerging variants of the virus. Here we report the development and performance benchmarking of an inexpensive (approximately US$0.30 per test) assay for the rapid (sample-to-answer time within 30 min) colorimetric detection of SARS-CoV-2 variants. The assay, which we integrated into foldable paper strips, leverages nucleic acid strand-displacement reactions, the thermodynamic energy penalty associated with single-base-pair mismatches and the metal-ion-controlled enzymatic cleavage of urea to amplify the recognition of viral RNAs for the colorimetric readout of changes in pH via a smartphone. For 50 throat swab samples, the assay simultaneously detected the presence of SARS-CoV-2 and mutations specific to the SARS-CoV-2 variants Alpha, Beta and Gamma, with 100% concordance with real-time quantitative polymerase chain reaction and RNA sequencing. Customizable and inexpensive paper-based assays for the detection of viruses and their variants may facilitate viral surveillance.

Ratiometric Fluorescence Determination of Avian Influenza a Virus Subtype H1N1 DNA with Functionalized Quantum Dots and Gold Nanoparticles

A new type of ratiometric fluorescence sensor based on DNA functionalized quantum dots (DNA-QDs) and gold nanoparticles (AuNPs) was constructed for sensitive fluorescence determination of the avian influenza A virus subtype H1N1 DNA. Using the nucleic acid strand displacement reaction, the carboxyfluorescein-DNA (FAM-DNA) replaced DNA-QDs on the surface of DNA-AuNPs, so that the fluorescence of QDs increased and the fluorescence of FAM decreased. In the presence of H1N1 DNA, the ends of the DNA on the AuNPs-DNA-QDs conjugates were blocked, preventing strand displacement. The fluorescence intensity ratio of FAM to QDs (F 520/F 624) was proportional to the concentration of H1N1 DNA. The linear range was from 2.5 to 20.0 nM with a limit of detection of 0.52 nM. Furthermore, this sensor showed good selectivity. This strategy is a simple, accurate, and sensitive tool to detect DNA biomarkers.

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as "artificial transcription factors" to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitrogenetic devices for applications such as bio-sensing, disease detection, and data storage.

Enzyme-free amplified detection of cellular microRNA by light-harvesting fluorescent nanoparticle probes

Detection of cellular microRNA biomarkers is an emerging powerful tool in cancer diagnostics. Currently, it requires multistep tedious protocols based on molecular amplification of the RNA target, e.g. RT-qPCR. Here, we developed a one-step enzyme-free method for microRNA detection in cellular extracts based on light-harvesting nanoparticle (nanoantenna) biosensors. They amplify the fluorescence signal by effective Förster resonance energy transfer (FRET) from ultrabright dye-loaded polymeric nanoparticle to a single acceptor and thus convert recognition of one microRNA copy (through nucleic acid strand displacement) into a response of >400 dyes. The developed nanoprobes of 17–19 nm diameter for four microRNAs (miR-21, let-7f, miR-222 and miR-30a) exhibit outstanding brightness (up to 3.8 × 107 M−1cm−1) and ratiometric sequence-specific response to microRNA with the limit of detection (LOD) down to 1.3 pM (21 amol), equivalent to 24 RT-qPCR cycles. They enable quantitative detection of the four microRNAs in RNA extracts from five cancerous cell lines (human breast cancer - T47D and MCF7, head and neck cancer - CAL33 and glioblastoma - LNZ308 and U373) and two non-cancerous ones (Hek293 and MCF10A), in agreement with RT-qPCR. The results confirmed that let-7f and especially miR-21 are systematically overexpressed in all studied cancerous cell lines. These nanoparticle biosensors are compatible with low-cost portable fluorometers and small detection volumes (11 amol LOD), opening a route to rapid point-of-care cancer diagnostics.

Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions.

A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.

Principles and Applications of Nucleic Acid Strand Displacement Reactions.

Dynamic DNA nanotechnology, a subfield of DNA nanotechnology, is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addition, the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNA-based nanomachine (Yurke, B.; et al. Nature 2000, 406, 605-608). Since then, toehold-mediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnology has grown exponentially. Besides molecular machines, the field has produced enzyme-free catalytic systems, all DNA chemical oscillators and the most complex molecular computers yet devised. Enzyme-free catalytic systems can function as chemical amplifiers and as such have received considerable attention for sensing and detection applications in chemistry and medical diagnostics. Strand-displacement reactions have been combined with other enzymatically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol. 2015, 11, 287-294). Strand-displacement principles have also been applied in synthetic biology to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnology over the past years, the field now seems poised for practical application.

Nucleic Acid Strand Displacement with Synthetic mRNA Inputs in Living Mammalian Cells.

Strand displacement reactions are widely used in DNA nanotechnology as a building block for engineering molecular computers and machines. Here, we demonstrate that strand displacement-based probes can be triggered by RNA expressed in mammalian cells, thus taking a step toward adapting the DNA nanotechnology toolbox to a cellular environment. We systematically compare different probe architectures in order to identify a design that works robustly in living cells. Our optimized strand displacement probe combines chemically modified nucleic acids that enhance stability to degradation by cellular nucleases with structural elements that improve probe retention in the cytoplasm. We visualize probe binding to individual mRNA carrying 96 repeats of a target sequence in the 3'UTR. We find that RNA counts based on live cell imaging using a strand displacement probe are comparable to counts from independent measurement based on fluorescence in situ hybridization experiments. We used probes with scrambled toeholds and scrambled binding domains to demonstrate that target recognition indeed occurs through toehold-mediated strand displacement. Our results demonstrate that strand displacement probes can work reliably in mammalian cells and lay the groundwork for future applications of such probes for live-cell imaging and molecular computing.

A Logic Programming Language for Computational Nucleic Acid Devices.

Computational nucleic acid devices show great potential for enabling a broad range of biotechnology applications, including smart probes for molecular biology research, in vitro assembly of complex compounds, high-precision in vitro disease diagnosis and, ultimately, computational theranostics inside living cells. This diversity of applications is supported by a range of implementation strategies, including nucleic acid strand displacement, localization to substrates, and the use of enzymes with polymerase, nickase, and exonuclease functionality. However, existing computational design tools are unable to account for these strategies in a unified manner. This paper presents a logic programming language that allows a broad range of computational nucleic acid systems to be designed and analyzed. The language extends standard logic programming with a novel equational theory to express nucleic acid molecular motifs. It automatically identifies matching motifs present in the full system, in order to apply a specified transformation expressed as a logical rule. The language supports the definition of logic predicates, which provide constraints that need to be satisfied in order for a given rule to be applied. The language is sufficiently expressive to encode the semantics of nucleic strand displacement systems with complex topologies, together with computation performed by a broad range of enzymes, and is readily extensible to new implementation strategies. Our approach lays the foundation for a unifying framework for the design of computational nucleic acid devices.

A kinetically controlled, isothermal method for the detection of single nucleotide mismatches

We describe an isothermal, enzyme-free method to detect single nucleotide differences between oligonucleotides of close homology. The approach exploits kinetic differences in toe-hold-mediated, nucleic acid strand-displacement reactions to detect single nucleotide polymorphisms (SNPs) with essentially “digital” precision. The theoretical underpinning, experimental analyses, predictability, and accuracy of this new method are reported. We demonstrate detection of biologically relevant SNPs and single nucleotide differences in the let-7 family of microRNAs. The method is adaptable to microarray formats, as demonstrated with on-chip detection of SNP variants involved in susceptibility to the therapeutic agents abacavir, Herceptin, and simvastatin.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis.

High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

While detection of microRNA with or without signal amplification is highly informative, nanosensors with high specificity for cell-specific RNA detection are rare.Methods: In this study, a tetrahedral DNA nanostructure (TDN) with a specific function was combined with gold nanoparticles (Au-NP) possessing fluorescence quenching effects and a large surface area to fabricate a fluorescence resonance energy transfer based nanosensor (Au-TDNN). The presence of miR-21 (target) can separate the fluorescent dye-labeled detection probe on Au-TDNNs from Au-NPs, which separates the donor and acceptor, thus inducing an intensive fluorescence signal. High specificity for discerning point mutation targets was achieved by rationally designing the nucleic acid strand displacement reaction to occur spontaneously with ΔG0 ≈ 0 based on thermodynamic parameters; under this condition, slight thermodynamic changes caused by base mismatch exert significant effects on hybridization yield.Results: Chemically synthesized DNA of three single-base-changed analogues of target, let-7d, and miR-200b were tested. A discrimination factor (DF) of 15.4 was produced by the expected detection probe on Au-NPs for proximal single-base mismatch. As the control group, the DF produced by an ordinary detection probe on Au-NPs only reached 2.4. The feasibility of the proposed strategy was also confirmed using hepatocyte cancer cells (HepG2).Conclusion: This improved nanosensor opens a new avenue for the specific and easy detection of microRNA in live cells.

A Robust Molecular Network Motif for Period-Doubling Devices.

Life is sustained by a variety of cyclic processes such as cell division, muscle contraction, and neuron firing. The periodic signals powering these processes often direct a variety of other downstream systems, which operate at different time scales and must have the capacity to divide or multiply the period of the master clock. Period modulation is also an important challenge in synthetic molecular systems, where slow and fast components may have to be coordinated simultaneously by a single oscillator whose frequency is often difficult to tune. Circuits that can multiply the period of a clock signal (frequency dividers), such as binary counters and flip-flops, are commonly encountered in electronic systems, but design principles to obtain similar devices in biological systems are still unclear. We take inspiration from the architecture of electronic flip-flops, and we propose to build biomolecular period-doubling networks by combining a bistable switch with negative feedback modules that preprocess the circuit inputs. We identify a network motif and we show it can be "realized" using different biomolecular components; two of the realizations we propose rely on transcriptional gene networks and one on nucleic acid strand displacement systems. We examine the capacity of each realization to perform period-doubling by studying how bistability of the motif is affected by the presence of the input; for this purpose, we employ mathematical tools from algebraic geometry that provide us with valuable insights on the input/output behavior as a function of the realization parameters. We show that transcriptional network realizations operate correctly also in a stochastic regime when processing oscillations from the repressilator, a canonical synthetic in vivo oscillator. Finally, we compare the performance of different realizations in a range of realistic parameters via numerical sensitivity analysis of the period-doubling region, computed with respect to the input period and amplitude. Our mathematical and computational analysis suggests that the motif we propose is generally robust with respect to specific implementation details: functionally equivalent circuits can be built as long as the species-interaction topology is respected. This indicates that experimental construction of the circuit is possible with a variety of components within the rapidly expanding libraries available in synthetic biology.

Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene

In the present study, we proposed a novel dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) based signal amplification system for highly sensitive determination of tumor suppressor genes. The system primarily consisted of a signaling hairpin probe (SHP), a label-free hairpin probe (LHP) and an initiating primer (IP). The presence of target DNA was able to induce one CNDP through continuous process of ligation, polymerization and nicking, leading to extensively accumulation of two nicked triggers (NT1 and NT2). Intriguingly, the NT1 could directly hybridize SHP, while the NT2 could act as the target analog to induce another CNDP. The resulting dual-CNDP contributed the striking signal amplification, and only a very weak blank noise existed since the ligation template of target was not involved. In this case, the target could be detected in a wide linear range (5 orders of magnitude), and a low detection limit (78 fM) was obtained, which is superior to most of the existing fluorescent methods. Moreover, the dual-CNDP sensing system provided a high selectivity towards target DNA against mismatched target and was successfully applied to analysis of target gene extracted from cancer cells or in human serum-contained samples, indicating its great potential for practical applications.

Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits.

Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers' attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.