Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

?29 DNA polymerase (?29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ?29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ?29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

Arrest of Rolling Circle Amplification by Protein‐Binding DNA Aptamers

Certain DNA polymerases, such as ?29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super‐long single‐stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand‐displacement ability of these polymerases. In this work, the ability of ?29DNAP to carry out RCA over circular templates containing a protein‐binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein‐binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.     

Protein-Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer-Containing DNA Primer

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.     

Engineering Micrometer-Sized DNA Tracks for High-Speed DNA Synthesis and Biosensing

φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote “micrometer-sized DNA track” (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer-binding domain at its 3′ end and ≈1000 repeating sequence units at its 5′ end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1 pM let-7a in 10 minutes.     

Optimal DNA Templates for Rolling Circle Amplification Revealed by In Vitro Selection

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC‐rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.     

Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification （RCA） technique. In the presence of DNA adenine methylation （Dam） MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons （detection probes）, resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.     

CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.     

An Autonomous Bio‐barcode DNA Machine for Exponential DNA Amplification and Its Application to the Electrochemical Determination of Adenosine Triphosphate

A novel autonomous bio‐barcode DNA machine that is driven by template‐dependent DNA replication is developed to exponentially amplify special DNA sequences. Combined with a DNA aptamer recognition element, the DNA machine can be further applied in the aptamer‐based, amplified analysis of small molecules. As a model analyte, adenosine triphosphate (ATP) is determined by using the DNA machine system in combination with a DNA aptamer recognition strategy and differential pulse anodic stripping voltammetry (DPASV). Under the optimum conditions, detection limits as low as 2.8×10^?17 M (3σ) for target DNA and 4.7×10^?9 M (3σ) for ATP are achieved. The satisfactory determination of ATP in K562 leukemia cell and Ramos Burkitt’s lymphoma cell reveal that this protocol possesses good selectivity and practicality. As a promising biomolecular device, this DNA machine may have an even broader application in the rapidly developing field of nanobiotechnology.     

Growth and Origami Folding of DNA on Nanoparticles for High‐Efficiency Molecular Transport in Cellular Imaging and Drug Delivery

A novel three‐dimensional (3D) superstructure based on the growth and origami folding of DNA on gold nanoparticles (AuNPs) was developed. The 3D superstructure contains a nanoparticle core and dozens of two‐dimensional DNA belts folded from long single‐stranded DNAs grown in situ on the nanoparticle by rolling circle amplification (RCA). We designed two mechanisms to achieve the loading of molecules onto the 3D superstructures. In one mechanism, ligands bound to target molecules are merged into the growing DNA during the RCA process (merging mechanism). In the other mechanism, target molecules are intercalated into the double‐stranded DNAs produced by origami folding (intercalating mechanism). We demonstrated that the as‐fabricated 3D superstructures have a high molecule‐loading capacity and that they enable the high‐efficiency transport of signal reporters and drugs for cellular imaging and drug delivery, respectively.     

Dynamic,Bioresponsive Hydrogels via Changes in DNA Aptamer Conformation

DNA aptamers are integrated into synthetic hydrogel networks with the aim of creating hydrogels that undergo volume changes when exposed to target molecules. Specifically, single‐stranded DNA aptamers in cDNA‐bound, extended state are incorporated into hydrogel networks as cross‐links, so that the nanoscale conformational change of DNA aptamers upon binding to target molecules will induce macroscopic volume decreases of hydrogels. Hydrogels incorporating adenosine triphosphate (ATP)–binding aptamers undergo controllable volume decreases of up to 40.3 ± 4.6% when exposed to ATP, depending on the concentration of DNA aptamers incorporated in the hydrogel network, temperature, and target molecule concentration. Importantly, this approach can be generalized to aptamer sequences with distinct binding targets, as demonstrated here that hydrogels incorporating an insulin‐binding aptamer undergo volume changes in response to soluble insulin. This work provides an example of bioinspired hydrogels that undergo macroscopic volume changes that stem from conformational shifts in resident DNA‐based cross‐links.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

Simple and Sensitive Electrochemical DNA Detection of Primer Generation‐Rolling Circle Amplification

A new electrochemical sequence‐specific DNA detection platform based on primer generation‐rolling circle amplification (PG‐RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG‐RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG‐RCA amplicons. End‐point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon‐bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon‐bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point‐of‐care and on‐site testing. Real‐time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin.     

DNA Aptamers are Functional Molecular Recognition Sensors in Protic Ionic Liquids

The function and structural changes of an AMP molecular aptamer beacon and its molecular recognition capacity for its target, adenosine monophosphate (AMP), was systematically explored in solution with a protic ionic liquid, ethylammonium nitrate (EAN). It could be proven that up to 2 M of EAN in TBS buffer, the AMP molecular aptamer beacon was still capable of recognizing AMP while also maintaining its specificity. The specificity was proven by using the guanosine monophosphate (GMP) as target; GMP is structurally similar to AMP but was not recognized by the aptamer. We also found that in highly concentrated EAN solutions the overall amount of double stranded DNA formed, as well as its respective thermal stability, diminished gradually, but surprisingly the hybridization rate (k_h) of single stranded DNA was significantly accelerated in the presence of EAN. The latter may have important implications in DNA technology for the design of biosensing and DNA‐based nanodevices in nonconventional solvents, such as ionic liquids.     

A G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

Ultrasensitive Fluorescence Polarization Aptasensors Based on Exonuclease Signal Amplification and Polystyrene Nanoparticle Amplification

Here, we combine T7 exonuclease (T7 Exo) signal amplification and polystyrene nanoparticle (PS NP) amplification to develop novel fluorescence polarization (FP) aptasensors. The binding of a target/open aptamer hairpin complex or a target/single‐stranded aptamer complex to dye‐labeled DNA bound to PS NPs, or the self‐assembly of two aptamer subunits (one of them labeled with a dye) into a target/aptamer complex on PS NPs leads to the cyclic T7 Exo‐catalyzed digestion of the dye‐labeled DNA or the dye‐labeled aptamer subunit. This results in a substantial decrease in the FP value for the amplified sensing process. Our newly developed aptasensors exhibit a sensitivity five orders of magnitude higher than that of traditional homogeneous aptasensors and a high specificity for the target molecules. These distinct advantages of our proposed assay protocol make it a generic platform for the design of amplified aptasensors for ultrasensitive detection of various target molecules.     

Circular DNA by “Bis‐Click” Ligation: Template‐Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides

A highly effective and convenient “bis‐click” strategy was developed for the template‐independent circularization of single‐stranded oligonucleotides by employing copper(I)‐assisted azide–alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7‐deaza‐2′‐deoxyadenosine and 2′‐deoxyuridine residues with different side chains were used in solid‐phase synthesis with phosphoramidite chemistry. The bis‐click ligation of linear 9‐ to 36‐mer oligonucleotides with 1,4‐bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9‐mers. Compared with linear duplexes, circular bis‐click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis‐click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.