Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs are used to develop AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

An Autonomous Nonenzymatic Concatenated DNA Circuit for Amplified Imaging of Intracellular ATP.

A robust ATP aptasensor has been successfully constructed for intracellular imaging via the autonomous nonenzymatic cascaded hybridization chain reaction (Ca-HCR) circuit. This compact aptasensor is easily assembled by integrating the sensing module and amplification module, and is furtherly introduced for selective adenosine triphosphate (ATP) assay and for the sensitive tracking of varied ATP expressions in living cells. The ATP-targeting aptamer-encoded sensing module can specifically recognize ATP and release the initiator strand for successively motivating the two-layered HCR (hybridization chain reaction) circuit via the FRET transduction mechanism. The synergistic reaction acceleration of the two HCRs contributes to the high signal gain (amplification efficiency of N2). The whole reaction process was modeled and simulated by MATLAB to deeply explore the underlying molecular reaction mechanism, implying that the cascade HCR is sufficient enough to guarantee the ATP-recognition and amplification processes. The Ca-HCR-amplified aptasensor shows high sensitivity and selectivity for in vitro ATP assay, and can monitor these varied ATP expressions in living cells via intracellular imaging technique. Furthermore, the present aptasensor can be easily extended for monitoring other low-abundance biomarkers, which is especially important for precisely understanding these related biological processes.

Fluorometric determination of ssDNA based on functionalized magnetic microparticles and DNA supersandwich self-assemblies.

A method is described for the determination of DNA via nucleic acid amplification by using nucleic acid concatemers that result from DNA supersandwich self-assemblies (SSAs). The method employs two auxiliary probes to form self-assembled biotin SSAs. These exhibit strong fluorescence if labeled with intercalator SYBR Green I. In the presence of the target (as exemplified for a 30-mer), streptavidin is released from the surface of the functionalized magnetic microparticles (FMMPs) by competitive hybridization on the surface. However, the SSA products do not conjugate to the FMMPs. This leads to a large amount of SYBR Green I intercalated into the concatemers and eventually results in amplified fluorescence in the supernate. The SSA products can be prepared beforehand, and amplification therefore can be completed within 50 min. The method is more efficient than any other conventional amplification. The detection limit for the 30-mer is 26.4 fM which is better by a factor of 10 compared to other amplification methods. Conceivably, the method can be further extended to the determination of a wide variety of targets simply by replacing the sequences of the probes. Finally, this rapid and highly sensitive method was employed for detection of Ebola virus gene (≈30-mer) and ATP in spiked serum with satisfactory results. Graphical abstract A high sensitivity and efficiency bioassay is described based on functionalized magnetic microparticles and DNA supersandwich self-assemblies.

DNA concatemer-silver nanoparticles as a signal probe for electrochemical prostate-specific antigen detection.

In this study, we report a metallobioassay for ultrasensitive electrochemical detection of prostate-specific antigen (PSA) based on DNA hybridization chain reaction (HCR) for amplifying the signal, which is derived from silver nanoparticles (Ag NPs) on DNA concatemers. The assay mainly consists of primary antibody (Ab1), secondary antibody (Ab2) with primer, and a signal probe. In the presence of PSA, a sandwich structure with DNA concatemers was formed, and numerous Ag NPs were loaded on the DNA concatemers, resulting in a strong signal, which appeared within the applied potential (-0.2 V to 0.3 V) in the phosphate-buffered saline (PBS). Differential pulse voltammetry (DPV) was employed to evaluate the analytical performance. Under optimal conditions, the DPV peak current of Ag NPs at about +0.09 V (vs. SCE) increased linearly as the logarithm of PSA concentration increased from 0.1 pg mL-1 to 75 ng mL-1, and the detection limit of PSA was estimated to be 0.033 pg mL-1 at the signal to noise ratio of 3. In addition, the assay was evaluated with human serum samples, and satisfying results were obtained, indicating that the assay can achieve PSA detection in the serum sample.

Selection of self-priming molecular replicators.

Self-priming amplification of oligonucleotides is possible based on foldback of 3' ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.

Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.

Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.

Adaption of a Solid-State Nanopore to Homogeneous DNA Organization Verification and Label-Free Molecular Analysis without Covalent Modification.

Recent advances have shown increasing designs of nucleic acid organizations via controlling the thermodynamics and kinetics of oligonucleotides. Nevertheless, deeper understanding and further applications of these DNA nanotechnologies are majorly hampered by the lack of effective analytical methodologies that are competent enough to investigate them. To deliver a potential solution, here we developed an innovative exploration that employed the emerging nanopore technique to characterize DNA organization at the single-molecule level and in completely homogeneous condition without covalent modification. With the help of counting and profiling the translocation-induced current drop of a DNA assembly structure passing through a conical glass nanopore (CGN), we have directly verified the formation of the individual double-helix concatemer generated from our model, hybridization chain reaction (HCR). Due to the ultrasensitivity of the nanopore technology, those concatemers that were difficult to observe on a conventional electrophoresis image were brought to light. The translocation duration time also provided the approximate length and folding information for the concatemers. These advantages were proven also applicable to structures with more sophisticated folding behaviors. Eventually, when coupling with an upstream reaction, CGN was further turned to a universal detector that was capable of even detecting other nucleic acid organization behaviors as well as targets that were unable to generate huge products. All of these results are expected to promote deeper study and applications of the nanopore technique in the field of nucleic acid nanotechnology.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based on DNA Hybridization Chain Reaction Amplifying Signal.

In this work, a novel electrochemical immunoassay protocol has been reported for simultaneous determination of multiple tumor biomarkers based on DNA hybridization chain reaction (HCR) for signal amplification. Alpha-fetoprotein (AFP) and prostate specific antigen (PSA) were selected as model biomarkers. The immunoassay protocol contained primary antibodies immobilized on gold nanoparticles (Au NPs), secondary antibodies conjugated with DNA concatemer from HCR of primer, auxiliary probe, and signal probe labeled with signal molecules (methyleneblue (MB) and ferrocene (Fc)). In the presence of target biomarkers, the sandwich immunocomplex was formed between the primary antibodies and secondary antibodies bioconjugates carrying numerous signal molecules. As a result, two well-resolved reduction peaks, one was at -0.35 V (corresponding to MB) and other was at 0.33 V (corresponding to Fc; both vs SCE), were obtained in differential pulse voltammetry, and peak currents changed were related to the level of biomarkers. Under optimal conditions, the electrochemical immunoassay exhibited a wide linear response range (0.5 pg mL(-1) to 50 ng mL(-1)) and low detection limits (PSA, 0.17 pg mL(-1); AFP, 0.25 pg mL(-1)) (at S/N = 3). In addition, the immunoassay was evaluated by analyzing simulate human serum sample, and the recoveries obtained were within 99.4-107.6% for PSA and 97.9-108.2% for AFP, indicating the immnuoassay could be applied to the simultaneous detection of AFP and PSA in human serum samples.

Isothermal RNA detection through the formation of DNA concatemers containing HRP-mimicking DNAzymes on the surface of gold nanoparticles.

An enzyme-free signal amplification strategy for colorimetric detection of RNA molecules was developed. The detecting process was started by the hybridization of the target RNA via two helper oligonucleotides to bi-functionalized (initiator and linker oligonucleotides-modified) gold nanoparticles. Afterwards, in presence of two auxiliary oligonucleotides, the nanoparticle-confined initiators triggered the formation of DNA concatemers containing hemin-binding aptamers through a modified hybridization chain reaction [HCR] strategy. In addition to the mediatory role, the helper oligonucleotides, with a toehold-mediated strand displacement [TMSD] ability, helped unwind the secondary structures of the RNA molecule and provided a binding site for a biotinylated capture probe. Upon binding, the complex was harvested on the streptavidin-coated microwell, and subsequently the formation of HRP-mimicking DNAzymes was stimulated by adding hemin molecules. The assay was successfully employed for detecting target nucleic acids, which bears secondary structures, in isothermal conditions (room temperature) without heat denaturation. The assay detected DNA molecule with LOD value of 1 pM with the ability to differentiate a spurious target containing a single-nucleotide substitution. On real RNA samples, the assay truly discriminated Escherichia coli O157:H7's 16s rRNA from closely related bacteria with a detection limit of ≥ 5 × 10(5)CFU. Compared to enzyme-based signal amplifications, the assay can detect the target RNA with a sensitivity similar to the one already found for RT-PCR, NASBA, and RT-LAMP assays. Overall, the analytical platform eliminates the need for enzymatic reactions, heat denaturation, and complicated instruments during the detection process.

Nanorings from Concatemeric DNA: Chemical Modification Drives Nanostructure Formation.

Self-assembly of DNA concatemers from native duplexes and those containing non-nucleotidic bridges of varying polarity composed of repeating oligo(ethylene glycol) phosphates -O(CH2CH2O)(n)PO2- or α,Ω-alkanediol phosphates -O(CH2)10OPO2(-)- units was compared. The structures obtained were characterised by polyacrylamide gel electrophoresis, enzymatic digestion and AFM. Our results have revealed that chemically-modified duplexes favour self-termination of concatemer growth and yield up to 35% of nanosized DNA rings.

Concatenated hERG1 tetramers reveal stoichiometry of altered channel gating by RPR-260243.

Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates repolarization of action potentials in cardiomyocytes. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR) slows deactivation and attenuates inactivation of hERG1 channels. A detailed understanding of the molecular mechanism of hERG1 agonists such as RPR may facilitate the design of more selective and potent compounds for prevention of arrhythmia associated with abnormally prolonged ventricular repolarization. RPR binds to a hydrophobic pocket located between two adjacent hERG1 subunits, and, hence, a homotetrameric channel has four identical RPR binding sites. To investigate the stoichiometry of altered channel gating induced by RPR, we constructed and characterized tetrameric hERG1 concatemers containing a variable number of wild-type subunits and subunits containing a point mutation (L553A) that rendered the channel insensitive to RPR, ostensibly by preventing ligand binding. The slowing of deactivation by RPR was proportional to the number of wild-type subunits incorporated into a concatenated tetrameric channel, and four wild-type subunits were required to achieve maximal slowing of deactivation. In contrast, a single wild-type subunit within a concatenated tetramer was sufficient to achieve half of the maximal RPR-induced shift in the voltage dependence of hERG1 inactivation, and maximal effect was achieved in channels containing three or four wild-type subunits. Together our findings suggest that the allosteric modulation of channel gating involves distinct mechanisms of coupling between drug binding and altered deactivation and inactivation.

Site-resolved structural energetics of the T7 concatemer junction.

The concatemer junction is a conserved sequence of 8 bp, which is strategically located at the junction between the head-to-tail repeats of genomic DNA in T7 and related bacteriophages. The RNA polymerase pauses at this site to recruit the machinery necessary for cleavage of the concatemer into single genome DNA. During pausing, the transcription bubble collapses and the transcription RNA-DNA hybrid is shortened to only 3 bp. This work addresses the question of the role of the nucleic acid components of the transcription elongation complex in this collapse of the transcription bubble. The nucleic acid structures investigated are the DNA-DNA duplex structure present at the concatemer junction when the DNA is not transcribed and the RNA-DNA hybrid formed when the concatemer junction is transcribed. The structural energetics of each base pair in the two structures is characterized using imino proton exchange and nuclear magnetic resonance spectroscopy. The results show that 5 bp in the DNA-DNA duplex at the concatemer junction site are significantly more stable than the corresponding base pairs in the RNA-DNA hybrid that forms when the site is transcribed. Because of their energetic preference for the DNA-DNA duplex, these 5 bp favor the collapse of the transcription bubble. Four of the 5 bp with enhanced stability in the DNA-DNA duplex are located in the downstream half of the concatemer junction site. This location suggests that only after the entire concatemer junction is transcribed can the RNA-DNA hybrid accumulate sufficient structural destabilization to trigger the dissociation of the RNA and the switch of the DNA template strand from the hybrid structure to the DNA-DNA double-helical structure.

Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

Construction and modeling of concatemeric DNA multilayers on a planar surface as monitored by QCM-D and SPR.

The sequential hybridization of a 534 base pair DNA concatemer layer was monitored by QCM-D and SPR, and the QCM-D data were analyzed by Voigt viscoelastic models. The results show that Voigt-based modeling gives a good description of the experimental data but only if shear viscosity and elasticity are allowed to depend on the shear frequency. The derived layer thickness, shear viscosity and elasticity of the growing film give a representation of the DNA film in agreement with known bulk properties of DNA, and reveal a maximum in film viscosity when the molecules in the layer contain 75 base pairs. The experimental data during construction of a 3084 bp DNA concatemer layer were compared to predictions of the QCM-D response of a 1 µm thick film of rod-like polymers. A predicted nonmonotonous variation of dissipation with frequency (added mass) is in qualitative agreement with the experiments, but with a quantitative disagreement which likely reflects that the flexibility of such long DNA molecules is not included in the model.

Non-enzymatic DNA cleavage reaction induced by 5-ethynyluracil in methylamine aqueous solution and application to DNA concatenation.

DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5'-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.

Hemin/G-quadruplex-based DNAzyme concatamers as electrocatalysts and biolabels for amplified electrochemical immunosensing of IgG1.

Hemin/G-quadruplex-based DNAzyme concatamers were utilized as electrocatalysts and biolabels to construct a sandwich-type electrochemical immunosensor for sensitive detection of IgG1 (as a model analyte).

Gold nanoparticles enhance DNA damage induced by anti-cancer drugs and radiation.

The chemotherapeutic agent cisplatin was chemically linked to pGEM-3Zf(-) plasmid DNA to produce a cisplatin-DNA complex, Gold nanoparticles, which bind electrostatically to pure DNA, could also be added to this complex. Dry films of pure plasmid DNA and DNA-cisplatin, DNA-gold nanoparticles and DNA-cisplatin-gold nanoparticles complexes were bombarded by 60 keV electrons. The yields of single- and double-strand breaks were measured as a function of exposure by electrophoresis. From a comparison of such yields from the different type of films, we found that the binding of only one gold nanoparticle to a plasmid-cisplatin complex containing 3197 base pairs increases by a factor of 3 the efficiency of the chemotherapeutic agent cisplatin to produce double-strand breaks in irradiated DNA. Furthermore, adding two cisplatin molecules and one gold nanoparticle to DNA enhances radiation-induced DSBs by a factor of 7.5. A number of phenomena could contribute to this huge enhancement, including the higher density of low-energy electrons and reactive species around the gold nanoparticles and the weakening of bonds adjacent to cisplatin in the DNA backbone. The addition of gold nanoparticles to cisplatin and other platinum agents may therefore provide interesting avenues of research to improve the treatment of cancer by concomitant chemoradiation.

DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum.

Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasite's intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of approximately 27 bp and a K(d) of approximately 63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU-DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.

Programming biomolecular self-assembly pathways.

In nature, self-assembling and disassembling complexes of proteins and nucleic acids bound to a variety of ligands perform intricate and diverse dynamic functions. In contrast, attempts to rationally encode structure and function into synthetic amino acid and nucleic acid sequences have largely focused on engineering molecules that self-assemble into prescribed target structures, rather than on engineering transient system dynamics. To design systems that perform dynamic functions without human intervention, it is necessary to encode within the biopolymer sequences the reaction pathways by which self-assembly occurs. Nucleic acids show promise as a design medium for engineering dynamic functions, including catalytic hybridization, triggered self-assembly and molecular computation. Here, we program diverse molecular self-assembly and disassembly pathways using a 'reaction graph' abstraction to specify complementarity relationships between modular domains in a versatile DNA hairpin motif. Molecular programs are executed for a variety of dynamic functions: catalytic formation of branched junctions, autocatalytic duplex formation by a cross-catalytic circuit, nucleated dendritic growth of a binary molecular 'tree', and autonomous locomotion of a bipedal walker.