Hybridization Of Complementary DNA Strands Research Articles

Full Site-Specific Addressability in DNA Origami-Templated Silica Nanostructures.

DNA nanotechnology allows for the fabrication of nanometer-sized objects with high precision and selective addressability as a result of the programmable hybridization of complementary DNA strands. Such structures can template the formation of other materials, including metals and complex silica nanostructures, where the silica shell simultaneously acts to protect the DNA from external detrimental factors. However, the formation of silica nanostructures with site-specific addressability has thus far not been explored. Here, it is shown that silica nanostructures templated by DNA origami remain addressable for post silicification modification with guest molecules even if the silica shell measures several nm in thickness. The conjugation of fluorescently labeled oligonucleotides is used to different silicified DNA origami structures carrying a complementary ssDNA handle as well as DNA-PAINT super-resolution imaging to show that ssDNA handles remain unsilicified and thus ensure retained addressability. It is also demonstrated that not only handles, but also ssDNA scaffold segments within a DNA origami nanostructure remain accessible, allowing for the formation of dynamic silica nanostructures. Finally, the power of this approach is demonstrated by forming 3D DNA origami crystals from silicified monomers. These results thus present a fully site-specifically addressable silica nanostructure with complete control over size and shape.

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis.

Vesicle Fusion Mediated by Solanesol-Anchored DNA

Fusion between two lipid bilayers is one of the central processes in cell biology, playing a key role in endocytosis, exocytosis, and vesicle transport. We have previously developed a model system that uses the hybridization of complementary DNA strands to model the formation of the SNARE four-helix bundle that mediates synaptic vesicle fusion and used it to study vesicle fusion to a tethered lipid bilayer. Using single vesicle assays, 70% of observed fusion events in the DNA-lipid system are arrested at the hemifusion stage, whereas only 5% eventually go to full fusion. This may be because the diglycerol ether that anchors the DNA in the membrane spans only half the bilayer: upon hemifusion and mixing of the outer leaflets, the DNA-lipid is free to diffuse into the target membrane and away from the vesicle. Here, we test the hypothesis that the length of the membrane anchor may impact the outcome by comparing single leaflet-spanning DNA-lipid mediated vesicle fusion with fusion mediated by DNA anchored by solanesol, a C45 isoprenoid of sufficient length to span the bilayer. When the solanesol anchor was present on the incoming vesicles, target membrane, or both, ∼2–3 times as much full fusion was observed as in the DNA-lipid mediated system, as measured by lipid mixing or content transfer. These results indicate that a transmembrane anchor increases the efficiency of full fusion.

DNA-assisted decoration of single-walled carbon nanotubes with gold nanoparticles for applications in surface-enhanced Raman scattering imaging of cells

Single-walled carbon nanotubes (SWNTs) are 1D nanostructures with distinct physical and chemical properties that have shown great promise for applications in many fields, including biomedicine. Since for biomedical application the water solubility is crucial and SWNTs have low solubility, various methods (including polymer and biopolymer wrapping, chemical modifications) have been developed to solubilize and disperse them in water. Due to their unique optical properties such as photoluminescence in the NIR and strong resonant Raman signatures, they can be used as nanoprobes in biomedical imaging and phototherapies. Furthermore, decoration of SWNTs with noble metal nanoparticles will induce an excellent surface-enhanced Raman scattering (SERS) effect of the nanoparticles-SWNTs composites, with applications in cell imaging. Herein, we present a new and facile strategy for the DNA-assisted decoration of SWNTs with gold nanoparticles (AuNPs) and their application in SERS imaging. By ultrasonication at room temperature of SWNTs with AuNPs functionalized with synthetic DNA, SWNT-AuNPs nanocomposites with enhanced Raman signal were obtained. Among the important advantages of the proposed method are the presence of the free DNA overhangs around the SWNT-AuNPs suitable for post-synthetic modification of nanocomposite through hybridization of complementary DNA strands containing molecules of interest attached by well-developed bio-conjugation chemistry.

High-Performance Three-Dimensional Tubular Nanomembrane Sensor for DNA Detection.

We report an ultrasensitive label-free DNA biosensor with fully on-chip integrated rolled-up nanomembrane electrodes. The hybridization of complementary DNA strands (avian influenza virus subtype H1N1) is selectively detected down to attomolar concentrations, an unprecedented level for miniaturized sensors without amplification. Impedimetric DNA detection with such a rolled-up biosensor shows 4 orders of magnitude sensitivity improvement over its planar counterpart. Furthermore, it is observed that the impedance response of the proposed device is contrary to the expected behavior due to its particular geometry. To further investigate this difference, a thorough model analysis of the measured signal and the electric field calculation is performed, revealing enhanced electron hopping/tunneling along the DNA chains due to an enriched electric field inside the tube. Likewise, conformational changes of DNA might also contribute to this effect. Accordingly, these highly integrated three-dimensional sensors provide a tool to study electrical properties of DNA under versatile experimental conditions and open a new avenue for novel biosensing applications (i.e., for protein, enzyme detection, or monitoring of cell behavior under in vivo like conditions).

Wireframe and Tensegrity DNA Nanostructures

CONSPECTUS: Not only can triangulated wireframe network and tensegrity design be found in architecture, but it is also essential for the stability and organization of biological matter. Whether the scaffolding material is metal as in Buckminster Fuller's geodesic domes and Kenneth Snelson's floating compression sculptures or proteins like actin or spectrin making up the cytoskeleton of biological cells, wireframe and tensegrity construction can provide great stability while minimizing the material required. Given the mechanical properties of single- and double-stranded DNA, it is not surprising to find many variants of wireframe and tensegrity constructions in the emerging field of DNA nanotechnology, in which structures of almost arbitrary shape can be built with nanometer precision. The success of DNA self-assembly relies on the well-controlled hybridization of complementary DNA strands. Consequently, understanding the fundamental physical properties of these molecules is essential. Many experiments have shown that double-stranded DNA (in its most commonly occurring helical form, the B-form) behaves in a first approximation like a relatively stiff cylindrical beam with a persistence length of many times the length of its building blocks, the base pairs. However, it is harder to assign a persistence length to single-stranded DNA. Here, normally the Kuhn length is given, a measure that describes the length of individual rigid segments in a freely jointed chain. This length is on the order of a few nucleotides. Two immediate and important consequences arise from this high flexibility: single-stranded DNA is almost always present in a coiled conformation, and it behaves, just like all flexible polymers in solution, as an entropic spring. In this Account, we review the relation between the mechanical properties of DNA and design considerations for wireframe and tensegrity structures built from DNA. We illustrate various aspects of the successful evolution of DNA nanotechnology starting with the construction of four-way junctions and then allude to simple geometric objects such as the wireframe cube presented by Nadrian Seeman along with a variety of triangulated wireframe constructions. We examine DNA tensegrity triangles that self-assemble into crystals with sizes of several hundred micrometers as well as prestressed DNA origami tensegrity architecture, which uses single-stranded DNA with its entropic spring behavior as tension bearing components to organize stiff multihelix bundles in three dimensions. Finally, we discuss emerging applications of the aforementioned design principles in diverse fields such as diagnostics, drug delivery, or crystallography. Despite great advances in related research fields like protein and RNA engineering, DNA self-assembly is currently the most accessible technique to organize matter on the nanoscale, and we expect many more exciting applications to emerge.

Mechanics and Energetics of DNA Hybridization on Single-Walled Carbon Nanotubes Explored Using Adaptive Biasing Force Calculations

Hybrid nanostructures consisting of single-walled carbon nanotubes (swCNs) coated with single stranded DNA (ssDNA) are of great interest due to their numerous potential applications in nanotechnology, medicine, and homeland security. Recent experiments have demonstrated that DNA-CN hybrids can detect the hybridization of complementary DNA strands, paving the way for applications in DNA sequencing and genetic testing. However, the molecular mechanisms remain poorly understood. Previous molecular dynamics (MD) simulations studying DNA-CN self-assembly have found that adsorbed DNA bases are poorly positioned to hybridize with complementary DNA strands. Here, we apply the adaptive biasing force (ABF) method to all-atom MD models, and find that DNA bases must desorb from the swCN sidewall and suffer significant energy penalties to hybridize with complementary DNA. This agrees with the extremely slow hybridization time scales in fluorescence experiments, as well as observations made using DNA-CN transistor devices. We present the free energy landscape for two model systems and compare the energy required to hybridize a G-C versus an A-T pair. These results reveal significant impediments to rapid DNA hybridization on the swCN surface, and further our understanding of the mechanism by which hybridization gradually occurs—essential knowledge for the advancement of nanotechnology based on DNA-CN.

Coarse-Grained Modeling of DNA-Vesicle Systems with the Martini Force Field

Computational modeling of DNA is a prime example of both recent advances in force field development as well as lingering difficulties in MD simulations. Coarse-grained models reduce the number of particles in the system and increase the available time and length scales of MD simulations [1]. However, most of the coarse-grained DNA models handle just DNA or, at most, only a few other classes of biomolecules which limits the range of applications for which they can be used. We are developing a coarse-grained model for DNA that is compatible with the Martini force field [2] and, thus, allows simulation studies of various DNA/biomolecular systems. We present the implementation of the current Martini DNA model and compare it to atomistic simulations and experimental results. We applied our model to study the behavior of DNA-copolymers in lipid membranes and vesicles. This work complements the work of our experimental collaborators working on selective fusion of vesicles, based on block copolymers containing single-stranded DNA that are placed on the surfaces of the vesicles. The selectivity in fusion is based on the hybridization of complementary DNA strands. Our Martini simulations provide near-atomic detail on the DNA-polymer-membrane interactions that is used to guide the experimental work.[1] H. Ingolfsson, C.A. Lopez, J.J. Uusitalo, D.H. de Jong, S. Gopal, X. Periole, S.J. Marrink. The power of coarse-graining in biomolecular simulations. WIREs Comput. Mol. Sci., in press, 2013. DOI:10.1002/wcms.1169.[2] S.J. Marrink, D.P. Tieleman. Perspective on the Martini model. Chem. Soc. Rev., 42, 6801-6822, 2013.

A New Method of Immobilizing Antibody Probes on Immunochips Based on DNA Hybridization

Abstract A new method for immobilizing antibodies on immunochips was developed based on hybridization of complementary DNA strands in this study. In this method, each kind of antibodies is tagged with oligonucleotides of a specific sequence; on the other hand, complementary strands are synthesized on a solid substrate. Antibodies are subsequently immobilized onto the solid surface via a hybridization process. The feasibility of this method was first verified on magnetic beads surface. An immunochip against transgenic Bt Cry1Ac protein was further developed and the characteristics of antibody assembly were analyzed by Cy3 labeled secondary antibody tracking. The sensitivity of the formed array was tested by detecting the gradient concentrations of Bt Cry1Ac protein solutions. The result showed that the chip using this immobilization method achieved specific and uniform assembly of antibody probes, little non-specific adsorption, as well as low detection limit of 0.01–0.05 ng mL−1. Finally, the feasibility of regenerating antibody arrays using dissociation of bound DNA strand was also validated.

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Several laboratories have reported the occurrence of a split or n − 1 peak at the vWA locus in PowerPlex ® 16 and PowerPlex ® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n − 10/ n − 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n − 10/ n − 18 artifact in PowerPlex ® 16 and PowerPlex ® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex ® 16, PowerPlex ® Y, PowerPlex ® ES, AmpFlSTR ® Profiler Plus ® ID, AmpFlSTR ® Cofiler ®, and AmpFlSTR ® SGM Plus ® kits.

Detection of cystic fibrosis related DNA targets using AC field focusing of magnetic labels and spin-valve sensors

Over the past few years the concept of using magnetic field sensors for biological applications in particular, the development of magnetoresistive biochips and biosensors, has generated increasing interest from laboratories and companies. A spin-valve sensor based biochip was used to detect cystic fibrosis related DNA targets for the purpose of developing an affordable diagnostic chip and detection system. The strategy is based on the AC magnetic field focusing technique. This method consists of the attraction, concentration and manipulation of magnetically-labelled target DNA within on-chip u-shaped current line regions surface functionalized with a cystic fibrosis-related DNA probe. Cystic fibrosis related probes were immobilized on the oxide surface and 250 nm diameter non-remanent magnetic particles were functionalized with cystic fibrosis related DNA targets complementary or non-complementary to the immobilized probes. The hybridization of the target is detected using a u-shaped spin-valve sensor fabricated within the line structure. The proximity of probe and target at the spin-valve sensor surface promotes the hybridization of complementary DNA strands. In this way, hybridization occurs in relatively short times, (5-25 minutes), in comparison with conventional hybridization approaches (3 to 12 hours), as limited by diffusion of the target DNA in solution. Magnetic labels bound to the sensor surface through the hybridization of complementary DNA strands have a magnetic stray field that changes the resistance of sensors enabling detection of the hybridization in real-time. Results show a discernable difference in sensor response after washing when using complementary or non-complementary DNA targets. The use of complementary target DNA resulted in distinct hybridization signals and the binding of the particles in the sensor area was verified by visual inspection. In addition, it was observed that hybridization signals decreased slightly after the more stringent wash indicating that non-specifically or weakly bound labels were washed away. The use of non-complementary target DNA resulted in negligible sensor response after washing and no particles were observed in the sensor area.

ELECTRICAL EFFECTS OF DNA MOLECULES ON SILICON FIELD EFFECT TRANSISTOR

Much research has been devoted to the field of DNA detection for biotechnology and medical diagnostics. Conventionally, this has involved lab-scale large instruments such as fluorescent microscope, with DNA binding and tagging. Recently, however, the possibility of label-free, rapid, sensitive and miniaturized DNA detection electronically has attracted increasing interest in the field. To investigate the feasibility of DNA detection by the semiconductor field effect without tagging or binding agents, we studied the effects of DNA molecules directly on the gate oxide of field effect transistors (FET). DNA solution was deposited onto the gate region of conventional silicon FET devices without gate metal, and current-voltage measurements were performed. Our work showed that the presence of DNA molecules in an aqueous solution on the gate decreased channel current I ds and increased threshold voltage. With increasing concentration in the solution, the threshold voltage increased monotonically. We observed that the channel current was reduced systematically during the hybridization of complementary DNA strands. The mechanism that induced the threshold voltage shift is attributed to the negative charges the DNA molecules carry and to changes in work function (Fermi Energy). Our results showed the possibility that field effect transistors may provide a vehicle for a label-free, miniaturized and semiconductor-based DNA detector.

Electric field pulse assisted covalent immobilization and hybridization of DNA in the nanosecond time scale

AbstractA single, square, voltage pulse is used to accelerate both the immobilization kinetics of DNA molecules on a functionalized thin film silicon dioxide surface and the hybridization of complementary DNA strands to immobilized DNA molecules. The voltage pulse is applied to an integrated thin-film metal electrode beneath the functionalized surface. The duration and magnitude of the voltage pulse are compatible with silicon microelectronics circuits. During immobilization, covalent thiol bonding to the functionalized surface occurs during a single pulse lasting only 100 ns. Hybridization to the immobilized complementary strand occurs during 100 μs pulses.

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand tranfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo.

Comb-type cationic copolymer expedites DNA strand exchange while stabilizing DNA duplex.

The accelerating effect of cationic substances on the DNA strand exchange reaction between a 20 bp DNA duplex and its complementary single strand was studied. A polycationic comb-type copolymer, that consists of a poly(L-lysine) backbone and a dextran graft chain (PLL-g-Dex) and known to stabilize triplex DNA, expedites the strand exchange reaction under physiological relevant conditions. Electrostatically a small excess of the copolymer let to a 300-1500-fold increase in the DNA strand exchange while large excess of spermine or cetyltrimethylammonium bromide, a cationic detergent known to promote markedly hybridization of complementary DNA strands, shows only a slight effect. The efficacy of the copolymer was not affected by a 10 mM Mg2+ concentration. Notably the copolymer promotes the strand exchange reaction while it stabilizes double-stranded DNA. The stabilization of strand exchange intermediates consisting of the parent duplex and the single strand by the copolymer is believed to be responsible for the observed acceleration behavior.

Acceleration of DNA strand exchange by polycation comb-type copolymer.

The accelerating effect of cationic substances on DNA strand exchange reaction between 20 bp DNA duplex and its complementary single strand was studied. A comb-type polycationic copolymer which is composed of poly (L-lysine) backbone and dextran graft chain (PLL-g-Dex) and known to stabilize triplex DNA expedites the strand exchange reaction under physiological relevant conditions. Electrostatically small excess of the copolymer increased DNA strand exchange rate by 300-fold while large excess of spermine or cethyltrimethylammonium bromide, cationic detergent known to promote markedly hybridization of complementary DNA strands, showed slight effect. It should be noted that the copolymer promotes the strand exchange reaction while it stabilizes double stranded DNA.