DNA-network-templated self-assembly of silver nanoparticles and their application in surface-enhanced Raman scattering

A large-scale lambda-DNA network on a mica surface was successfully fabricated with a simple method. Silver nanoparticles capped with the cationic surfactant cetyltrimethylammonium bromide (CTAB) were self-assembled onto a two-dimensional DNA network template by electrostatic interaction and formed nanoporous silver films, which can be used as active surface-enhanced raman scattering (SERS) substrates. Two probe molecules, Rhodamine 6G (R6G) and 4-aminothiophenol (4-ATP), were studied on these substrates with very low concentrations, and great enhancement factors for R6G (0.21 x 10(10)-4.09 x 10(11)) and 4-ATP (approximately 1.70 x 10(5)) were observed. It was found that the enhancement ability was affected by the DNA concentration and the electrostatic absorption time of the CTAB-stabilized silver nanoparticles on the DNA strands. These SERS substrates formed by the self-assembly of silver nanoparticles on DNA network also show good stability and reproducibility in our experiments.     

Stretching of single DNA molecules complexed with restriction endonuclease by Langmuir-Blodgett method

We have proposed a new technique for stretching single double-stranded DNA molecules on solid substrates by the Langmuir–Blodgett (LB) method. The polyion complex monolayer of a cationic amphiphile and DNA molecules formed at the air–water interface was transferred on a clean glass substrate. Vertical lifting up of the glass substrate provided the transferred monolayer consisting the stretched individual DNA molecules aligned parallel to the lifting direction on the glass. The DNA molecules complexed with the restriction endonuclease (EcoRI) were employed for stretching by using this method. Fluorescence images of the transferred monolayer showed that the EcoRI-binding DNA molecules could be stretched and immobilized on the glass substrate. A specific sequence of DNA recognized by EcoRI was detected as spatial positions of the stretched DNA molecules.     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I

Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule F?rster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations--open, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3'-5' exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair.     

6MAP, a fluorescent adenine analogue, is a probe of base flipping by DNA photolyase

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) in the presence of visible light. How PL recognizes and binds its substrate is still not well understood. Fluorescent nucleic acid base analogues are powerful probes of DNA structure. We have used the fluorescent adenine analogue 6MAP, a pteridone, to probe the local double helical structure of the CPD substrate when bound by photolyase. Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability. Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted. 6MAP shows a different quenching pattern compared to 2-aminopurine, another important adenine analogue, although both probes show that the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes. We also show that 6MAP/CPD duplexes are substrates for PL. Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory. Using these results, the F?rster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern. These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe. However, no experimental evidence for this process was observed in the steady-state emission spectra.     

Time-resolved events on the reaction pathway of transcript initiation by a single-subunit RNA polymerase: Raman crystallographic evidence

The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking nucleoside triphosphate (NTP) substrates and divalent cations into the RNAP and promoter DNA complex crystal, where the phosphodiester bond formation is completed in about 40 min. This slow reaction allowed us to monitor the changes of the RNAP and DNA conformations as well as bindings of substrate and metal through Raman spectra taken every 5 min. Recently published snapshot X-ray crystal structures along the same reaction pathway assisted the spectroscopic assignments of changes in the enzyme and DNA, while isotopically labeled NTP substrates allowed differentiation of the Raman spectra of bases in substrates and DNA. We observed that substrates are bound at 2-7 min after soaking is commenced, the O-helix completes its conformational change, and binding of both divalent metals required for catalysis in the active site changes the conformation of the ribose triphosphate at position +1. These are followed by a slower decrease of NTP triphosphate groups due to phosphodiester bond formation that reaches completion at about 15 min and even slower complete release of the divalent metals at about 40 min. We have also shown that the O-helix movement can be driven by substrate binding only. The kinetics of the in crystallo nucleotidyl transfer reaction revealed in this study suggest that soaking the substrate and metal into the RNAP-DNA complex crystal for a few minutes generates novel and uncharacterized intermediates for future X-ray and spectroscopic analysis.     

Electrostatic-assembly metallized nanoparticles network by DNA template

Eighteen-nanometer gold and 3.5-nm silver colloidal particles closely packed by cetyltrimethylammonium bromide (CTAB) to form its positively charged shell. The DNA network was formed on a mica substrate firstly. Later, CTAB-capped gold or silver colloidal solutions were cast onto DNA network surface. It was found that the gold or silver nanoparticles metallized networks were formed owing to the electrostatic-driven template assembling of positive charge of CTAB-capped gold and silver particles on the negatively charged phosphate groups of DNA molecules by the characterizations of AFM, XPS and UV-vis. This method may provide a novel and simple way to studying nanoparticles assembly conjugating DNA molecules and offer some potential promising applications in nanocatalysis, nanoelectronics, and nanosensor on the basis of the fabricated metal nanoparticles network.     

Collagen adsorption and structure on polymer surfaces observed by atomic force microscopy

The structure and adsorption patterns of type I and type III collagen were imaged on various polymer substrates with atomic force microscopy. Type I collagen had higher adsorption on polystyrene than on a series of polymethacrylates and formed a network of tightly, interwoven strands. Upon adsorption to different polymethacrylates, with varying side chain lengths, the collagen molecules formed long, branching fibrils. Types I and III collagen had different adsorption patterns, in some cases, on the identical substrate material. For example, instead of forming a tightly packed network, type III forms long, branching fibers on the polystyrene surface. On other materials, such as poly(n-butyl methacrylate), the two types of collagen showed similar adsorption pattern and structure. Adsorbed collagen was also imaged on various blends of polystyrene and polymethacrylates to determine how the polymer surface chemical structure and surface topography mediates protein adsorption.     

Rational Engineering of a Dynamic,Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy-driven DNA nanomachine with three-dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy-driven catalytic reaction of intramolecular toehold-mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye-labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction-based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity-induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

Effect of pH and applied potential on the adsorption of DNA on highly oriented pyrolytic graphite electrodes. Atomic force microscopy surface characterisation

Single-stranded and double-stranded DNA electrochemical biosensors prepared by adsorption during 3 min on HOPG, with or without an applied potential, at pH 5.3 and 7.0, were characterised by MAC mode AFM. During adsorption DNA condenses on the substrate forming complex network films with pores exposing the HOPG surface. The thin films formed in pH 5.3 acetate buffer always presented a better coverage of the HOPG surface with DNA molecules than films formed in pH 7.0 phosphate buffer. The application of a positive potential of 300 mV during adsorption enhanced the robustness and stability of the DNA films with the formation of bigger network holes and a more condensed and compact self-assembled DNA lattice. The knowledge of the morphology of adsorbed DNA on electrode surfaces explains non-specific adsorption on the electrode surface and can be used to improve and develop DNA-electrochemical biosensors.     

Rational Engineering of a Dynamic,Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy‐driven DNA nanomachine with three‐dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy‐driven catalytic reaction of intramolecular toehold‐mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye‐labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction‐based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity‐induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

On sub-T(g) dewetting of nanoconfined liquids and autophobic dewetting of crystallites

The glass transition temperature (T(g)) of thin films is reduced by nanoconfinement, but it is also influenced by the free surface and substrate interface. To gain more insights into their contributions, dewetting behaviors of n-pentane, 3-methylpentane, and toluene films are investigated on various substrates as functions of temperature and film thickness. It is found that monolayers of these molecules exhibit sub-T(g) dewetting on a perfluoro-alkyl modified Ni substrate, which is attributable to the evolution of a 2D liquid. The onset temperature of dewetting increases with film thickness because fluidity evolves via cooperative motion of many molecules; sub-T(g) dewetting is observed for films thinner than 5 monolayers. In contrast, monolayers wet substrates of graphite, silicon, and amorphous solid water until crystallization occurs. The crystallites exhibit autophobic dewetting on the substrate covered with a wetting monolayer. The presence of premelting layers is inferred from the fact that n-pentane crystallites disappear on amorphous solid water via intermixing. Thus, the properties of quasiliquid formed on the crystallite surface differ significantly from those of the 2D liquid formed before crystallization.     

Hydrophobic amino acid and single-atom substitutions increase DNA polymerase selectivity

DNA polymerase fidelity is of immense biological importance due to the fundamental requirement for accurate DNA synthesis in both replicative and repair processes. Subtle hydrogen-bonding networks between DNA polymerases and their primer/template substrates are believed to have impact on DNA polymerase selectivity. We show that deleting defined interactions of that kind by rationally designed hydrophobic substitution mutations can result in a more selective enzyme. Furthermore, a single-atom replacement within the DNA substrate through chemical modification, which leads to an altered acceptor potential and steric demand of the DNA substrate, further increased the selectivity of the developed systems. Accordingly, this study about the impact of hydrophobic alterations on DNA polymerase selectivity--enzyme and substrate wise--further highlights the relevance of shape complementary and polar interactions on DNA polymerase selectivity.     

An Efficient Bead‐captured Denaturation Method for Preparing Long Single‐stranded DNA

Previous methods to prepare single‐stranded DNA (ssDNA ) substrates are limited to short DNA lengths and inefficient. We have developed an efficient and rapid method to prepare long ssDNA substrates (up to 4000 nt) based on the denaturation of the bead‐captured DNA substrates, with the individual steps optimized. Immobilization of the targeted DNA substrates on the antibody‐modified beads allows easy separation of the denatured targeted ssDNA strand. This method also allows the recovery of the captured strand, making it possible to obtain two ssDNA strands from the same duplex DNA . Within 20 min, 80 nM of the 200 nt ssDNA strand could be obtained from its duplex DNA.     

Complete plastic nanofluidic devices for DNA analysis via direct imprinting with polymer stamps

Development of all polymer-based nanofluidic devices using replication technologies, which is a prerequisite for providing devices for a larger user base, is hampered by undesired substrate deformation associated with the replication of multi-scale structures. Therefore, most nanofluidic devices have been fabricated in glass-like substrates or in a polymer resist layer coated on a substrate. This letter presents a rapid, high fidelity direct imprinting process to build polymer nanofluidic devices in a single step. Undesired substrate deformation during imprinting was significantly reduced through the use of a polymer stamp made from a UV-curable resin. The integrity of the enclosed all polymer-based nanofluidic system was verified by a fluorescein filling experiment and translocation/stretching of λ-DNA molecules through the nanochannels. It was also found that the funnel-like design of the nanochannel inlet significantly improved the entrance of DNA molecules into nanochannels compared to an abrupt nanochannel/microfluidic network interface.     

New dialkyldithiophosphinic acid self-assembled monolayers (SAMs): influence of gold substrate morphology on adsorbate binding and SAM structure

We report the fabrication and characterization of new self-assembled monolayers (SAMs) formed from dihexadecyldithiophosphinic acid [(C(16))(2)DTPA] molecules on gold substrates. In these SAMs, the ability of the (C(16))(2)DTPA headgroup to chelate to the gold surface depends on the morphology of the gold substrate. Gold substrates fabricated by electron-beam evaporation (As-Dep gold) consist of ～50-nm grains separated by deep grain boundaries (～10 nm). These grain boundaries inhibit the chelation of (C(16))(2)DTPA adsorbates to the surface, producing SAMs in which there is a mixture of monodentate and bidentate adsorbates. In contrast, gold substrates produced by template stripping (TS gold) consist of larger grains (～200-500 nm) with shallower grain boundaries (<2 nm). On these substrates, the low density of shallow grain boundaries allows (C(16))(2)DTPA molecules to chelate to the surface, producing SAMs in which all molecules are bidentate. The content of bidentate adsorbates in (C(16))(2)DTPA SAMs formed on As-Dep and TS gold substrates strongly affects the SAM properties: Alkyl chain organization, wettability, frictional response, barrier properties, thickness, and thermal stability all depend on whether a SAM has been formed on As-Dep or TS gold. This study demonstrates that substrate morphology has an important influence on the structure of SAMs formed from these chelating adsorbates.     

A recoverable enzymatic microgel based on biomolecular recognition

The enzyme beta-galactosidase has been immobilized through incorporation into a selectively soluble microgel, prepared from DNA, biotinylated peptide nucleic acid (PNA), and the protein avidin. The enzyme was conjugated to avidin, allowing it to be integrated directly into the microgel network. Efficient hydrolysis of a small-molecule substrate occurred at 37 degrees , but cooling and centrifuging led to precipitation of the microgels and product separation. The microgels were then reconstituted by adding fresh buffer and shaking. The enzymatic activity was completely recovered through repeated cycles. This method should be generalizable to a wide variety of other enzymes and substrates.     

Attachment of Single-Stranded DNA to Certain SERS-Active Gold and Silver Substrates: Selected Practical Tips

Layers formed from single-stranded DNA on nanostructured plasmonic metals can be applied as “working elements” in surface–enhanced Raman scattering (SERS) sensors used to sensitively and accurately identify specific DNA fragments in various biological samples (for example, in samples of blood). Therefore, the proper formation of the desired DNA layers on SERS substrates is of great practical importance, and many research groups are working to improve the process in forming such structures. In this work, we propose two modifications of a standard method used for depositing DNA with an attached linking thiol moiety on certain SERS-active structures; the modifications yield DNA layers that generate a stronger SERS signal. We propose: (i) freezing the sample when forming DNA layers on the nanoparticles, and (ii) when forming DNA layers on SERS-active macroscopic silver substrates, using ω-substituted alkanethiols with very short alkane chains (such as cysteamine or mercaptopropionic acid) to backfill the empty spaces on the metal surface unoccupied by DNA. When 6-mercapto-1-hexanol is used to fill the unoccupied places on a silver surface (as in experiments on standard gold substrates), a quick detachment of chemisorbed DNA from the silver surface is observed. Whereas, using ω-substituted alkanethiols with a shorter alkane chain makes it possible to easily form mixed DNA/backfilling thiol monolayers. Probably, the significantly lower desorption rate of the thiolated DNA induced by alkanethiols with shorter chains is due to the lower stabilization energy in monolayers formed from such compounds.     

Fuzzy DNA Strand Displacement: A Strategy to Decrease the Complexity of DNA Network Design

Toehold‐mediated DNA strand displacement endows DNA nanostructures with dynamic response capability. However, the complexity of sequence design dramatically increases as the size of the DNA network increases. We attribute this problem to the mechanism of toehold‐mediated strand displacement, termed exact strand displacement (ESD), in which one input strand corresponds to one specific substrate. In this work, we propose an alternative to toehold‐mediated DNA strand displacement, termed fuzzy strand displacement (FSD), in which one‐to‐many and many‐to‐one relationships are established between the input strand and the substrate, to reduce the complexity. We have constructed four modules, termed converter, reporter, fuzzy detector, and fuzzy trigger, and demonstrated that a sequence pattern recognition network composed of these modules requires less complex sequence design than an equivalent network based on toehold‐mediated DNA strand displacement.     

Cationic lipid absorption on titanium: a counterion-mediated bilayer-to-lipid-tubule-network transition

By carefully tuning the screening effects of monovalent salt in solution, it is possible to control the formation of different lipid morphologies on a titanium dioxide substrate. A transition from an absorbed vesicle layer to fluid lipid bilayers is observed as a function of monovalent salt concentration in solution, and, above a threshold salt concentration, a network of lipid tubules with a fascinating distorted-hexagonal structure is formed in two dimensions on the substrate. We present fluorescence microscopy data for these different morphologies, confirming the connectivity and fluidity of the bilayers and tubules.