Binding feasibility and vibrational characteristics of single-strand spacer-added DNA and protein complexes

The information capacity of double-crossover (DX) tiles was successfully increased beyond a binary representation to higher base representations. By controlling the length and the position of DNA hairpins on the DX tile, ternary and senary (base-3 and base-6) digit representations were realized and verified by atomic force microscopy (AFM). Also, normal mode analysis (NMA) was carried out to study the mechanical characteristics of each structure.

The information capacity of DNA double-crossover (DX) tiles was successfully increased beyond a binary representation to higher base representations. By controlling the length and the position of DNA hairpins on the DX tile, ternary and senary (base-3 and base-6) digit representations were realized and verified by atomic force microscopy. Also, normal mode analysis was carried out to study the mechanical characteristics of each structure.

This work proposed a new sensing strategy for protease detection by converting a homogeneous assay into a surface-tethered electrochemical analysis. Streptavidin (SA), a tetramer protein, was used as the sensing unit based on the SA-biotin coupling chemistry. Caspase-3 was used as the model analyte, and a biotinylated peptide with a sequence of biotin-GDEVDGK-biotin was designed as the substrate. Specifically, the peptide substrate could induce an assembly of SA to form (SA-biotin-GDEVDGK-biotin)n aggregates through SA-biotin interactions, which was confirmed by atomic force microscopy (AFM). The peptide substrate-induced assembly of SA was facilely initiated on an electrode-liquid surface by modification of the electrode with SA. The in situ formation of (SA-biotin-GDEVDGK-biotin)n aggregates created an insulating layer, thus limiting the electron transfer of ferricyanide. Once the peptide substrate was cleaved into two shorter fragments (biotin-GDEVD and GK-biotin) by caspase-3, the resulting products would compete with biotin-GDEVDGK-biotin to bind SA proteins immobilized on the electrode surface and distributed in a solution, thus preventing the in situ formation of (SA-biotin-GDEVDGK-biotin)n assemblies. With the simple principle of the substrate-induced assembly of SA, a dual-signal amplification was achieved with improved sensitivity. Taking advantage of high sensitivity, simple principle, and easy operation, this method can be augmented to design various surface-tethered biosensors for practical applications.

Nature manifests diverse and complicated patterns through efficient physical, chemical, and biological processes. One of the approaches to generate complex patterns, as well as simple patterns, is the use of the cellular automata algorithm. However, there are certain limitations to produce such patterns experimentally due to the difficulty of finding candidate programmable building blocks. Here, we demonstrated the feasibility of generating an ocellated lizard skin-like pattern by simulation considering the probabilistic occurrence of cells and constructed the simulation results on DNA lattices via bottom-up self-assembly. To understand the similarity between the simulated pattern (SP) and the observed pattern (OP) of lizard skin, a unique configuration scheme (unit configuration was composed of 7 cells) was conceived. SPs were generated through a computer with a controlling population of gray and black cells in a given pattern. Experimental patterns (EPs) on DNA lattices, consisting of double-crossover (DX) tiles without and with protruding hairpins, were fabricated and verified through atomic force microscopy (AFM). For analyzing the similarity of the patterns, we introduced deviation of the average configuration occurrence for SP and EP with respect to OP, i.e., σα(SO) and σα(EO). The configuration and deviation provide characteristic information of patterns. We recognized that the minimum values of <σα(SO)> and <σα(EO)> occurred when 50% (55%) of black cells in given SPs (DX tiles with hairpins in given EPs) appeared to be most similar to the OP. Our study provides a novel platform for the applicability of DNA molecules to systematically demonstrate other naturally existing complex patterns or processes with ease.

Construction of various nanostructures with nanometre-scale precision through various DNA building blocks depends upon self-assembly, base-pair complementarity and sequence programmability. During annealing, unit tiles are formed by the complementarity of base pairs in each strand. Enhancement of growth of target lattices is expected if seed lattices (i.e. boundaries for growth of target lattices) are initially present in a test tube during annealing. Although most processes for annealing DNA nanostructures adopt a one-step high temperature method, multi-step annealing provides certain advantages such as reusability of unit tiles and tuneability of lattice formation. We can construct target lattices effectively (through multi-step annealing) and efficiently (via boundaries) by multi-step annealing and combining boundaries. Here, we construct efficient boundaries made of single, double, and triple double-crossover DNA tiles for growth of DNA lattices. Two unit double-crossover DNA tile-based lattices and copy-logic implemented algorithmic lattices were introduced to test the growth of target lattices on boundaries. We used multi-step annealing to tune the formation of DNA crystals during fabrication of DNA crystals comprised of boundaries and target lattices. The formation of target DNA lattices was visualized using atomic force microscopy (AFM). The borders between boundaries and lattices in a single crystal were clearly differentiable from AFM images. Our method provides way to construct various types of lattices in a single crystal, which might generate various patterns and enhance the information capacity in a given crystal.

Target-oriented cellular automata with computation are the primary challenge in the field of DNA algorithmic self-assembly in connection with specific rules. We investigate the feasibility of using the principle of cellular automata for mathematical subjects by using specific logic gates that can be implemented into DNA building blocks. Here, we connect the following five representative elementary functions: (i) enumeration of multiples of 2, 3, and 4 (demonstrated via R094, R062, and R190 in 3-input/1-output logic rules); (ii) the remainder of 0 and 1 (R132); (iii) powers of 2 (R129); (iv) ceiling function for n/2 and n/4 (R152 and R144); and (v) analogous pattern of annihilation (R184) to DNA algorithmic patterns formed by specific rules. After designing the abstract building blocks and simulating the generation of algorithmic lattices, we conducted an experiment as follows: designing of DNA tiles with specific sticky ends, construction of DNA lattices via a two-step annealing method, and verification of expected algorithmic patterns on a given DNA lattice using an atomic force microscope (AFM). We observed representative patterns, such as horizontal and diagonal stripes and embedded triangles, on the given algorithmic lattices. The average error rates of individual rules are in the range of 8.8% (R184) to 11.9% (R062), and the average error rate for all the rules was 10.6%. Interpretation of elementary functions demonstrated through DNA algorithmic patterns could be extended to more complicated functions, which may lead to new insights for achieving the final answers of functions with experimentally obtained patterns.

The fast and extensivegeneration of patterns using specific algorithmsis a major challenge in the field of DNA algorithmic self-assembly.Turing machines (TMs) are simple computable machines that executecertain algorithms using carefully designed logic gates. We investigateTuring algorithms for the generation of patterns on algorithmic latticesusing specific logic gates. Logic gates can be implemented into Turingbuilding blocks. We discuss comprehensive methods for designing Turingbuilding blocks to demonstrate an M-state and N-color Turing machine (M–N TM). The M-state and N-color (M–N = 1–1,2–1, and 1–2) TMs generate Turing patterns that canbe fabricated via DNA algorithmic self-assembly. The M–N TMs require two-input and three-outputlogic gates. We designed the head, tape, and transition rule tilesto demonstrate TMs for the 1–1, 2–1, and 1–2Turing algorithms. By analyzing the characteristics of the Turingpatterns, we classified them into two classes (DL and DR for statesgrown diagonally to the left and right, respectively) for the 1–1TM, three for the 2–1 TM, and nine for the 1–2 TM. Amongthese, six representative Turing patterns generated using rules R11-0and R11-1 for 1–1 TM, R21-01 and R21-09 for 2–1 TM,and R12-02 and R12-08 for 1–2 TM were constructed with DNAbuilding blocks. Turing patterns on the DNA lattices were visualizedby atomic force microscopy. The Turing patterns on the DNA latticeswere similar to those simulated patterns. Implementing the Turingalgorithms into DNA building blocks, as demonstrated via DNA algorithmicself-assembly, can be extended to a higher order of state and colorto generate more complicated patterns, compute arithmetic operations,and solve mathematical functions.

The effect of airway motion and breathing phase during imaging on CFD simulations of respiratory airflow

Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using "Self-assembling UNiversal (SUN) anchors" formed by DNA analog oligonucleotides, synthesized with an extension, a "sticky-end" that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.

What Came First: The Helix or the H2O?

Structural stability and electrical characteristic of DNA lattices doped with lanthanide ions

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

Molecular Dynamics Study for Streptavidin Mutant With/Without Biotin Analog

The ability of streptavidin (SA) to simultaneously bind four biotins is often used in linker layers, where a biotinylated molecule is linked to a biotin-functionalized surface via SA. For biosensor and array applications, it is desirable that the SA linker layer be stable to drying and rehydration. In this study it was observed that a significant decrease in binding capacity of a SA layer occurred when that layer was dried. For this study a SA linker layer was constructed by binding SA to a biotin-containing alkylthiolate monolayer (BAT/OEG) self-assembled onto gold. Its stability after drying was investigated using surface plasmon resonance (SPR). Approximately a quarter of the SA layer was removed from the BAT/OEG surface upon drying and rehydration, suggesting disruption of SA-biotin binding when dry. This resulted in the dried SA layer losing approximately 40% of its biotinylated ferritin (BF) binding capacity. Coating the layer with trehalose before drying was found to inhibit the loss of SA from the BAT/OEG surface. SPR showed that the trehalose-protected SA linker layer retained approximately 91% of its original BF binding capacity after drying and rehydration. Atomic force microscopy, which was used to image individual surface-bound SA and BF molecules, qualitatively confirmed these observations.

APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.