Rational Design of pH‐Responsive DNA Motifs with General Sequence Compatibility

Reconfigurable molecular events are key to molecular machines. In response to external cues, molecular machines rearrange/change their structures to perform certain functions. Such machines exist in nature, for example cell surface receptors, and have been artificially engineered. To be able to build sophisticated and efficient molecular machines for an increasing range of applications, constant efforts have been devoted to developing new mechanisms of controllable structural reconfiguration. Herein, we report a general design principle for pH‐responsive DNA motifs for general DNA sequences (not limited to triplex or i‐motif forming sequences). We have thoroughly characterized such DNA motifs by polyacrylamide gel electrophoresis (PAGE) and fluorescence spectroscopy and demonstrated their applications in dynamic DNA nanotechnology. We expect that it will greatly facilitate the development of DNA nanomachines, biosensing/bioimaging, drug delivery, etc.     

Dynamic Reconfigurable DNA Nanostructures,Networks and Materials

DNA nanotechnology relies on the structural and functional information encoded in nucleic acids. Specifically, the sequence-guided reconfiguration of nucleic acids by auxiliary triggers provides a means to develop DNA switches, machines and stimuli-responsive materials. The present Review addresses recent advances in the construction and applications of dynamic reconfigurable DNA nanostructures, networks and materials. Dynamic transformations proceeding within engineered origami frames or between origami tiles, and the triggered dynamic reconfiguration of scaled supramolecular origami structures are addressed. The use of origami frameworks to assemble dynamic chiroplasmonic optical devices and to operate switchable chemical processes are discussed. Also, the dynamic operation of DNA networks is addressed, and the design of “smart” stimuli-responsive all-DNA materials and their applications are introduced. Future perspectives and applications of dynamic reconfigurable DNA nanostructures are presented.     

The concept of molecular machinery is useful for design of stimuli-responsive gene delivery systems in the mammalian cell

Since the first generation of molecular machines including photoresponsive crown ethers and its analogues was reported by Shinkai et al., a huge number of molecular machines exhibiting dynamic chemical and physical functions have been designed and developed. On the other hand, non-viral vectors are desired to possess conflicting properties to associate with DNA until reaching the nucleus as their final destination and dissociate from DNA there. In other words, non-viral vectors should work as a sort of molecular machinery. To overcome this dilemma, recently, much attention is focused on the development of the intelligent vectors, also called as ‘stimuli responsive vectors’ working as molecular machines. In this review, stimulus responsive gene delivery systems in which some structural factors and/or physiological properties are regulated in response to extracellular signals such as redox, pH, ultrasound, light, temperature, etc. are introduced as a new generation of non-viral vectors. These extracellular signals such as ultrasound, light, and temperature can be potent stimuli capable of site-, timing-, and duration-specific gene expression. This is a paper selected for “HGCS Japan Award of Excellence 2006”.     

Ratiometric DNA Walking Machine for Accurate and Amplified Bioassay

DNA walking machines opened new avenues for the biosensing and demonstrated great success in the past few years. Since DNA machines are mainly nonequilibrium systems driven by dynamic interactions, the matrix effects on DNA machines is a bottleneck and more intricate than common DNA-mediated assays, especially for complicated physiological samples. Herein, to realize an accurate and reliable quantitative machine, a ratiometric DNA walking machine was developed in human serums and cell lysates based on the elemental isotope ratio measurement. The target DNA-triggered walking machine converted and amplified biological signals into mass spectrometric signal ratios (¹⁹⁷Au/¹¹⁵In) via a burnt-bridge mechanism. Under the optimized conditions, the limit of detection (LOD, 3σ) was 8 fM for target DNA, with a dynamic linear range of 0.05–0.7 pM. The ratiometric DNA walking machine was directly applied in human serum samples with satisfactory recoveries of 94 to 105 %, demonstrating an excellent stability and a high accuracy. Combining the aptamer-based specific recognition, the proposed DNA machine is expected to be a versatile platform for other targets, such as small biomolecules and proteins.     

Towards artificial muscles at the nanometric level

The authors describe their study of molecular systems suited to the fabrication of machines and (rotory or linear) motors at the molecular level. They indicate that a future application of these molecular'muscles'could be in the area of information storage and processing.     

Artificial molecular-level machines

The concept of "machine" can be extended to the molecular level by designing supramolecular species capable of performing mechanical-like movements as a consequence of an appropriate energy supply. Molecular-level machines operate via electronic and nuclear rearrangements, for example, through some kind of chemical reaction. Like macroscopic machines, they are characterized by: (i) the kind of energy input supplied to make them work, (ii) the kind of movement performed by their components, (iii) the way in which their operation can be controlled and monitored, (iv) the possibility to repeat the operation at will and establish a cyclic process, (v) the time scale needed to complete a cycle of operation, and (vi) the function performed. A crucial issue is that concerning energy supply. Artificial machines powered by chemical energy ("fuels") produce waste products whose accumulation compromises the operation of the machine unless they are removed from the system. Photochemical and electrochemical energy inputs, however, can be used to make a machine work without formation of waste products. Examples of chemically, electrochemically, and photochemically powered machines investigated in our laboratory are reviewed, and future directions for the construction of novel machines are illustrated. The two most interesting kinds of applications of molecular-level machines are related to the mechanical aspect, which can be exploited, for example, for molecular-level transportation purposes, and the logic aspect, which can be exploited for information processing at the molecular level and, in the long run, for the construction of molecular level (chemical) computers.     

Platinum-DNA Origami Hybrid Structures in Concentrated Hydrogen Peroxide

The DNA origami technique allows fast and large-scale production of DNA nanostructures that stand out with an accurate addressability of their anchor points. This enables the precise organization of guest molecules on the surfaces and results in diverse functionalities. However, the compatibility of DNA origami structures with catalytically active matter, a promising pathway to realize autonomous DNA machines, has so far been tested only in the context of bio-enzymatic activity, but not in chemically harsh reaction conditions. The latter are often required for catalytic processes involving high-energy fuels. Here, we provide proof-of-concept data showing that DNA origami structures are stable in 5 % hydrogen peroxide solutions over the course of at least three days. We report a protocol to couple these to platinum nanoparticles and show catalytic activity of the hybrid structures. We suggest that the presented hybrid structures are suitable to realize catalytic nanomachines combined with precisely engineered DNA nanostructures.     

An Exonuclease III‐Powered,On‐Particle Stochastic DNA Walker

DNA‐based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)‐powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)‐based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt‐bridge mechanism. The operation of this Exo III‐propelled DNA walker was monitored in real time and at the single‐particle resolution using total internal reflection fluorescence microscopy (TIRF). We further interrogated the morphological effect of the 3D track on the nuclease activity, which suggested that the performance of the DNA walker was critically dependent upon the DNA density and the track conformation. Finally, we demonstrated potential bioanalytical applications of this SNA‐based stochastic DNA walker by exploiting movement‐triggered cascade signal amplification.     

An Exonuclease III‐Powered,On‐Particle Stochastic DNA Walker

DNA‐based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)‐powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)‐based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt‐bridge mechanism. The operation of this Exo III‐propelled DNA walker was monitored in real time and at the single‐particle resolution using total internal reflection fluorescence microscopy (TIRF). We further interrogated the morphological effect of the 3D track on the nuclease activity, which suggested that the performance of the DNA walker was critically dependent upon the DNA density and the track conformation. Finally, we demonstrated potential bioanalytical applications of this SNA‐based stochastic DNA walker by exploiting movement‐triggered cascade signal amplification.     

DNAzymes for sensing, nanobiotechnology and logic gate applications

Catalytic nucleic acids (DNAzymes or ribozymes) are selected by the systematic evolution of ligands by exponential enrichment process (SELEX). The catalytic functions of DNAzymes or ribozymes allow their use as amplifying labels for the development of optical or electronic sensors. The use of catalytic nucleic acids for amplified biosensing was accomplished by designing aptamer-DNAzyme conjugates that combine recognition units and amplifying readout units as in integrated biosensing materials. Alternatively, "DNA machines" that activate enzyme cascades and yield DNAzymes were tailored, and the systems led to the ultrasensitive detection of DNA. DNAzymes are also used as active components for constructing nanostructures such as aggregated nanoparticles and for the activation of logic gate operations that perform computing.     

Editorial: DNA-based nanoarchitectures and nanomachines

The emerging area of DNA-based architectures and machines promises exciting opportunities and will impact on the future of DNA structures in nanobiotechnology.     

Mechanically interlocked molecules incorporating cucurbituril and their supramolecular assemblies

Mechanically interlocked molecules incorporating cucurbituril (CB[6]) as a molecular 'bead' and their supramolecular assemblies are described. An efficient synthesis of 1D, 2D and 3D polyrotaxanes with high structural regularity and molecular necklaces has been achieved by a combination of self-assembly and coordination chemistry. The functional aspects of these interlocked molecules and their supramolecular assemblies, including molecular machines and switches based on [2]rotaxanes, a 2D polyrotaxane with large cavities and channels, pseudorotaxane-terminated dendrimers, and interaction of pseudorotaxanes containing polyamines and CB[6] with DNA are also described.     

DNA-Topology Simplification by Topoisomerases

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.     

A high-performance multilane microdevice system designed for the DNA forensics laboratory

We report preliminary testing of "GeneTrack", an instrument designed for the specific application of multiplexed short tandem repeat (STR) DNA analysis. The system supports a glass microdevice with 16 lanes of 20 cm effective length and double-T cross injectors. A high-speed galvanometer-scanned four-color detector was specially designed to accommodate the high elution rates on the microdevice. All aspects of the system were carefully matched to practical crime lab requirements for rapid reproducible analysis of crime-scene DNA evidence in conjunction with the United States DNA database (CODIS). Statistically significant studies demonstrate that an absolute, three-sigma, peak accuracy of 0.4-0.9 base pair (bp) can be achieved for the CODIS 13-locus multiplex, utilizing a single channel per sample. Only 0.5 microL of PCR product is needed per lane, a significant reduction in the consumption of costly chemicals in comparison to commercial capillary machines. The instrument is also designed to address problems in temperature-dependent decalibration and environmental sensitivity, which are weaknesses of the commercial capillary machines for the forensics application.     

A family of novel DNA sequencing instruments based on single-photon detection

We have developed a family of high-performance capillary DNA sequencing instruments based on a novel multicolor fluorescent detection technology. This technology is based on two technical innovations: the multilaser excitation of fluorescence of labeled DNA fragments and the "color-blind" single-photon detection of modulated fluorescence. Our machines employ modern digital and broadband techniques that are essential for achieving superior instrument performance. We discuss the design and testing results for several versions of the automated single lane DNA sequencers, as well as our approach to scaling up to multilane instruments.     

Dynamic range of fluorescence detection and base-calling accuracy in DNA sequencer based on single-photon counting.

Recently, we developed a family of high-performance automated capillary DNA sequencing instruments based on a single-photon detection of fluorescently labeled DNA fragments. Our machines employ digital and broadband techniques, essential for achieving superior instrument sensitivity and dynamic range. In the present paper, we discuss limitations of the instrument's performance caused by the nonlinearity of single-photon detectors as well as methods for nonlinearity compensation which increase the detection dynamic range and base-calling accuracy.     

Structure and function of noncanonical nucleobases

DNA and RNA contain, next to the four canonical nucleobases, a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m(5) dC, hm(5) dC, f(5) dC und ca(5) dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified non-canonical nucleosides in both DNA and RNA.