Micro- and nanofluidics for DNA analysis

Miniaturization to the micrometer and nanometer scale opens up the possibility to probe biology on a length scale where fundamental biological processes take place, such as the epigenetic and genetic control of single cells. To study single cells the necessary devices need to be integrated on a single chip; and, to access the relevant length scales, the devices need to be designed with feature sizes of a few nanometers up to several micrometers. We will give a few examples from the literature and from our own research in the field of miniaturized chip-based devices for DNA analysis, including dielectrophoresis for purification of DNA, artificial gel structures for rapid DNA separation, and nanofluidic channels for direct visualization of single DNA molecules.     

The DNA double helix fifty years on

This year marks the 50th anniversary of the proposal of a double helical structure for DNA by James Watson and Francis Crick. The place of this proposal in the history and development of molecular biology is discussed. Several other discoveries that occurred in the middle of the twentieth century were perhaps equally important to our understanding of cellular processes; however, none of these captured the attention and imagination of the public to the same extent as the double helix. The existence of multiple forms of DNA and the uses of DNA in biological technologies is presented. DNA is also finding increasing use as a material due to its rather unusual structural and physical characteristics as well as its ready availability.     

Dynamics and biological relevance of epigenetic N6-methyladenine DNA modification in eukaryotic cells

DNA methylation represents a major type of DNA modifications that play key roles in diverse biological processes. With the recent development of highly selective and sensitive bioanalytical techniques,N⁶-methyladenine（6mA） has been characterized as an important internal DNA modification dynamically occurring in multiple eukaryotes including humans. Increasing evidence has indicated that 6mA may act as a novel epigenetic modification involved in regulation of development, stress respon...     

Biological consequences associated with DNA oxidation mediated by singlet oxygen.

Singlet oxygen is a major oxidative species that can be generated by numerous biological processes such as photosensitization. This oxidant can react with deoxyguanosine and with guanine in deoxyribonucleic acid (DNA) leading to the induction of at least four different reaction products such as 4,8-dihydro-4-hydroxy-8-oxodeoxyguanosine and 7,8-dihydro-8-oxodeoxyguanosine. The induction of true single-stranded breaks in the oxidated DNA is still a matter of controversy and is not yet clearly established. This paper focuses mainly on several biological consequences which can be associated with the induction of DNA lesions by singlet oxygen. Oxidated DNA loses its transformation efficiency probably because unrepaired lesions can partially inhibit DNA replication. Mutagenesis is one of the main effects induced by guanine oxidation products. Molecular analysis of mutated genes reveals that G to T transversions are the most frequent mutations; these are probably introduced in DNA by misincorporation of deoxyadenosine monophosphate (dAMP) opposite to the lesion. Efficient repair of these oxidated guanine residues can take place via specific glycosylase, endonuclease or the SOS network. However, the data concerning the toxicity of singlet oxygen for eukaryotic cells are not frequent enough in the literature to draw a clear picture of the effects of this activated species in several biologically revelant phenomena.     

DNA-mediated electron transfer from a modified base to ethidium: π-stacking as a modulator of reactivity

Background: The DNA double helix is composed of an array of aromatic heterocyclic base pairs and, as a molecular π-stack, represents a novel system for studying long-range electron transfer. Because many base damage and repair processes result from electron-transfer reactions, the ability of DNA to mediate charge transport holds important biological implications. Seemingly contradictory conclusions have been drawn about electron transfer in DNA from the many different studies that have been carried out. These studies must be reconciled so that this phenomenon can be understood both at a fundamental level and in the context of biological systems.Results: The photoinduced oxidation of a modified base, 7-deazaguanine, has been examined as a function of distance, sequence, and base stacking in DNA duplexes covalently modified with ethidium. Over ethidium/deazaguanine separations of 6–27 Å, the photooxidation reaction proceeded on a subnanosecond time scale, and the quenching yield exhibited a shallow distance dependence. The efficiency of the reaction was highly sensitive to small changes in base composition. Moreover, the overall distance-dependence of the reaction is sensitive to sequence, despite the constancy of photoexcited ethidium as acceptor.Conclusions: The remarkable efficiency of deazaguanine photooxidation by intercalated ethidium over long distances provides new evidence for fast electron-transfer pathways through DNA. By varying sequence as well as reactant separation, this work provides the first experimental demonstration of the importance of reactant stacking in the modulation of long-range DNA-mediated electron transfer.     

Folding dynamics of cytochrome C using pulse radiolysis

Pulse radiolysis is a powerful method to realize real-time observation of various redox processes, which induces various structural and functional changes occurring in biological systems. However, its application has been mainly limited to studies of the redox reactions of rather smaller biological systems such as DNA because of an undesired reaction due to various free radicals generated by pulse radiolysis. For application of pulse radiolysis to generate plenty of redox reactions of biological systems, selective redox reactions induced by electron pulses have to be developed. In this study, we report that in the presence of the high concentration of the denaturant, guanidine HCl (GdHCl), the selective reduction of the oxidized cytochrome c (Cyt c) takes place in time scales of a few microseconds by the electron transfer from the guanidine radical that is formed by the fast reaction of e(aq)(-) with GdHCl, consequently leading to folding kinetics of Cyt c. By providing insight into the folding dynamics of Cyt c, we show that the pulse radiolysis technique can be used to track the folding dynamics of various biomolecules in the presence of a denaturant including GdHCl.     

A study of the behavior of histone H1 and its complex with DNA by the spin-label method

The behavior of the tyrosine-72 residue of histone H1 has been studied by the spin-label method as a function of the ionic strength of the solution and of the temperature and on its interaction with DNA. It has been shown that in the formation of complexes of histone H1 with DNA the globular part of the protein not directly interacting with the nucleic acid retains a definite conformation enabling it to participate in various processes taking place in the chromatin.     

Nucleic Acid-based Enzyme Cascades—Current Trends and Future Perspectives

The natural micro- and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.     

Programmable Assembly of DNA-protein Hybrid Structures

DNA is the genetic information carrier for most known living organisms on Earth,while proteins are the functional component that carry out most biological processes.Many natural machineries are DNA-protein hybrid complexes to cooperatively and efficiently conduct sophisticated biological tasks.It has drawn increasing interest to the research field to construct artificial DNA-protein hybrid structures towards a variety of applications including biological studies,nanofabrication,biomedical research,etc.In this regard,here in this report we reviewed the up-to-date progress on making DNA-protein hybrid structures,with a particular focus on DNA nanotechnology-enabled programmable assembly of DNA-protein hybrid structures.     

PETITE INDUCTION IN YEAST, Saccharomyces cerevisiae, BY PHOTOACTIVATION OF 3-A-ZIDO-6-A-MINO-10-M-ETHYLACRIDINIUM CHLORIDE

Abstract— The photoinduction of petite colonies and cell toxicity in non-growing yeast, Saccharomyces cerevisiae , by 3-a-zido-6-a-mino-10-m-ethylacridinium chloride (AAMAC) has been examined. The results presented here indicate that mitochondrial DNA damage in resting yeast which occurs following irradiation of AAMAC-treated cells for short time periods is probably mediated through a covalent adduct between AAMAC and DNA. Furthermore, the photoreaction which contributes to biological activity is dependent on the presence of oxygen. Pre-irradiated AAMAC, which no longer exhibited the short-term photo-induction of biological effects showed a second biological activity. In this case longer irradiation times, e.g. 30 min, were required to induce petites for resting yeast. Again there was a strong dependence on the presence of oxygen. These results suggest that both processes may be effected through oxygen intermediates (photodynamic processes).     

Optical fluid and biomolecule transport with thermal fields

A long standing goal is the direct optical control of biomolecules and water for applications ranging from microfluidics over biomolecule detection to non-equilibrium biophysics. Thermal forces originating from optically applied, dynamic microscale temperature gradients have shown to possess great potential to reach this goal. It was demonstrated that laser heating by a few Kelvin can generate and guide water flow on the micrometre scale in bulk fluid, gel matrices or ice without requiring any lithographic structuring. Biomolecules on the other hand can be transported by thermal gradients, a mechanism termed thermophoresis, thermal diffusion or Soret effect. This molecule transport is the subject of current research, however it can be used to both characterize biomolecules and to record binding curves of important biological binding reactions, even in their native matrix of blood serum. Interestingly, thermophoresis can be easily combined with the optothermal fluid control. As a result, molecule traps can be created in a variety of geometries, enabling the trapping of small biomolecules, like for example very short DNA molecules. The combination with DNA replication from thermal convection allows us to approach molecular evolution with concurrent replication and selection processes inside a single chamber: replication is driven by thermal convection and selection by the concurrent accumulation of the DNA molecules. From the short but intense history of applying thermal fields to control fluid flow and biological molecules, we infer that many unexpected and highly synergistic effects and applications are likely to be explored in the future.     

Constant Cwall ultrafiltration process control

Ultrafiltration processes normally operate with constant transmembrane pressure. The tradition of such control derives from its inherent simplicity. Both fundamental and practical considerations suggest, however, that ultrafiltration processes should be controlled by maintaining a constant wall concentration (C_w) of fully retained solutes. Since protein sieving, solubility, and adsorption losses as well as time and area optimization are dependent on C_w, we investigated a control strategy using constant C_w instead of constant transmembrane pressure. We explored three different strategies for such control and evaluated the theoretical and industrial implications for single solute systems. The effects of solute wall concentration on process time and product yield were also evaluated. Implementation of this technology required the development of a novel methodology for determination of mass transfer coefficients. The use of C_w technology also led to the development of new optimization schemes for both concentration and diafiltration. Industrial scale processes using constant C_w control have been successfully implemented on several recombinant DNA derived human protein pharmaceuticals. Constant C_w control has eliminated variability in process time, enhanced product yields, and provided insurance of tight protein product quality specifications. Optimum process design based on fundamental filtration theory has replaced empirical development procedures.     

Photochemical approach to probing different DNA structures

The various conformations of DNA--the A, B, and Z forms, the protein-induced DNA kink, and the G-quartet form--are thought to play important biological roles in processes such as DNA replication, gene expression and regulation, and the repair of DNA damage. The investigation of local DNA conformational changes associated with biological events is therefore essential for understanding the function of DNA. In this Minireview, we discuss the use of photochemical dehalogenation of 5-halouracil-containing DNA to probe the structure of DNA. Hydrogen abstraction by the resultant uracil-5-yl radicals is atom-specific and highly dependent on the structure of the DNA, suggesting that this photochemical approach could be applied as a probe of DNA conformations in living cells.     

Controlled rotation of biological micro- and nano-particles in microvortices

Micrometer-sized re-circulating flows generated in a microfluidic system are used to drive the controlled rotation of biological particles of both micro- and nano-meter scale dimensions. This technique is independent of the intrinsic nature of the particle, and possesses the potential to rotate particles at high rates. We demonstrate in such microvortices the orientation control of single DNA molecules, and the axial rotation of biological cells in which the cellular contents were visibly affected by rotation.     

DNA single strands tethered to fused quartz/water interfaces studied by second harmonic generation

Second harmonic generation (SHG) is used to study oligonucleotides at aqueous/solid interfaces for the first time. Detailed thermodynamic state information for interfacial DNA single strands, namely, the interfacial charge density, the interfacial potential, and the change in the interfacial energy density, is obtained. The phosphate groups on the DNA backbone serve as intrinsic labels that do not require DNA modification other than surface attachment. This approach is broadly applicable for the investigation of DNA during its interaction with biological targets, as well as charged biopolymers in general, and has important implications for predicting and controlling macromolecular interactions, improving biodiagnostics, and understanding life processes.     

Vibration-actuated drop motion on surfaces for batch microfluidic processes

When a liquid drop is subjected to an asymmetric lateral vibration on a nonwettable surface, a net inertial force acting on the drop causes it to move. The direction and velocity of the drop motion are related to the shape, frequency, and amplitude of vibration, as well as the natural harmonics of the drop oscillation. Aqueous drops can be propelled through fluidic networks connecting various unit operations in order to carry out batch processing at the miniature scale. We illustrate the integration of several unit operations on a chip: drop transport, mixing, and thermal cycling, which are precursor steps to carrying out advanced biological processes at microscale, including cell sorting, polymerase chain reaction, and DNA hybridization.     

Femtochemistry: Atomic-Scale Dynamics of the Chemical Bond Using Ultrafast Lasers (Nobel Lecture) Copyright((c)) The Nobel Foundation 2000. We thank the Nobel Foundation, Stockholm, for permission to print this lecture

Over many millennia, humankind has thought to explore phenomena on an ever shorter time scale. In this race against time, femtosecond resolution (1 fs=10(-15) s) is the ultimate achievement for studies of the fundamental dynamics of the chemical bond. Observation of the very act that brings about chemistry-the making and breaking of bonds on their actual time and length scales-is the wellspring of the field of femtochemistry, which is the study of molecular motions in the hitherto unobserved ephemeral transition states of physical, chemical, and biological changes. For molecular dynamics, achieving this atomic-scale resolution using ultrafast lasers as strobes is a triumph, just as X-ray and electron diffraction, and, more recently, STM and NMR spectroscopy, provided that resolution for static molecular structures. On the femtosecond time scale, matter wave packets (particle-type) can be created and their coherent evolution as a single-molecule trajectory can be observed. The field began with simple systems of a few atoms and has reached the realm of the very complex in isolated, mesoscopic, and condensed phases, as well as in biological systems such as proteins and DNA structures. It also offers new possibilities for the control of reactivity and for structural femtochemistry and femtobiology. This anthology gives an overview of the development of the field from a personal perspective, encompassing our research at Caltech and focusing on the evolution of techniques, concepts, and new discoveries.     

Programming DNA-Based Systems through Effective Molarity Enforced by Biomolecular Confinement

The fundamental concept of effective molarity is observed in a variety of biological processes, such as protein compartmentalization within organelles, membrane localization and signaling paths. To control molecular encountering and promote effective interactions, nature places biomolecules in specific sites inside the cell in order to generate a high, localized concentration different from the bulk concentration. Inspired by this mechanism, scientists have artificially recreated in the lab the same strategy to actuate and control artificial DNA-based functional systems. Here, it is discussed how harnessing effective molarity has led to the development of a number of proximity-induced strategies, with applications ranging from DNA-templated organic chemistry and catalysis, to biosensing and protein-supported DNA assembly.