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Efficient fabrication of structurally and functionally diverse nanomolecular devices and machines by organizing separately prepared DNA origami building blocks into a larger structure is limited by origami attachment yields. A general method that enables attachment of origami building blocks using 'sticky ends' at very high yields is demonstrated. Two different rectangular origami monomers are purified using agarose gel electrophoresis conducted in solute containing 100 × 10-3 m NaCl, a treatment that facilitates the dissociation of most of the incorrectly hybridized origami structures that form through blunt-end interactions during the thermal annealing process and removes these structures as well as excess strands that otherwise interfere with the desired heterodimerization reaction. Heterodimerization yields of gel-purified monomers are between 98.6% and 99.6%, considerably higher than that of monomers purified using the polyethylene glycol (PEG) method (88.7-96.7%). Depending on the number of PEG purification rounds, these results correspond to about 4- to 25-fold reduction in the number of incorrect structures observed by atomic force microscopy. Furthermore, the analyses of the incorrect structures observed before and after the heterodimerization reactions and comparison of the purification methods provide valuable information on the reaction mechanisms that interfere with heterodimerization.

Organizing DNA origami building blocks into higher order structures is essential for fabrication of large structurally and functionally diverse devices and molecular machines. Unfortunately, the yields of origami building block attachment reactions are typically not sufficient to allow programed assembly of DNA devices made from more than a few origami building blocks. To investigate possible reasons for these low yields, a detailed single-molecule fluorescence study of the dynamics of rectangular origami dimerization and origami dimer dissociation reactions is conducted. Reactions kinetics and yields are investigated at different origami and ion concentrations, for different ion types, for different lengths of bridging strands, and for the "sticky end" and "weaving welding" attachment techniques. Dimerization yields are never higher than 86%, which is typical for such systems. Analysis of the dynamic data shows that the low yield cannot be explained by thermodynamic instability or structural imperfections of the origami constructs. Atomic force microscopy and gel electrophoresis evidence reveal self-dimerization of the origami monomers, likely via blunt-end interactions made possible by the presence of bridging strands. It is suggested that this mechanism is the major factor that inhibits correct dimerization and means to overcome it are discussed.

Realization of bioinspired molecular machines that can perform many and diverse operations in response to external chemical commands is a major goal in nanotechnology, but current molecular machines respond to only a few sequential commands. Lack of effective methods for introduction and removal of command compounds and low efficiencies of the reactions involved are major reasons for the limited performance. We introduce here a user interface based on a microfluidics device and single-molecule fluorescence spectroscopy that allows efficient introduction and removal of chemical commands and enables detailed study of the reaction mechanisms involved in the operation of synthetic molecular machines. The microfluidics provided 64 consecutive DNA strand commands to a DNA-based motor system immobilized inside the microfluidics, driving a bipedal walker to perform 32 steps on a DNA origami track. The microfluidics enabled removal of redundant strands, resulting in a 6-fold increase in processivity relative to an identical motor operated without strand removal and significantly more operations than previously reported for user-controlled DNA nanomachines. In the motor operated without strand removal, redundant strands interfere with motor operation and reduce its performance. The microfluidics also enabled computer control of motor direction and speed. Furthermore, analysis of the reaction kinetics and motor performance in the absence of redundant strands, made possible by the microfluidics, enabled accurate modeling of the walker processivity. This enabled identification of dynamic boundaries and provided an explanation, based on the "trap state" mechanism, for why the motor did not perform an even larger number of steps. This understanding is very important for the development of future motors with significantly improved performance. Our universal interface enables two-way communication between user and molecular machine and, relying on concepts similar to that of solid-phase synthesis, removes limitations on the number of external stimuli. This interface, therefore, is an important step toward realization of reliable, processive, reproducible, and useful externally controlled DNA nanomachines.

We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET-ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker's leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed in situ structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology.

The stability of the nucleosome core particle (NCP) is believed to play a major role in regulation of gene expression. To understand the mechanisms that influence NCP stability, we studied stability and dissociation and association kinetics under different histone protein (NCP) and NaCl concentrations using single-pair Förster resonance energy transfer and alternating laser excitation techniques. The method enables distinction between folded, unfolded, and intermediate NCP states and enables measurements at picomolar to nanomolar NCP concentrations where dissociation and association reactions can be directly observed. We reproduced the previously observed nonmonotonic dependence of NCP stability on NaCl concentration, and we suggest that this rather unexpected behavior is a result of interplay between repulsive and attractive forces within positively charged histones and between the histones and the negatively charged DNA. Higher NaCl concentrations decrease the attractive force between the histone proteins and the DNA but also stabilize H2A/H2B histone dimers, and possibly (H3/H4)2 tetramers. An intermediate state in which one DNA arm is unwrapped, previously observed at high NaCl concentrations, is also explained by this salt-induced stabilization. The strong dependence of NCP stability on ion and histone concentrations, and possibly on other charged macromolecules, may play a role in chromosomal morphology.

Prebiotic chemistry presumably took place before formation of an oxygen-rich atmosphere and thus under conditions of intense short wavelength UV irradiation. Therefore, the UV photochemical stability of the molecular building blocks of life may have been an important selective factor in determining the eventual chemical makeup of critical biomolecules. To investigate the role of UV irradiation in base-pairing we have studied guanine (G) and cytosine (C) base pairs in the absence of the RNA backbone. We distinguished base-pair structures by IR–UV hole-burning spectroscopy as well as by high-level correlated ab initio calculations. The Watson–Crick structure exhibits broad UV absorption, in stark contrast to other GC structures and other base-pair structures. This broad absorption may be explained by a rapid internal conversion that makes this specific base pair arrangement uniquely photochemically stable.

The vibronic spectra of laser desorbed and jet cooled guanine (G) adenine (A), and cytosine (C) consist of bands from four, two and two major tautomers respectively, as revealed by UV-UV and IR-UV double resonance spectroscopy. The vibronic spectrum of adenine around 277 nm consists of weak nπ* and strong ππ* transitions, based on IR-UV and deuteration experiments. Precise ionization potentials of G and A were determined with 2-color, 2-photon ionization. We also measured vibronic and IR spectra of several base pairs. GC exhibits a HNH ... OH/NH ... N/C=O ... HNH bonding similar to the Watson-Crick GC base pair but with C as enol tautomer. One GG isomer exhibits non-symmetric hydrogen bonding with HNH ... N/NH ... N/C=O ... HNH interactions. A second observed GG isomer has a symmetrical hydrogen bond arrangement with C=O ... NH/NH ... O=C bonding. Two CC isomers were observed with symmetrical C=O ... NH/NH ... O=C bonding and nonsymmetrical C=O ... HNH/NH ... N interaction, respectively. Guanosine (Gs), 2-DeoxyGs und 3-DeoxyGs each exhibit only one isomer in the investigated wavelength range around 290 nm with a strong intramolecular sugar(5-OH) ... enolguanine(3-N) hydrogen bond.

We report resonant two-photon ionization spectra of laser desorbed, jet cooled, cytosine, 1-methyl cytosine, 5-methyl cytosine, and dimers of these. Unlike other pyrimidine bases, cytosine exhibits vibronic spectra with sharp features in two spectral regions, separated by about 5000 cm 1 . We interpret these as being due to two tautomeric forms, one keto and one enol. The dimers absorb at wavelengths that are intermediate between those of the two monomer forms. By UV–UV hole burning we determined the numbers of isomers contributing to each spectrum and by delayed two color ionization we determined triplet lifetimes. We observed hydrogen transfer between bases both in collisions between monomers and after photo-excitation in clusters. 2002 Published by Elsevier Science B.V.

We present R2PI, IR–UV and UV–UV double resonance measurements of the guanine–cytosine (G–C) dimer formed in a supersonic jet. We show that there is only one isomer of G–C in the investigated wavelength range from 33200 to 34100 cm−1. We assigned the observed G–C isomer to a specific structure, based on comparisons of the IR spectra of the G and C monomers with the G–C dimer in the range of the OH and NH stretching vibrations and ab initio-calculated vibrational frequencies and dimer stabilities. The cluster exhibits an HNH⋯O/NH⋯N/CO⋯HNH bonding similar to the Watson–Crick G–C base pair bonding but with C as the enol tautomer. We did not observe any keto–keto or enol–enol G–C dimers in the investigated wavelength region.

In this paper we present detailed R2PI spectra with IR–UV and UV–UV double resonance measurements of the guanine dimer (GG) and its methylated derivatives. We show that there are two isomers of GG in the investigated wavelength range from 32565 to 33600 cm−1. We were able to assign the two isomers to specific structures based on comparison of the intermolecular vibronic patterns of the dimers with and without methylation, on analysis of the IR spectra in the range of the OH and NH stretching vibrations and on comparison with ab initio calculated dimer stabilities and vibrational frequencies. In both structures both guanine moieties are in the keto tautomeric form, even though the enol tautomers are also present in the beam. One isomer exhibits nonsymmetric hydrogen bonding with HNH⋯N, NH⋯N and CO⋯HNH interactions (K9K7-2). The other isomer has a symmetrical hydrogen bond arrangement with CO⋯NH/NH⋯OC bonding (K9K7-1). The most stable guanine dimer forms CO⋯NH/NH⋯OC hydrogen bonds and has C2h symmetry (K9K9-1). Due to its strong exciton splitting the allowed S0–S2 transition is outside the investigated spectral range. We did not observe any keto–enol or enol–enol dimers in the investigated wavelength region. The calculations predict these dimers to be considerably less stable.

In this work, the successful operation of a dynamic DNA device constructed from two DNA origami building blocks is reported. The device includes a bipedal walker that strides back and forth between the two origami tiles. Two different DNA origami tiles are first prepared separately; they are then joined together in a controlled manner by a set of DNA strands to form a stable track in high yield as confirmed by single-molecule fluorescence (SMF). Second, a bipedal DNA motor, initially attached to one of the two origami units and operated by sequential interaction with "fuel" and "antifuel" DNA strands, moves from one origami tile to another and then back again. The operational yield, measured by SMF, was similar to that of a motor operating on a similar track embedded in a single origami tile, confirming that the transfer across the junction from one tile to the other does not result in dissociation that is any more than that of steps on a single tile. These results demonstrate that moving parts can reliably travel from one origami unit to another, and it demonstrates the feasibility of dynamic DNA molecular machines that are made of more than a single origami building block. This study is a step toward the development of motors that can stride over micrometer distances.

The vibronic spectrum of laser desorbed and jet cooled guanine consists of bands from three different tautomers of guanine as revealed by UV–UV and IR–UV double resonance spectroscopy. 1-methylguanine, in which the Keto–Enol tautomerism is blocked, shows hole burning spectra from the 9H-and 7H-Keto form. A comparison of the vibronic pattern of the different tautomers demonstrates that the vibronic spectrum built on the redmost guanine band at 32 870 cm−1 (electronic origin 0) can be traced back to the 9H-Enol tautomer, while the spectra built on the origins at 0+404 cm−1 and 0+1044 cm−1 stem from the two Keto tautomers. The IR–UV double resonance spectra of the OH-and NH-stretch vibrations of the different tautomers support this assignment. The UV and IR spectra can be partly assigned by comparison with ab initio calculated vibrational frequencies and with the help of deuteration experiments.

We present R2PI spectra of a series of purine nucleobases, nucleosides, and related derivatives, including adenine, guanine, guanosine, 2-aminopurine, 2,6-diaminopurine, 9-ethyladenine, and 9-methy...

Here we provide high resolution study of DNA hairpin dynamics achieved by probability distribution analysis (PDA) of diffusion-based single-molecule Förster resonance energy transfer (sm-FRET) histograms. The opening and closing rates of three hairpins both free and attached to DNA origami were determined. The agreement with rates previously obtained using the total internal reflection (TIRF) technique and between free hairpins and hairpins attached to origami validated the PDA and demonstrated that the origami had no influence on the hairpin dynamics. From comparison of rates of four DNA hairpins, differing only in stem sequence, and from comparison with rates calculated using nearest-neighbor method and standard transition state theory, we conclude that the unfolding reaction resembles that of melting of DNA duplex with a corresponding sequence and that the folding reaction depends on counterion concentration and not on stem sequence. Our validation and demonstration of the PDA method will encourage its implementation in future high-resolution dynamic studies of freely diffusing biomolecules.

While numerous DNA-based molecular machines have been developed in recent years, high operational yield and speed remain a major challenge. To understand the reasons for the limited performance, and to find rational solutions, we applied single-molecule fluorescence techniques and conducted a detailed study of the reactions involved in the operation of a model system comprised of a bipedal DNA walker that strides on a DNA origami track powered by interactions with fuel and antifuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo-first-order antifuel binding mechanism leading to a rapid and complete leg-lifting, indicating that the fuel-removal reaction is not responsible for the 1% operational yield observed after six steps. Analysis of the leg-placing reactions showed that although increased concentrations of fuel increase the reaction rate, they decrease the yield by consecutively binding the motor and leading to an undesirable trapped state. Recognizing this, we designed asymmetrical hairpin-fuels that by regulating the reaction hierarchy avoid consecutive binding. Motors operating with the improved fuels show 74% yield after 12 consecutive reactions, a dramatic increase over the 1% observed for motors operating with nonhairpin fuels. This work demonstrates that studying the mechanisms of the reactions involved in the operation of DNA-based molecular machines using single-molecule fluorescence can facilitate rationally designed improvements that increase yield and speed and promote the applicability of DNA-based machines.

We introduce an extended version of oxDNA, a coarse-grained model of deoxyribonucleic acid (DNA) designed to capture the thermodynamic, structural, and mechanical properties of single- and double-stranded DNA. By including explicit major and minor grooves and by slightly modifying the coaxial stacking and backbone-backbone interactions, we improve the ability of the model to treat large (kilobase-pair) structures, such as DNA origami, which are sensitive to these geometric features. Further, we extend the model, which was previously parameterised to just one salt concentration ([Na(+)] = 0.5M), so that it can be used for a range of salt concentrations including those corresponding to physiological conditions. Finally, we use new experimental data to parameterise the oxDNA potential so that consecutive adenine bases stack with a different strength to consecutive thymine bases, a feature which allows a more accurate treatment of systems where the flexibility of single-stranded regions is important. We illustrate the new possibilities opened up by the updated model, oxDNA2, by presenting results from simulations of the structure of large DNA objects and by using the model to investigate some salt-dependent properties of DNA.

The vibronic spectrum of the adenine ± thymine (A ± T) base pair was obtained byone-color resonant two-photon ionization (R2PI) spectroscopyin a free jet of thermallyevaporated A and T under conditions favorable for formation of small clusters. The onset of the spectrum at 35 064 cm 1 exhibits a large red shift relative to the ±* origin of 9H-adenine at 36 105 cm 1 . The IR ± UV spectrum was assigned to cluster structures with HNH ¥¥¥ OC/N ¥¥¥ HN hydrogen bonding bycomparison with the IR spectra of A and T monomers and with ab initio calculated vibrational spectra of the most stable A ± T isomers. The Watson ± Crick A ± T base pair is not the most stable base-pair structure at different levels of ab initio theory, and its vibrational spectrum is not in agreement with the observed experimental spectrum. Experiments with methylated A and T were performed to further support the structural assignment.

The two intertwined strands of DNA are held together through base pairing—the formation of hydrogen bonds between bases located opposite each other on the two strands. DNA replication and transcription involve the breaking and re-forming of these hydrogen bonds, but it is difficult to probe these processes directly. For example, conventional DNA spectroscopy1,2,3 is dominated by solvent interactions, crystal modes and collective modes of the DNA backbone; gas-phase studies, in contrast, can in principle measure interactions between individual molecules in the absence of external effects, but require the vaporization of the interacting species without thermal degradation4,5,6,7,8,9. Here we report the generation of gas-phase complexes comprising paired bases, and the spectroscopic characterization of the hydrogen bonding in isolated guanine–cytosine (G–C) and guanine–guanine (G–G) base pairs. We find that the gas-phase G–C base pair adopts a single configuration, which may be Watson–Crick, whereas G–G exists in two different configurations, and we see evidence for proton transfer in the G–C pair, an important step in radiation-induced DNA damage pathways10. Interactions between different bases and between bases and water molecules can also be characterized by our approach, providing stringent tests for high-level ab initio computations that aim to elucidate the fundamental aspects of nucleotide interactions11,12,13.

Figure 1. REMPI spectrum of jet-cooled guanine. The spectrum consists of four parts, as indicated by the horizontal arrows, that were recorded independently. In each part the ionization laser fluence varies such that it is highest in the center of the respective range. The peaks are not normalized for laser fluence. The top panel shows IR data from ref 8, plotted relative to the lowest energy peak in the REMPI spectrum for comparison.