DNA bipedal motor walking dynamics: an experimental and theoretical study of the dependency on step size.

We present a detailed coarse-grained computer simulation and single molecule fluorescence study of the walking dynamics and mechanism of a DNA bipedal motor striding on a DNA origami. In particular, we study the dependency of the walking efficiency and stepping kinetics on step size. The simulations accurately capture and explain three different experimental observations. These include a description of the maximum possible step size, a decrease in the walking efficiency over short distances and a dependency of the efficiency on the walking direction with respect to the origami track. The former two observations were not expected and are non-trivial. Based on this study, we suggest three design modifications to improve future DNA walkers. Our study demonstrates the ability of the oxDNA model to resolve the dynamics of complex DNA machines, and its usefulness as an engineering tool for the design of DNA machines that operate in the three spatial dimensions.

Self-Assembly of DNA Origami Heterodimers in High Yields and Analysis of the Involved Mechanisms.

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.

Study of DNA Origami Dimerization and Dimer Dissociation Dynamics and of the Factors that Limit Dimerization.

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.

Photon-by-Photon Hidden Markov Model Analysis for Microsecond Single-Molecule FRET Kinetics.

The function of biological macromolecules involves large-scale conformational dynamics spanning multiple time scales, from microseconds to seconds. Such conformational motions, which may involve whole domains or subunits of a protein, play a key role in allosteric regulation. There is an urgent need for experimental methods to probe the fastest of these motions. Single-molecule fluorescence experiments can in principle be used for observing such dynamics, but there is a lack of analysis methods that can extract the maximum amount of information from the data, down to the microsecond time scale. To address this issue, we introduce H2MM, a maximum likelihood estimation algorithm for photon-by-photon analysis of single-molecule fluorescence resonance energy transfer (FRET) experiments. H2MM is based on analytical estimators for model parameters, derived using the Baum-Welch algorithm. An efficient and effective method for the calculation of these estimators is introduced. H2MM is shown to accurately retrieve the reaction times from ∼1 s to ∼10 µs and even faster when applied to simulations of freely diffusing molecules. We further apply this algorithm to single-molecule FRET data collected from Holliday junction molecules and show that at low magnesium concentrations their kinetics are as fast as ∼104 s-1. The new algorithm is particularly suitable for experiments on freely diffusing individual molecules and is readily incorporated into existing analysis packages. It paves the way for the broad application of single-molecule fluorescence to study ultrafast functional dynamics of biomolecules.