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Register a new userRandomness and optimality in enhanced DNA ligation with crowding effects
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Enzymatic ligation is essential for the synthesis of long DNA. However, the number of ligated products exponentially decays as the DNA synthesis proceeds in a random manner. The controlling of ligation randomness is of importance to suppress exponential decay and demonstrate an efficient synthesis of long DNA. Here, we report the analysis of randomness in sequential DNA ligations, named qPCR-based STatistical Analysis of Randomness (qPCR-bSTAR), by a probability distribution of ligated DNA concentration. We show that the exponential decay is suppressed in a solution of another polymer and DNA ligation is activated at an optimal crowded condition. Theoretical model of kinetic ligation explains that intermolecular attraction due to molecular crowding can be involved in the optimal balance of the ligation speed and the available ligase. Our finding indicates that crowding effects enhance the synthesis of long DNA that retains large genetic information.
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Coordinating functional parts to operate in concert is essential for machinery. In gear trains, meshed gears are compactly interlocked, working together to impose rotation or translation. In photosynthetic systems, a variety of biological entities in the thylakoid membrane interact with each other, converting light energy into chemical energy. However, coordinating individual parts to carry out regulated and coordinated motion within an artificial nanoarchitecture poses challenges, owing to the requisite control on the nanoscale. Here, we demonstrate DNA-directed nanosystems, which comprise hierarchically-assembled DNA origami filaments, fluorophores, and gold nanocrystals. These individual building blocks can execute independent, synchronous, or joint motion upon external inputs. These are optically monitored in situ using fluorescence spectroscopy, taking advantage of the sensitive distance-dependent interactions between the gold nanocrystals and fluorophores positioned on the DNA origami. Our work leverages the complexity of DNA-based artificial nanosystems with tailored dynamic functionality, representing a viable route towards technomimetic nanomachinery.
We perform a spatially resolved simulation study of an AND gate based on DNA strand displacement using several lengths of the toehold and the adjacent domains. DNA strands are modelled using a coarse-grained dynamic bonding model {[}C. Svaneborg, Comp. Phys. Comm. 183, 1793 (2012){]}. We observe a complex transition path from the initial state to the final state of the AND gate. This path is strongly influenced by non-ideal effects due to transient bubbles revealing undesired toeholds and thermal melting of whole strands. We have also characterized the bound and unbound kinetics of single strands, and in particular the kinetics of the total AND operation and the three distinct distinct DNA transitions that it is based on. We observe a exponential kinetic dependence on the toehold length of the competitive displacement operation, but that the gate operation time is only weakly dependent on both the toehold and adjacent domain length. Our gate displays excellent logical fidelity in three input states, and quite poor fidelity in the fourth input state. This illustrates how non-ideality can have very selective effects on fidelity. Simulations and detailed analysis such as those presented here provide molecular insights into strand displacement computation, that can be also be expected in chemical implementations.
Biological materials are self-assembled with near-atomic precision in living cells, whereas synthetic 3D structures generally lack such precision and controllability. Recently, DNA nanotechnology, especially DNA origami technology, has been useful in the bottom-up fabrication of well-defined nanostructures ranging from tens of nanometres to sub-micrometres. In this Primer, we summarize the methodologies of DNA origami technology, including origami design, synthesis, functionalization and characterization. We highlight applications of origami structures in nanofabrication, nanophotonics and nanoelectronics, catalysis, computation, molecular machines, bioimaging, drug delivery and biophysics. We identify challenges for the field, including size limits, stability issues and the scale of production, and discuss their possible solutions. We further provide an outlook on next-generation DNA origami techniques that will allow in vivo synthesis and multiscale manufacturing.
We investigate the distribution of bubble lifetimes and bubble lengths in DNA at physiological temperature, by performing extensive molecular dynamics simulations with the Peyrard-Bishop-Dauxois (PBD) model, as well as an extended version (ePBD) having a sequence-dependent stacking interaction, emphasizing the effect of the sequences guanine-cytosine (GC)/adenine-thymine (AT) content on these distributions. For both models we find that base pair-dependent (GC vs AT) thresholds for considering complementary nucleotides to be separated are able to reproduce the observed dependence of the melting temperature on the GC content of the DNA sequence. Using these thresholds for base pair openings, we obtain bubble lifetime distributions for bubbles of lengths up to ten base pairs as the GC content of the sequences is varied, which are accurately fitted with stretched exponential functions. We find that for both models the average bubble lifetime decreases with increasing either the bubble length or the GC content. In addition, the obtained bubble length distributions are also fitted by appropriate stretched exponential functions and our results show that short bubbles have similar likelihoods for any GC content, but longer ones are substantially more likely to occur in AT-rich sequences. We also show that the ePBD model permits more, longer-lived, bubbles than the PBD system.
Single-molecule experiments provide new insights into biological processes hitherto not accessible by measurements performed on bulk systems. We report on a study of the kinetics of a triple-branch DNA molecule with four conformational states by pulling experiments with optical tweezers and theoretical modelling. Three distinct force rips associated with different transitions between the conformational states are observed in the folding and unfolding trajectories. By applying transition rate theory to a free energy model of the molecule, probability distributions for the first rupture forces of the different transitions are calculated. Good agreement of the theoretical predictions with the experimental findings is achieved. Furthermore, due to our specific design of the molecule, we found a useful method to identify permanently frayed molecules by estimating the number of opened basepairs from the measured force jump values.
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