Search query: A DNA- fuelled molecular machine made of DNA

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abstract: Molecular recognition between complementary strands of DNA allows construction on a nanometre length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semiconductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible for example, a nanomechanical switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chemically modified DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymatic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel' The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addition of auxiliary strands of 'fuel' DNA; each cycle produces a duplex DNA waste product.
author_names: Bernard Yurke

Andrew J Turberfield

Allen P Mills

Friedrich C Simmel

Jennifer Neumann
title: A DNA fuelled molecular machine made of DNA
venue: Nature
year: 2000

abstract: Molecular recognition between complementary strands of DNA allows construction on a nanometre length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semiconductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possibleDfor example, a nanomechanical switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chemically modi(r)ed DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymatic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as `fuel' The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addition of auxiliary strands of `fuel' DNA; each cycle produces a duplex DNA waste product. Single strands of DNA composed of complementary sequences of the bases adenine, cytosine, guanine and thymine (A, C, G and T) hybridize to form a stable duplex (double helix) bound together by hydrogen bonds between complementary base pairs (A+ T and C+ G) Our machine is prepared by mixing stoichiometric quantities of three strands, A, B and C, in SPSC buffer (see Methods) at 20 8C to give a (r)nal concentration of 1 mM; the base sequences chosen for the three strands are given in Fig. 1. The structure and operation of the machine are shown in Fig. 2. Strand A consists of two 18 base sequences which hybridize with complementary sequences at the ends of strands B and C to form two stiff arms; the hinge is formed from a four base single stranded region of A between the regions hybridized to strands B and C. In the machine's rest state, the remaining unhybridized 24 base portions of the 42 base strands B and C dangle oppily from the ends of the tweezers: double stranded DNA has a persistence length of the order of 100 base pairs, whereas at 1 M salt concentration singlestranded DNA has a persistence length of about 1 nm (ref. 16) or approximately three bases. Strand A is labelled at the 59 and 39 ends with dyes TET (59 tetrachlorouorescein phosphoramidite) and TAMRA (carboxytetramethylrhodamine) respectively. When TET is excited by the 514.5 nm emission of an argon ion laser, it uoresces with a peak emission wavelength of 536 nm; this emission is quenched by resonant intramolecular energy transfer from TET to TAMRA (a longer wavelength dye whose absorption band overlaps the emission band of TET) with an ef(r)ciency that decreases rapidly as the distance between the dyes increases. Fluorescence quenching is used as an indicator to titrate strands B and C against A; as half of A is straightened from a random coil by hybridization with B (or C) the mean separation between the dye groups on A increases leading to a (r)vefold increase in the uorescence intensity. The cumulative effect of hybridization with both B and C is a sevenfold increase in uorescence intensity in the rest state. The assembled tweezers are opened and closed with fuel strands F and FA. The 56 base closing strand F consists of two consecutive 24base sections, which are complementary to the dangling ends of B and C, with an additional 8 base overhang section. Figure 2b shows how closing strand F hybridizes with the free ends of strands B and C, pulling the ends of the tweezers together. The average free energy change associated with the hybridization of a complementary base pair is 78 meV 1.8 kcal mol) at 20 8C (ref. 19) and the separation between base pairs in single stranded DNA is 0.43 nm (ref. 20) giving an average closing force of about 15 pN which is consistent with that required to pull apart double stranded DNA. This is at the upper end of the range of measured forces exerted by singlegroup kinesin and myosin motors. The second component of the fuel is removal strand FA, the complement of F: the additional free energy gained when the overhang (single stranded in the initial state) hybridizes with FA ensures that when a stoichiometric quantity of FA is added it removes F from the machine to form a doublestranded waste product FFA and returns the tweezers to the open state. Hybridization between the closing and removal fuel strands is expected to occur (r)rst at the exposed overhang and to proceed by branch migration, a random walk of the junction between the region of F newly hybridized to the removal strand FA and the region still hybridized to the tweezer components B and C; this branch migration continues until both B and C have been completely displaced and the FFA duplex diffuses away. The random walk occurs suf(r)ciently quickly that the rate limiting step in such strand displacement reactions is the endothermic nucleation of a region in which complementary bases are joined (in this case, the nucleation of hybridization between the F and FA strands) this
author_names: Nathalie Capron

Francesco G Gatti

FrancEois Kajzar

David A Leigh

Francesco Zerbetto

Songwei Zhang
title: A DNA fuelled molecular machine made of DNA

abstract: The predictable and specific hybridization of complementary strands of DNA allows construction on a nanometre length scale: (i) DNA tags may be used to organize the assembly of colloidal particles; (ii) DNA may be used as a structural material in its own right to make complicated objects, e.g. ordered arrays of tiles, linked rings and polyhedra; (iii) DNA may act as a template to direct the growth of semiconductor nanocrystals and metal wires. These are static structures: Seeman and co workers have produced an active device, a nanomechanical switch whose conformation is changed by inducing a B Z transition (from a rightto a left handed double helix) by changing the composition of the buffer solution. Conformational changes caused by the binding of oligonucleotides or other small molecules have been shown to change the enzymatic activity of ribozymes We report the construction of a new class of active nanostructure: a machine in which DNA is used not only as a structural material but also as a fuel.
author_names: Bernard Yurke

Andrew J Turberfield Mills
title: A molecular machine made and powered by DNA
year: 2000

abstract: Significance The circadian clock is an internal molecular 24 h timer that is critical to life on Earth. We describe a series of artificial intelligence (AI) and machine learning (ML) based approaches that enable more cost effective analysis and insight into circadian regulation and function. Throughout the manuscript, we illuminate what is inside the ML "black box" via explanation or interpretation of predictive ML models. Using this interpretation of our models, we derive biological insights into why a prediction was made, alongside accurate predictions. Most innovatively, we use only DNA sequence features for accurate circadian gene expression prediction. Using explainable AI, we define possible, responsible regulatory elements as we make these predictions; this critically requires no prior knowledge of regulatory elements. The circadian clock is an important adaptation to life on Earth. Here, we use machine learning to predict complex, temporal, and circadian gene expression patterns in Arabidopsis. Most significantly, we classify circadian genes using DNA sequence features generated de novo from public, genomic resources, facilitating downstream application of our methods with no experimental work or prior knowledge needed. We use local model explanation that is transcript specific to rank DNA sequence features, providing a detailed profile of the potential circadian regulatory mechanisms for each transcript. Furthermore, we can discriminate the temporal phase of transcript expression using the local, explanation derived, and ranked DNA sequence features, revealing hidden subclasses within the circadian class. Model interpretation/explanation provides the backbone of our methodological advances, giving insight into biological processes and experimental design. Next, we use model interpretation to optimize sampling strategies when we predict circadian transcripts using reduced numbers of transcriptomic timepoints. Finally, we predict the circadian time from a single, transcriptomic timepoint, deriving marker transcripts that are most impactful for accurate prediction; this could facilitate the identification of altered clock function from existing datasets.
author_names: Laura-Jayne Gardiner

Rachel Rusholme-Pilcher

Joshua Colmer

Hannah Rees

Juan Manuel Crescente

Anna Paola Carrieri

Susan Duncan

Edward O Pyzer-Knapp

Ritesh Krishna
title: Interpreting machine learning models to investigate circadian regulation and facilitate exploration of clock function
venue: Proceedings of the National Academy of Sciences
year: 2021

abstract: Significance DNA mismatch repair (MMR) is critical to avoid mutations that can lead to genetic disease, cancer, and death. The MMR system is evolutionarily conserved from bacteria to humans and is an example of a remarkable molecular machine. Here we show that this machine operates unidirectionally with respect to the chromosome of Escherichia coli. The most likely explanation for this directionality is that the MMR machinery is associated with the complex responsible for DNA replication. We suggest that this association facilitates the mechanism of strand discrimination in MMR. Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5' to 3' and the 3' to 5' directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3 bp insertion deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication.
author_names: A M Mahedi Hasan

David R F Leach
title: Chromosomal directionality of DNA mismatch repair in Escherichia coli
venue: Proceedings of the National Academy of Sciences
year: 2015

abstract: Significance Tailed bacteriophages and herpes viruses use powerful molecular machines to package their genomes into a viral capsid using ATP as fuel. Recent biophysical and structural studies have provided a detailed picture of mechanochemistry of DNA packaging. However, little is known about the packaging initiation step owing to its transient nature. Here, we reconstituted the bacteriophage T4 DNA packaging machine and imaged individual packaging events in real time. We discovered that initiations occur in bursts and through multiple pathways, including direct capture of DNA by the capsid portal, and they require rapid input of energy, analogous to the cranking of an engine. This system opens a new window into the mechanism of viral genome packaging initiation and the evolution of icosahedral viruses. Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.
author_names: Reza Vafabakhsh

Kiran Kondabagil

Tyler M Earnest

Kyung Suk Lee

Zhihong Zhang

Li Dai

Karin A Dahmen

Venigalla B Rao

Taekjip Ha
title: Single molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4
venue: Proceedings of the National Academy of Sciences
year: 2014

abstract: In this work, biomolecules, such as membrane proteins, lipids, and DNA, were identified and their spatial distribution was mapped within a single Escherichia coli cell by Raman hyperspectral imaging. Raman spectroscopy allows direct, nondestructive, rapid, and cost effective analysis of biological samples, minimizing the sample preparation and without the need of chemical label or immunological staining. Firstly, a comparison between an air dried and a freeze dried cell was made, and the principal vibrational modes associated to the membrane and nucleic acids were identified by the bacterium's Raman chemical fingerprint. Then, analyzing the Raman hyperspectral images by multivariate statistical analysis, the bacterium biological status was investigated at a subcellular level. Principal components analysis (PCA) was applied for dimensionality reduction of the spectral data, then spectral unmixing was performed by multivariate curve resolution alternating least squares (MCR ALS) Thanks to multivariate data analysis, the DNA segregation and the Z ring formation of a replicating bacterial cell were detected at a sub micrometer level, opening the way to real time molecular analysis that could be easily applied on in vivo or ex vivo biological samples, avoiding long preparation and analysis process.
author_names: Giulia Barzan

Alessio Sacco

Luisa Mandrile

Andrea Mario Giovannozzi

Chiara Portesi

Andrea Mario Rossi
title: Hyperspectral Chemical Imaging of Single Bacterial Cell Structure by Raman Spectroscopy and Machine Learning
year: 2021

abstract: The anti tumor potency of poly(ADP ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single strand DNA breaks through PARP2 trapping, potentiates PARPi induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.
author_names: Charlotte Blessing

Imke K Mandemaker

Claudia Gonzalez-Leal

Julia Preisser

Adrian Schomburg

Andreas G Ladurner
title: The Oncogenic Helicase ALC1 Regulates PARP Inhibitor Potency by Trapping PARP2 at DNA Breaks.
venue: Molecular cell
year: 2020

abstract: Machines undergo repeated energy (fuel) driven cycling between various functional and structural states. Such cycling involves motions of the machine parts, which take place in a highly coordinated manner in time and space. This description applies not only to man made machines but also to the molecular machines that mediate many of the key processes in all forms of life [1] Examples include protein synthesis by the ribosome, DNA unwinding by helicases during replication, protein degradation by the proteasome and protein folding by chaperones. The coordinated movements of most biomolecular machines are achieved by allosteric regulation of ATP binding and hydrolysis. Consequently, a molecular level understanding of how the essential machines of life work requires invoking and further developing allosteric theory. Hence, the motivation for the Royal Society Discussion Meeting entitled 'Allostery and molecular machines' that took place in June 2017. The meeting brought together scientists interested in allostery, in general, with others who are studying specific biomolecular machines of interest. The meeting led to many interesting discussions and resulted in this special issue of the Philosophical Transactions B In 1965, Monod, Wyman Changeux published a seminal paper [2] that described a so called 'concerted model' (referred to here and generally as the MWC model) for cooperative ligand binding by oligomeric proteins. This issue contains a paper by one of the co authors of this landmark paper in which the MWC model is explained in brief and its application to the nicotinic acetylcholine receptor is then described [3] The MWC model remains important but its elegance has come at the price of several restrictive assumptions. One key assumption is that cooperativity is due to a shift in equilibrium between different quaternary
author_names: George H Lorimer

Amnon Horovitz

Tom C B McLeish
title: Allostery and molecular machines
venue: Philosophical Transactions of the Royal Society B: Biological Sciences
year: 2018

abstract: Molecular machines have previously been designed that are propelled by DNAzymes1 3, protein enzymes4 6 and strand displacement7 9. These engineered machines typically move along precisely defined one and two dimensional tracks. Here, we report a DNA walker that uses hybridisation to drive walking on DNA coated microparticle surfaces. Through purely DNA:DNA hybridisation reactions, the nanoscale movements of the walker can lead to the generation of a single stranded product and the subsequent immobilisation of fluorescent labels on the microparticle surface. This suggests that the system could be of use in analytical and diagnostic applications, similar to how strand exchange reactions in solution have been used for transducing and quantifying signals from isothermal molecular amplification assays10,11. The walking behaviour is robust and the walker can take more than 30 continuous steps. The traversal of an unprogrammed, inhomogeneous surface is also due entirely to autonomous decisions made by the walker, behaviour analogous to amorphous chemical reaction network computations12,13 that have been shown to lead to pattern formation14 17.
author_names: Cheulhee Jung

Peter B Allen

Andrew D Ellington
title: A stochastic DNA walker that traverses a microparticle surface
venue: Nature nanotechnology
year: 2016